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PerfeCTa qPCR ToughMix

Tough-tested real-time PCR
Features & Benefits
  • ToughMix additives neutralize PCR inhibitors to ensure reliable assay performance with a spectrum of starting materials including: clinical specimens, plants, soil, environmental or complex food matrices
  • Superior assay sensitivity and target precision with AccuStart™ II enzyme technology – maximum-yielding, engineered Taq DNA polymerase with stringent, ultrapure antibody hotstart
  • Easy-to-use 2X concentrated master mixes with AccuVue™ plate loading dye and optimized passive reference dye for simplified reaction setup
  • Supports efficient vortex mixing with proprietary anti-foaming technology
  • Consistent and reliable performance with exceptional reagent stability (uninhibited after 20X freeze-thaw or 30 days at 22°C)

 

PerfeCTa qPCR ToughMix is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.

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PerfeCTa qPCR ToughMix

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PerfeCTa qPCR ToughMix ROX

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PerfeCTa qPCR ToughMix Low ROX

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PerfeCTa qPCR ToughMix UNG

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Part Number:  95138-012
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PerfeCTa qPCR ToughMix UNG ROX

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Kit Size:  1250 x 20 μL rxns (10 x 1.25 mL)
Part Number:  95139-012
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PerfeCTa qPCR ToughMix UNG Low ROX

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Description

PerfeCTa qPCR ToughMix is a 2X concentrated ready-to-use reaction cocktail for PCR amplification of DNA templates that relieves several types of PCR inhibition commonly encountered with crude extracts, environmental specimens, plant tissues, animal tissues, and complex food matrices. This robust real-time qPCR reagent provides maximum sensitivity and PCR efficiency with a variety of fluorogenic probe chemistries, including TaqMan® hydrolysis probes. The only user-supplied components are primers, probe(s), and DNA template. Pre-blended with inert AccuVue plate loading dye to help minimize pipette errors during setup and provides visual confirmation of thorough mixing. A key component of PerfeCTa qPCR ToughMix is an ultra pure, highly processive thermostable DNA polymerase that is combined with high avidity monoclonal antibodies. This proprietary polymerase mix is highly resistant to PCR inhibitors and provides an extremely stringent automatic hot-start allowing reaction assembly, and temporary storage, at room temperature prior to PCR amplification. PerfeCTa qPCR ToughMix delivers exceptional performance with either fast or conventional PCR cycling protocols. UNG containing versions are blended with Uracil N-glycosylase to eliminate potential post-PCR carryover contamination associated with routine molecular testing.
Details

Details

Contents

Single-tube, 2X concentrated reagent containing:

  • Reaction buffer with optimized concentrations of molecular-grade MgCl2, dATP, dCTP, dGTP, dTTP.
  • AccuStart II Taq DNA Polymerase.
  • Inert AccuVue dye.
  • Proprietary enzyme stabilizers and performance-enhancing additives.
  • Titrated reference dye (if applicable).
Instrument Capability

Instrument Capability

ROX

  • Applied Biosystems 5700
  • Applied Biosystems 7000
  • Applied Biosystems 7300
  • Applied Biosystems 7700
  • Applied Biosystems 7900
  • Applied Biosystems 7900HT
  • Applied Biosystems 7900 HT Fast
  • Applied Biosystems StepOne™
  • Applied Biosystems StepOnePlus™

Low ROX

  • Applied Biosystems 7500
  • Applied Biosystems 7500 Fast
  • Stratagene Mx3000P®
  • Stratagene Mx3005P™
  • Stratagene Mx4000™
  • Applied Biosystems ViiA 7
  • Applied Biosystems QuantStudio™ (all models)
  • Douglas Scientific IntelliQube®

No ROX

  • Quantabio Q
  • BioRad CFX
  • Roche LightCycler 480
  • QIAGEN Rotor-Gene Q
  • Agilent AriaMx
  • Azure Cielo™
  • Other

Bio-Rad iCycler iQ systems

  • BioRad iCycler iQ™
  • BioRad MyiQ™
  • BioRad iQ™5
Customer Testimonials

Customer Testimonials

PerfeCTa qPCR ToughMix

"This eliminates a costly and time consuming step to remove inhibitors manually, so it has saved us money on reagents and time for our small lab."

Max O. | Conservationist
PerfeCTa qPCR ToughMix

"Higher sensitivity, positives at a lower Cq. Great results with plant DNA with high inhibition level."

Paula S. | Lab Coordinator INIAV

Details

Contents

Single-tube, 2X concentrated reagent containing:

  • Reaction buffer with optimized concentrations of molecular-grade MgCl2, dATP, dCTP, dGTP, dTTP.
  • AccuStart II Taq DNA Polymerase.
  • Inert AccuVue dye.
  • Proprietary enzyme stabilizers and performance-enhancing additives.
  • Titrated reference dye (if applicable).

Instrument Capability

ROX

  • Applied Biosystems 5700
  • Applied Biosystems 7000
  • Applied Biosystems 7300
  • Applied Biosystems 7700
  • Applied Biosystems 7900
  • Applied Biosystems 7900HT
  • Applied Biosystems 7900 HT Fast
  • Applied Biosystems StepOne™
  • Applied Biosystems StepOnePlus™

Low ROX

  • Applied Biosystems 7500
  • Applied Biosystems 7500 Fast
  • Stratagene Mx3000P®
  • Stratagene Mx3005P™
  • Stratagene Mx4000™
  • Applied Biosystems ViiA 7
  • Applied Biosystems QuantStudio™ (all models)
  • Douglas Scientific IntelliQube®

No ROX

  • Quantabio Q
  • BioRad CFX
  • Roche LightCycler 480
  • QIAGEN Rotor-Gene Q
  • Agilent AriaMx
  • Azure Cielo™
  • Other

Bio-Rad iCycler iQ systems

  • BioRad iCycler iQ™
  • BioRad MyiQ™
  • BioRad iQ™5

Performance Data

Resources

Customer Profile Stories

Flyers

Product Manuals

Application Notes

CofA (PSFs)

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SDSs

Publications

Study of Shellfish Growing Area During Normal Harvesting Periods and Following Wastewater Overflows in an Urban Estuary With Complex Hydrography
Carlos J. A. Campos - 2024
Abstract
Viral testing combined with hydrographic studies is considered standard good practice in determining microbiological impacts on shellfish growing areas following wastewater overflows. In this study, norovirus genogroup I and II, indicators of viral contamination (F-RNA bacteriophage genogroup II (F-RNA GII), crAssphage, pepper mild mottle virus) and Escherichia coli were monitored during periods of normal harvesting and following overflows in two commercial shellfish growing areas in Otago Harbour (Aotearoa New Zealand). Dye tracing, drogue tracking and analysis of particle tracking modelling were also undertaken to assess the dispersion, dilution and time of travel of wastewater discharged from a pump station discharge that impacts the growing areas. Norovirus was not detected in any of the 218 shellfish samples tested. PMMoV and crAssphage were more prevalent than F-RNA GII as determined by RT-qPCR. The dye study indicated long residence time of the waters (≥5 days) in the embayment impacted by the discharge. No relationships were found between the concentrations of viral indicators or E. coli and wastewater dilution, distance between the discharge and the growing areas or time since the last overflow. For the three spills studied (≤327 m3), there was little evidence of microbiological impact on the growing areas. This was likely associated with a deep shipping channel that enhances water flushing in the harbour and reduces contaminant transport to the growing areas. We recommend flexibility in the approach for closure/reopening growing areas impacted by spills, particularly for small duration/volume spills and when norovirus is not present in the community.
IgM N-glycosylation correlates with COVID-19 severity and rate of complement deposition
Benjamin S. Haslund-Gourley - 2024
Abstract
The glycosylation of IgG plays a critical role during human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, activating immune cells and inducing cytokine production. However, the role of IgM N-glycosylation has not been studied during human acute viral infection. The analysis of IgM N-glycosylation from healthy controls and hospitalized coronavirus disease 2019 (COVID-19) patients reveals increased high-mannose and sialylation that correlates with COVID-19 severity. These trends are confirmed within SARS-CoV-2-specific immunoglobulin N-glycan profiles. Moreover, the degree of total IgM mannosylation and sialylation correlate significantly with markers of disease severity. We link the changes of IgM N-glycosylation with the expression of Golgi glycosyltransferases. Lastly, we observe antigen-specific IgM antibody-dependent complement deposition is elevated in severe COVID-19 patients and modulated by exoglycosidase digestion. Taken together, this work links the IgM N-glycosylation with COVID19 severity and highlights the need to understand IgM glycosylation and downstream immune function during human disease
Molecular surveillance of Kelch 13 polymorphisms in Plasmodium falciparum isolates from Kenya and Ethiopia
Brook Jeang - 2024
Abstract
Background Timely molecular surveillance of Plasmodium falciparum kelch 13 (k13) gene mutations is essential for monitoring the emergence and stemming the spread of artemisinin resistance. Widespread artemisinin resistance, as observed in Southeast Asia, would reverse significant gains that have been made against the malaria burden in Africa. The purpose of this study was to assess the prevalence of k13 polymorphisms in western Kenya and Ethiopia at sites representing varying transmission intensities between 2018 and 2022. Methods Dried blood spot samples collected through ongoing passive surveillance and malaria epidemiological studies, respectively, were investigated. The k13 gene was genotyped in P. falciparum isolates with high parasitaemia: 775 isolates from four sites in western Kenya (Homa Bay, Kakamega, Kisii, and Kombewa) and 319 isolates from five sites across Ethiopia (Arjo, Awash, Gambella, Dire Dawa, and Semera). DNA sequence variation and neutrality were analysed within each study site where mutant alleles were detected. Results Sixteen Kelch13 haplotypes were detected in this study. Prevalence of nonsynonymous k13 mutations was low in both western Kenya (25/783, 3.19%) and Ethiopia (5/319, 1.57%) across the study period. Two WHO-validated mutations were detected: A675V in three isolates from Kenya and R622I in four isolates from Ethiopia. Seventeen samples from Kenya carried synonymous mutations (2.17%). No synonymous mutations were detected in Ethiopia. Genetic variation analyses and tests of neutrality further suggest an excess of low frequency polymorphisms in each study site. Fu and Li’s F test statistic in Semera was 0.48 (P > 0.05), suggesting potential population selection of R622I, which appeared at a relatively high frequency (3/22, 13.04%). Conclusions This study presents an updated report on the low frequency of k13 mutations in western Kenya and Ethiopia. The WHO-validated R622I mutation, which has previously only been reported along the north-west border of Ethiopia, appeared in four isolates collected from eastern Ethiopia. The rapid expansion of R622I across Ethiopia signals the need for enhanced monitoring of the spread of drug-resistant P. falciparum parasites in East Africa. Although ACT remains currently efficacious in the study areas, continued surveillance is necessary to detect early indicators of artemisinin partial resistance.
High-resolution genomic analysis to investigate the impact of the invasive brushtail possum (Trichosurus vulpecula) and other wildlife on microbial water quality assessments
Marie Moine - 2024
Abstract
Escherichia coli are routine indicators of fecal contamination in water quality assessments. Contrary to livestock and human activities, brushtail possums (Trichosurus vulpecula), common invasive marsupials in Aotearoa/New Zealand, have not been thoroughly studied as a source of fecal contamination in freshwater. To investigate their potential role, Escherichia spp. isolates (n = 420) were recovered from possum gut contents and feces and were compared to those from water, soil, sediment, and periphyton samples, and from birds and other introduced mammals collected within the Mākirikiri Reserve, Dannevirke. Isolates were characterized using E. coli-specific real-time PCR targeting the uidA gene, Sanger sequencing of a partial gnd PCR product to generate a gnd sequence type (gST), and for 101 isolates, whole genome sequencing. Escherichia populations from 106 animal and environmental sample enrichments were analyzed using gnd metabarcoding. The alpha diversity of Escherichia gSTs was significantly lower in possums and animals compared with aquatic environmental samples, and some gSTs were shared between sample types, e.g., gST535 (in 85% of samples) and gST258 (71%). Forty percent of isolates gnd-typed and 75% of reads obtained by metabarcoding had gSTs shared between possums, other animals, and the environment. Core-genome single nucleotide polymorphism (SNP) analysis showed limited variation between several animal and environmental isolates (<10 SNPs). Our data show at an unprecedented scale that Escherichia clones are shared between possums, other wildlife, water, and the wider environment. These findings support the potential role of possums as contributors to fecal contamination in Aotearoa/New Zealand freshwater. Our study deepens the current knowledge of Escherichia populations in under-sampled wildlife. It presents a successful application of high-resolution genomic methods for fecal source tracking, thereby broadening the analytical toolbox available to water quality managers. Phylogenetic analysis of isolates and profiling of Escherichia populations provided useful information on the source(s) of fecal contamination and suggest that comprehensive invasive species management strategies may assist in restoring not only ecosystem health but also water health where microbial water quality is compromised.
FUNCTIONAL AND BIOCHEMICAL CHARACTERIZATION OF VIRAL AND HOST FACTORS INVOLVED IN THE ZIKA VIRUS REPLICATION CYCLE
M. Sc. Sarah Göllner - 2023
Abstract
rboviruses are one of the greatest threats to human health since they cause millions of infections each year. The mosquito-borne flavivirus Zika virus (ZIKV), which is listed as a priority disease, has been declared a public health emergency of global concern by the World Health Organization (WHO) in 2016. Even though most infections are asymptomatic or manifest only moderate symptoms, infections have been related to neurological disorders such as the GuillainBarré syndrome and microcephaly. Despite years of intensive research, there are presently no effective antiviral therapies or vaccines. Their development requires further studies of the fundamental principles of the ZIKV life cycle to provide the necessary knowledge for the development of effective therapeutic measures. Upon ZIKV infection, the cell’s endoplasmic reticulum (ER) is significantly remodeled resulting in the formation of defined ER membrane invaginations termed vesicle packets (VPs), which are thought to be the sites where viral RNA replication takes place. Since their formation is coupled to viral replication, it has been challenging to retrieve mechanistic information on VP biogenesis thus far, as perturbations that reduce viral replication inevitably impact VP formation. To overcome this hurdle, my first project was to develop an expression system supporting the formation of ZIKV VPs to enable research focusing entirely on their biogenesis and the factors involved. I was able to demonstrate that ZIKV VPs are forming upon expression of the viral replicase (NS1-NS5) via a construct comprising in addition the 5’ and 3’ untranslated region (UTRs). Ultrastructural characterization of VPs in transfected cells revealed that the VPs are morphologically identical to those produced in infected cells. This newly created system was given the name pIRO (plasmid-induced replication organelle). A thorough deletion mutagenesis study focusing on RNA elements contained in the 5’ and 3’UTR revealed that for VP formation the 5’UTR is largely dispensable. In contrast, the 3’UTR was required as deletion of distinct RNA elements contained therein decreased the efficiency of VP induction. In conclusion, I was able to show that ZIKV VP formation occurs independent of RNA elements with the size and morphology of VPs being independent of the length of the viral RNA. The second part of my PhD thesis was devoted to characterizing the mode-of action of a novel ZIKV NS4A inhibitor. This topic was a perfect fit to the first part of my thesis, given the essential role of NS4A in VP formation. The inhibitor was developed by a collaborator who did a large-scale cell-based high-content screen, resulting in the identification of (2E)-N-benzyl-3-(4- butoxyphenyl) prop-2-enamide (SBI-0090799). This compound exhibits potent and widespread antiviral activity against numerous ZIKV strains in vitro. Using a combination of biochemical, virological, and imaging-based techniques, I confirmed that SBI-0090799 inhibits ZIKV replication by blocking the de novo formation of ZIKV VPs. Resistance mutations mapping to NS4A rescued viral RNA replication and restored VP formation in cells treated with high concentrations of the compound. These findings suggest that SBI-0090799 perturbs VP formation by interfering with NS4A, either by preventing it from inducing ER membrane curvature or inhibiting it from binding viral or cellular factors contributing to VP formation. Thus, the mechanism of action of SBI-0090799 is comparable to the one of hepatitis C virus NS5A inhibitors that are in clinical use. iv The third part of my PhD study aimed to unravel the role of cholesterol in the viral replication cycle. Although it has already been shown that cholesterol plays a critical role in virus entry, particularly at the stage of viral envelope fusion with the endosome, as well as virion assembly, direct viral protein-cholesterol interactions have not been determined thus far. To fill this gap in knowledge I performed chemo-proteomics in ZIKV-infected human cells, using a photoactivatable and clickable cholesterol probe, to identify cholesterol binding viral proteins. I discovered that both the structural protein prM and its cleavage product, the M protein, can be efficiently cross-linked to cholesterol. Combining bioinformatics analyses and site-directed mutagenesis, alongside with cholesterol binding assays, I was able to show that the M protein has two functional cholesterol binding domains (CRAC motifs) in its transmembrane domains (TMD) 2 and 3 (CRAC2 and CRAC3, respectively). Using reverse genetics studies, I could show that the M protein’s ability to bind cholesterol is not required for the processing of the viral polyprotein, viral RNA replication, and subcellular localization of the uncleaved prM protein. However, mutations affecting the cholesterol binding motif 2 (CRAC2) significantly impaired viruses in their ability to infect cells having low cholesterol levels. Furthermore, I was able to show that complete exchange of the cholesterol binding motif 3 (CRAC3) severely affected virus particle assembly. In line with these results, atomistic molecular dynamics simulations confirmed cholesterol binding to membrane-associated wild type M protein, whereas M proteins containing mutations in CRAC2 and CRAC3 lost cholesterol interactions. In conclusion, I was able to uncover a bifunctional role for the cholesterol interaction of the M protein in the ZIKV life cycle: facilitating virus entry requiring CRAC2 and virus particle assembly requiring CRAC3.
Characterizing Neisseria meningitidis in Southern Vietnam between 2012 and 2021: A predominance of the chloramphenicol-resistant ST-1576 lineage
Thanh Van Phan - 2023
Abstract
Objectives Our goal was to describe Invasive Meningococcal Disease (IMD) in Southern Vietnam over the last ten years. We characterized 109 Neisseria meningitidis (Nm) strains in Southern Vietnam isolated between 1980s to 2021, that were collected from IMD (n=44), sexually transmitted infections (STI) (n=2), and healthy carriage (n=63). Methods IMD were confirmed by bacterial culture and/or real-time PCR at the national reference laboratory in Pasteur Institute of Ho Chi Minh City (PIHCM). Antimicrobial resistance (AMR) was determined on 31 IMD and 2 STI isolates with E-test for chloramphenicol (CHL), penicillin (PEN), ciprofloxacin (CIP), ceftriaxone (CRO), and rifampicin (RIF). Sequencing was performed for analyzing of multilocus-sequence-typing (MLST), porA, fetA, and antibiotic resistance genes, including gyrA, penA, and rpoB. Results The incidence rate during this period was 0.02 per 100,000 persons/year. Serogroup B accounted for over 90% of cases (50/54). ST-1576 were mainly responsible for IMD, 27/42 MLST profiles, and associated with CHL resistance. Resistance was prevalent among IMD isolates. Thirteen were resistant to CHL (MIC ≥ 16mg/L), twelve were intermediate to PEN (MIC between 0.19 and 0.5mg/L), and five were CIP-resistant (MIC between 0.19 and 0.5 mg/L). Particularly, one was non-susceptible to CRO (MIC at 0.125 mg/L), belonging to ST-5571 lineage. The resistance was due to carrying resistant alleles of penA and gyrA genes, and catP gene. Notably, seven isolates were resistant/non-susceptible to 2 or more antibiotics. Conclusion Our results suggest the persistence of the circulating ST-1576 in Southern Vietnam, with a spread of AMR across the community.
Zika virus prM protein contains cholesterol binding motifs required for virus entry and assembly
Sarah Goellner - 2023
Abstract
For successful infection of host cells and virion production, enveloped viruses, including Zika virus (ZIKV), extensively rely on cellular lipids. However, how virus protein–lipid interactions contribute to the viral life cycle remains unclear. Here, we employ a chemo-proteomics approach with a bifunctional cholesterol probe and show that cholesterol is closely associated with the ZIKV structural protein prM. Bioinformatic analyses, reverse genetics alongside with photoaffinity labeling assays, and atomistic molecular dynamics simulations identified two functional cholesterol binding motifs within the prM transmembrane domain. Loss of prM–cholesterol association has a bipartite effect reducing ZIKV entry and leading to assembly defects. We propose a model in which membrane-resident M facilitates cholesterol-supported lipid exchange during endosomal entry and, together with cholesterol, creates a platform promoting virion assembly. In summary, we identify a bifunctional role of prM in the ZIKV life cycle by mediating viral entry and virus assembly in a cholesterol-dependent manner.
Questionnaire study suggests grave consequences of infectious laryngotracheitis, infectious coryza and mycoplasmosis in small chicken flocks
Pernille Engelsen Etterlin - 2023
Abstract
Background A growing number of people in western countries keep small chicken flocks. In Sweden, respiratory disease is a common necropsy finding in chickens from such flocks. A respiratory real-time polymerase chain reaction (PCR) panel was applied to detect infectious laryngotracheitis virus (ILTV), Avibacterium paragallinarum (A. paragallinarum) and Mycoplasma gallisepticum (M. gallisepticum) in chickens from small flocks which underwent necropsy in 2017–2019 and had respiratory lesions. Owners (N = 100) of PCR-positive flocks were invited to reply to a web-based questionnaire about husbandry, outbreak characteristics and management. Results Response rate was 61.0%. The flocks were from 18 out of Sweden’s 21 counties indicating that respiratory infections in small chicken flocks are geographically widespread in Sweden. Among participating flocks, 77.0% were coinfected by 2–3 pathogens; 91.8% tested positive for A. paragallinarum, 57.4% for M. gallisepticum and 50.8% for ILTV. Larger flock size and mixed-species flock structure were associated with PCR detection of M. gallisepticum (P = 0.00 and P = 0.02, respectively). Up to 50% mortality was reported by 63.9% of respondents. Euthanasia of some chickens was carried out in 86.9% of the flocks as a result of the outbreaks. Full clinical recovery was reported by 39.3% of owners suggesting chronic infection is a major challenge in infected flocks. Live birds had been introduced in many flocks prior to outbreaks, which suggested these as an important source of infection. Following the outbreaks, 36.1% replaced their flocks with new birds and 9.8% ceased keeping chickens. Conclusions This study highlights the severity of respiratory outbreaks in small non-commercial chicken flocks and points to the need for more research and veterinary assistance to prevent and manage respiratory infections in small chicken flocks.
High-quality DNA isolation protocol for detection of Khapra beetle (Dermestidae: Trogoderma granarium Everts, 1898) in standard wheat germ trap
Xiaocheng Zhu - 2023
Abstract
Background Khapra beetle (Dermestidae: Trogoderma granarium Everts, 1898) is an internationally significant pest of grain crops and stored grain products. Wheat germ traps, routinely used in surveillance sampling of Khapra beetle provide feed-substrates used by the pest throughout its life cycle. However, Khapra beetle larvae, eggs and other traces of the pest, such as larval frass and exuviae, in wheat germ traps are difficult to sort and taxonomically identify. Additionally, high levels of polysaccharides in wheat germ can inhibit PCR based molecular detection of this pest captured in the traps. Methods and results We have developed a sensitive and low-cost protocol for extracting trace levels of Khapra beetle DNA from an entire wheat germ trap. Overnight digestion of entire trap contents in 6 mL of ATL buffer, followed by a 40 min lysis step was optimal for DNA extraction. Paired with reported qPCR assays, this protocol allows the detection of a few hairs of T. granarium in a typical 2-gram wheat germ trap. Conclusion This DNA extraction protocol makes it possible to perform a more rapid identification of the pest following wheat germ sample collection. The protocol has potential to improve international efforts for Khapra beetle surveillance.
Prevalence and Association of Campylobacter spp., Salmonella spp., and Blastocystis sp. in Poultry
Dr. Muriel Guyard - 2023
Abstract
Poultry and poultry meat are considered the most important sources of human campylobacteriosis and salmonellosis. However, data about the occurrence of Campylobacter and Salmonella concomitantly with intestinal protozoa such as Blastocystis sp. in poultry remain very scarce. Therefore, this study aimed to investigate the presence and possible interactions between these three microorganisms in fecal samples from 214 chickens collected either on farms or from live bird markets in Egypt. The results obtained showed that Campylobacter spp., Salmonella spp., and Blastocystis sp. were present in 91.6% (196/214), 44.4% (95/214), and 18.2% (39/214) of tested samples, respectively, highlighting an active circulation of these microorganisms. Moreover, a significant positive correlation was reported between the occurrence of Campylobacter spp. and Blastocystis sp. together with a significant negative correlation between Blastocystis sp. and Salmonella spp. This study confirms the association reported previously between Blastocystis sp. and Campylobacter spp. while disclosing an association between Blastocystis sp. and Salmonella spp.; it also highlights the need to improve studies on the interactions between bacteria and eukaryotes in the gut microbiota of poultry.
Dead-end hollow fiber ultrafiltration capture of environmental DNA for freshwater mussel (Unionidae) species detection with metabarcoding
Anna M. McKee - 2023
Abstract
Insufficient water sample volumes can be a limiting factor for detecting species with environmental DNA (eDNA) from aquatic habitats. We compared detections of freshwater mussel (Unionidae) communities using large water sample volumes and deadend hollow fiber ultrafiltration (D-HFUF or DEUF) with traditional eDNA filtration methods that use relatively small water sample volumes. Unionid species were detected in approximately 50-L D-HFUF eDNA samples with two mitochondrial DNA metabarcoding markers (COI and ND1) and compared to species detection results from eDNA captured from commonly used 1-L samples filtered with polyethersulfone (PES) filters at three lotic sites in Georgia and Missouri. Of the 431,560 COI and 1,035,472 ND1 reads from all environmental samples of both filter types that passed quality control, 95% (410,755 reads) of COI reads and 85% (883,472 reads) of ND1 reads were assigned to a unionid species. Nineteen different freshwater mussel species were detected across all D-HFUF samples, and 11 species were detected across all PES samples. Reads assigned to the genus Elliptio could not be resolved beyond the genus level with either marker. From D-HFUF samples, 15 and 16 mussel species were detected with the COI and ND1 markers, respectively. From PES samples, nine and seven species were detected with the COI and ND1 markers, respectively. More mussel species were detected at each site in D-HFUF samples than in PES samples regardless of whether results from both markers were combined or evaluated separately. Our results demonstrate the merit of further exploration and optimization of D-HFUF for capturing eDNA from high-volume water samples to facilitate detection of unionids and likely other aquatic organisms.
A validated and optimized environmental DNA and RNA assay to detect Arctic grayling (Thymallus arcticus)
Melissa D. Misutka - 2023
Abstract
Arctic grayling (Thymallus arcticus) is a salmonid fish of significant conservation value. However, conservation efforts are hindered by a lack of fundamental information regarding details such as current population distribution, migratory patterns, and natal habitats. In the current study, we designed, optimized, and field- and laboratoryvalidated an environmental DNA (eDNA) and environmental RNA (eRNA) assay for Arctic grayling biomonitoring. Using an in silico approach, a robust species-specific eDNA assay was generated, and filtering and extraction protocols were optimized for maximal eDNA yield. A Preserve, Precipitate, Lyse, Precipitate, and Purify (PPLPP) extraction method generated 70-fold higher eDNA yields than a column-based approach. Species-specificity relative to co-occurring salmonid fish was validated, and no significant amplification was noted for rainbow trout, brook trout, or mountain whitefish. Shedding rates of eDNA were around eight to nine times higher than those of eRNA, although the two types of nucleic acids decayed at similar rates. Shedding and decay rates were subsequently used to build detection probability models that account for pool size and water exchange rates. These data indicate that eDNA and eRNA are detectable in pools up to 32,500 m3 in volume and with water flow of less than 0.5 m3 s −1 when an Arctic grayling is present. This knowledge can be implemented when designing field sampling strategies. Finally, the assay successfully amplified Arctic grayling eDNA from field-collected samples, with signal strength indicating preferred Arctic grayling habitat or conditions that favored the concentration and retention of eDNA.
A validated and optimized environmental DNA and RNA assay to detect Arctic grayling (Thymallus arcticus)
Melissa D. Misutka - 2023
Abstract
Arctic grayling (Thymallus arcticus) is a salmonid fish of significant conservation value. However, conservation efforts are hindered by a lack of fundamental information regarding details such as current population distribution, migratory patterns, and natal habitats. In the current study, we designed, optimized, and field- and laboratoryvalidated an environmental DNA (eDNA) and environmental RNA (eRNA) assay for Arctic grayling biomonitoring. Using an in silico approach, a robust species-specific eDNA assay was generated, and filtering and extraction protocols were optimized for maximal eDNA yield. A Preserve, Precipitate, Lyse, Precipitate, and Purify (PPLPP) extraction method generated 70-fold higher eDNA yields than a column-based approach. Species-specificity relative to co-occurring salmonid fish was validated, and no significant amplification was noted for rainbow trout, brook trout, or mountain whitefish. Shedding rates of eDNA were around eight to nine times higher than those of eRNA, although the two types of nucleic acids decayed at similar rates. Shedding and decay rates were subsequently used to build detection probability models that account for pool size and water exchange rates. These data indicate that eDNA and eRNA are detectable in pools up to 32,500 m3 in volume and with water flow of less than 0.5 m3 s −1 when an Arctic grayling is present. This knowledge can be implemented when designing field sampling strategies. Finally, the assay successfully amplified Arctic grayling eDNA from field-collected samples, with signal strength indicating preferred Arctic grayling habitat or conditions that favored the concentration and retention of eDNA.
High-quality DNA isolation protocol for detection of Khapra beetle (Dermestidae: Trogoderma granarium Everts, 1898) in standard wheat germ trap
Xiaocheng Zhu - 2023
Abstract
Background Khapra beetle (Dermestidae: Trogoderma granarium Everts, 1898) is an internationally significant pest of grain crops and stored grain products. Wheat germ traps, routinely used in surveillance sampling of Khapra beetle provide feed-substrates used by the pest throughout its life cycle. However, Khapra beetle larvae, eggs and other traces of the pest, such as larval frass and exuviae, in wheat germ traps are difficult to sort and taxonomically identify. Additionally, high levels of polysaccharides in wheat germ can inhibit PCR based molecular detection of this pest captured in the traps. Methods and results We have developed a sensitive and low-cost protocol for extracting trace levels of Khapra beetle DNA from an entire wheat germ trap. Overnight digestion of entire trap contents in 6 mL of ATL buffer, followed by a 40 min lysis step was optimal for DNA extraction. Paired with reported qPCR assays, this protocol allows the detection of a few hairs of T. granarium in a typical 2-gram wheat germ trap. Conclusion This DNA extraction protocol makes it possible to perform a more rapid identification of the pest following wheat germ sample collection. The protocol has potential to improve international efforts for Khapra beetle surveillance.
Prevalence and Association of Campylobacter spp., Salmonella spp., and Blastocystis sp. in Poultry
Muriel Guyard-Nicodème - 2023
Abstract
Poultry and poultry meat are considered the most important sources of human campylobacteriosis and salmonellosis. However, data about the occurrence of Campylobacter and Salmonella concomitantly with intestinal protozoa such as Blastocystis sp. in poultry remain very scarce. Therefore, this study aimed to investigate the presence and possible interactions between these three microorganisms in fecal samples from 214 chickens collected either on farms or from live bird markets in Egypt. The results obtained showed that Campylobacter spp., Salmonella spp., and Blastocystis sp. were present in 91.6% (196/214), 44.4% (95/214), and 18.2% (39/214) of tested samples, respectively, highlighting an active circulation of these microorganisms. Moreover, a significant positive correlation was reported between the occurrence of Campylobacter spp. and Blastocystis sp. together with a significant negative correlation between Blastocystis sp. and Salmonella spp. This study confirms the association reported previously between Blastocystis sp. and Campylobacter spp. while disclosing an association between Blastocystis sp. and Salmonella spp.; it also highlights the need to improve studies on the interactions between bacteria and eukaryotes in the gut microbiota of poultry.
Wastewater surveillance of Mpox virus in Baltimore
Samendra P. Sherchan - 2023
Abstract
This study aimed to utilize wastewater surveillance for monitoring Mpox cases at a community level. Untreated wastewater samples were collected once a week from two wastewater treatment plants (A and B) in Baltimore City from July 27, 2022–September 22, 2022. The samples were concentrated via an adsorption–elution (AE) method and Polyethylene Glycol (PEG) precipitation method followed by quantitative polymerase chain reaction (qPCR). Monkeypox virus (MPXV) was detected in 89 % (8/9) samples from WWTP A and 55 % (5/9) samples from WWTP B with at least one concentration method. Higher detection rate in samples concentrated with PEG precipitation compared to AE method was observed, indicating that PEG precipitation is a more effective virus concentration method for MPXV. To our knowledge, this is the first study reporting the detection of MPXV in wastewater in Baltimore. The results highlight that wastewater surveillance could be used as a complementary early warning tool for monitoring future Mpox outbreaks.
Improved Dried Blood Spot PCR Assay for Universal Congenital Cytomegalovirus Screening in Newborns
Jean H. Kim - 2023
Abstract
Congenital cytomegalovirus (cCMV) is the most common perinatal infection, the leading cause of nongenetic sensorineural hearing loss, and one of the leading causes of neurodevelopmental impairment in the developed world. Early identification via newborn screening (NBS) would benefit the many undiagnosed infants who are either asymptomatic or mildly to moderately symptomatic, of whom 20% develop sequelae. The sensitivity of a recently developed PCR-based method to detect CMV in dried blood spots (DBS) is less than 80% and requires significantly more specimen than any other NBS test. We sought to improve the analytical sensitivity of the screening method by using droplet digital PCR and direct PCR and decreasing the amount of specimen utilized. The methods were tested with CMV-spiked filters, DBS from CMV-spiked cord blood, and DBS from neonates with cCMV. The results showed that the analytical sensitivity of all modified methods was equivalent to that of the reference method, with consistent CMV detection at high viral loads and inconsistent detection at low viral loads.
Diagnostic performance of NxTek Eliminate Malaria-Pf test for the detection of Plasmodium falciparum in school children with asymptomatic malaria
Abdissa Biruksew - 2023
Abstract
Background One of the major roadblocks to the falciparum malaria elimination programme is the presence of a portion of the population, such as school children, with asymptomatic malaria infection. Targeting such reservoirs of infections is critical to interrupting transmission and enhancing elimination efforts. The NxTek™ Eliminate Malaria Pf test is a highly sensitive rapid diagnostic test (hsRDT) for the detection of HRP-2. However, knowledge gaps exist in Ethiopia on the diagnostic performance of hsRDT for the detection of Plasmodium falciparum in school children with asymptomatic malaria. Methods A school-based cross-sectional study was conducted from September 2021 to January 2022 on 994 healthy school children (aged 6–15 years). Finger-pricked whole blood samples were collected for microscopy, hsRDT, conventional RDT (cRDT or SD Bioline Malaria Ag Pf/P.v), and QuantStudio™ 3 Real—Time PCR system (qPCR). The hsRDT was compared to cRDT and microscopy. qPCR and microscopy were used as reference methods. Results The prevalence of Plasmodium falciparum was 1.51%, 2.2%. 2.2% and 4.52%, by microscopy, hsRDT, cRDT and qPCR, respectively. Using qPCR as reference, the sensitivity of hsRDT was higher (48.89%) than the microscopy (33.3%), and showed 100% specificity and a positive predictive value (PPV). Microscopy showed similar specificity and PPV as hsRDT. Using microscopy as a reference, the diagnostic perforrmances of both hsRDT and cRDT were similar. Both RDTs demonstrated identical diagnostic performances in both comparison methods. Conclusions hsRDT has the same diagnostic performance as cRDT but improved diagnostic characteristics than microscopy for detection of P. falciparum in school children with asymptomatic malaria. It can be a useful tool for the national malaria elimination plan of Ethiopia.
Shiga toxin-producing Escherichia coli (STEC) and atypical enteropathogenic E. coli (aEPEC) in Swedish retail wheat flour
Robert Söderlund - 2023
Abstract
Wheat flour has been identified as the source of multiple outbreaks of gastrointestinal disease caused by shiga-toxin producing Escherichia coli (STEC). We have investigated the presence and genomic characteristics of STEC and related atypical enteropathogenic E. coli (aEPEC) in 200 bags of Swedish46 produced retail wheat flour, representing 87 products and 25 brands. Samples were enriched in modified TSB and screened with real-time PCR targeting stx1, stx2, eae, and the serogroups O157, O121, and O26. Isolation was performed by IMS for suspected STEC/aEPEC O157, O121 and O26, and by screening pools of colonies for other STEC. Real-time PCR after enrichment revealed 12% of samples to be positive for shiga toxin genes (stx1 and/or stx2) and 11% to be positive for intimin (eae). Organic production, small-scale production or whole grain did not significantly influence shiga toxin gene presence or absence in a generalized linear mixed model analysis. Eight isolates of STEC were recovered, all of which were intimin negative. Multiple serotype/sequence type/shiga-toxin subtype combinations were recovered which have also been found in flour samples in other European countries. Most STEC types recovered were associated with sporadic cases of STEC among humans in Sweden, but no types known to have caused outbreaks or severe cases of disease (i.e. haemolytic57 uraemic syndrome) were found. The most common finding was O187:H28 ST200 with stx2g, with possible links to cervid hosts. Wildlife associated with crop damage is a plausible explanation for at least some of the surprisingly high frequency of STEC in wheat flour.
At-home oral bacteria collection using a lollipop-based microfluidic device
Wan-chen Tu - 2023
Abstract
Our previous work introduced the CandyCollect,1 a lollipop-inspired saliva collection device for respiratory disease diagnostics. Here, we performed the first human subjects study using this device to capture bacteria in saliva; we demonstrate that the CandyCollect device can be used to collect salivary bacteria from healthy adults using Streptococcus mutans and Staphylococcus aureus as proof-of-concept commensal bacteria. We enrolled 14 healthy adults in a nationwide (USA) remote study in which participants were sent study packages containing CandyCollect devices and traditional commercially available oral swabs and spit tubes. Participants sampled themselves at home, completed usability and user preference surveys, and mailed the samples back to our laboratory for analysis by qPCR. S. mutans and S. aureus are not universally present in all healthy adults.2-5 Our results showed that for participants in which a given bacterium (S. mutans or S. aureus) was detected in one or both of the commercially available methods, CandyCollect devices had a 100% concordance with those results. Furthermore, the CandyCollect device was ranked the highest preference sampling method among the three sampling methods by 26 participants surveyed. We also showed that the CandyCollect device has a shelf life of up to 1 year at room temperature, a storage period that is convenient for clinics or patients to keep the CandyCollect device and use it any time. Taken together, we have demonstrated that the CandyCollect is a user-friendly saliva collection tool that has the potential to be incorporated into diagnostic assays in clinic visits and telemedicine.
Point-of-Care Testing for Sensitive Detection of the African Swine Fever Virus Genome
Ahmed Elnagar - 2022
Abstract
African swine fever (ASF) is a contagious viral hemorrhagic disease that affects domestic pigs and wild boar. The disease is notifiable to the World Organization of Animal Health (WOAH), and causes significant deaths and economic losses. There is currently no fully licensed vaccine available. As a result, early identification of the causative agent, ASF virus (ASFV), is crucial for the implementation of control measures. PCR and real-time PCR are the WOAH-recommended standard methods for the direct detection of ASFV. However, under special field conditions or in simple or remote field laboratories, there may be no sophisticated equipment or even stable electricity available. Under these circumstances, point-of-care systems can be put in place. Along these lines, a previously published, rapid, reliable, and electricity-free extraction method (TripleE) was used to isolate viral nucleic acid from diagnostic specimens. With this tool, nucleic acid extraction from up to eight diagnostic samples can be realized in one run in less than 10 min. In addition, the possibility of completely omitting viral DNA extraction was analyzed with so-called direct real-time PCR protocols using ASFV original samples diluted to 1:40 in RNase-free water. Furthermore, three real-time PCR cyclers, developed for use under field conditions (IndiField, Liberty16 and UF-300 GenecheckerTM), were comparatively applied for the sensitive high-speed detection of ASFV genomes, with overall PCR run times between 20 and 54 min. Depending on the viral DNA extraction/releasing method used and the point-of-care cycler applied, a total time for detection of 30 to 60 min for up to eight samples was feasible. As expected, the limitations in analytical sensitivity were positively correlated to the analysis time. These limitations are acceptable for ASFV diagnostics due to the expected high ASFV genome loads in diseased animals or carcasses.
A step forward for Shiga toxin-producing Escherichia coli identification and characterization in raw milk using longread metagenomics
Sandra Jaudou - 2022
Abstract
Shiga toxin-producing Escherichia coli (STEC) are a cause of severe human illness and are frequently associated with haemolytic uraemic syndrome (HUS) in children. It remains difficult to identify virulence factors for STEC that absolutely predict the potential to cause human disease. In addition to the Shiga-toxin (stx genes), many additional factors have been reported, such as intimin (eae gene), which is clearly an aggravating factor for developing HUS. Current STEC detection methods classically rely on real-time PCR (qPCR) to detect the presence of the key virulence markers (stx and eae). Although qPCR gives an insight into the presence of these virulence markers, it is not appropriate for confirming their presence in the same strain. Therefore, isolation steps are necessary to confirm STEC viability and characterize STEC genomes. While STEC isolation is laborious and time-consuming, metagenomics has the potential to accelerate the STEC characterization process in an isolation-free manner. Recently, short-read sequencing metagenomics have been applied for this purpose, but assembly quality and contiguity suffer from the high proportion of mobile genetic elements occurring in STEC strains. To circumvent this problem, we used long-read sequencing metagenomics for identifying eae-positive STEC strains using raw cow's milk as a causative matrix for STEC foodborne outbreaks. By comparing enrichment conditions, optimizing library preparation for MinION sequencing and generating an easy-to-use STEC characterization pipeline, the direct identification of an eae-positive STEC strain was successful after enrichment of artificially contaminated raw cow's milk samples at a contamination level as low as 5 c.f.u. ml−1. Our newly developed method combines optimized enrichment conditions of STEC in raw milk in combination with a complete STEC analysis pipeline from long-read sequencing metagenomics data. This study shows the potential of the innovative methodology for characterizing STEC strains from complex matrices. Further developments will nonetheless be necessary for this method to be applied in STEC surveillance.
An opinion on Wastewater-Based Epidemiological Monitoring (WBEM) with Clinical Diagnostic Test (CDT) for detecting high-prevalence areas of community COVID-19 Infections
Md. Aminul Islam - 2022
Abstract
Wastewater-Based Epidemiological Monitoring (WBEM) is an efficient surveillance tool during the COVID-19 pandemic as it meets all requirements of a complete monitoring system including early warning, tracking the current trend, prevalence of the disease, detection of genetic diversity as well asthe up-surging SARS-CoV-2 new variants with mutations from the wastewater samples. Subsequently, Clinical Diagnostic Test is widely acknowledged as the global gold standard method for disease monitoring, despite several drawbacks such as high diagnosis cost, reporting bias, and the difficulty of tracking asymptomatic patients (silent spreaders of the COVID-19 infection who manifest nosymptoms of the disease). In this current reviewand opinion-based study, we first propose a combined approach) for detecting COVID-19 infection in communities using wastewater and clinical sample testing, which may be feasible and effective as an emerging public health tool for the long-term nationwide surveillance system. The viral concentrations in wastewater samples can be used as indicatorsto monitor ongoing SARS-CoV-2 trends, predict asymptomatic carriers, and detect COVID-19 hotspot areas, while clinical sampleshelp in detecting mostlysymptomaticindividuals for isolating positive cases in communities and validate WBEM protocol for mass vaccination including booster doses for COVID-19.
Systematic stepwise screening of new microbial antagonists for biological control of European canker
G. Elena - 2022
Abstract
Neonectria ditissima is the causal agent of European canker. This pathogen causes cankers on apple branches and the main stem, which may lead to the loss of the whole tree. Chemical control is the essential component in disease management and no suitable biocontrol products are commercially available. This study aimed at selecting potential microbial antagonists against N. ditissima through a systematic stepwise screening program for the development of a new biocontrol product. A total of 520 potential candidates were isolated from apple branches showing canker symptoms. Important characteristics for application of Microbial Biological Control Agents were tested per each candidate: spore production, spore survival during storage, cold tolerance, drought tolerance and UV-B resistance. Isolates able to germinate and grow at human body temperature were excluded. A total of 252 candidates fulfilled the stablished criteria. All 520 candidates belonged to 44 different taxonomic groups, being the most abundant Alternaria spp. (22.2%), Aureobasidium pullulans (16.1%), Cladosporium spp. (9.5%) and Fusarium spp. (9.0%). Information on possible pathogenicity and toxicity for humans, animals, plants and the environment and on patents in biocontrol use was collected per each identified species. A total of 158 candidates belonging to species that did not show potential risks or patent conflicts were assessed by their antagonistic behaviour against N. ditissima in bioassays in planta. Each candidate was inoculated in ‘Elstar’ apple branches inoculated with N. ditissima 24 h before. Inoculated branches were incubated at 17 °C, 16 h light per day and RH>90%. After four weeks, canker symptom expression was visually assessed. The capacity of the candidates to reduce colonisation of N. ditissima in the branches was evaluated by quantifying N. ditissima DNA concentration using qPCR. Four candidates of Clonostachys rosea showed antagonistic properties; after four weeks of treatment, no canker symptoms or small bark cracks were observed in the inoculated branches and N. ditissima DNA was reduced by 90-99%. Following them, the branches inoculated with one candidate of Akanthomyces muscarius, A. pullulans and Cladosporium europaeum showed small bark cracks and small swollen bark and N. ditissima DNA was reduced by more than 90%. The systematic stepwise screening approach was a powerful strategy to find new MBCAs against N. ditissima with antagonistic properties that also fulfilled major criteria with regards to commercial production and safety, as well as ecological needs.
High-throughput microfluidic real-time PCR for the simultaneous detection of selected vector-borne pathogens in dogs in Bosnia and Herzegovina
Vito Colella - 2022
Abstract
A scarcity of information on the occurrence of zoonotic vector-borne pathogens (VBPs), alongside a lack of human and animal health authorities’ awareness of preexisting data, augment the risk of VBP infection for local people and limit our ability to establish control programs. This holds especially true in low-middle income countries such as Bosnia and Herzegovina (BiH). This dearth of information on zoonotic VBPs is bolstered by the inability of previously used diagnostic tests, including conventional molecular diagnostic methods, to detect the full spectrum of relevant pathogens. Considering this, we set out to apply a microfluidic qPCR assay capable of detecting 43 bacterial and protozoan pathogens from blood to accrue critical baseline data for VBPs occurrence in BiH. A total of 408 dogs were tested of which half were infected with at least one VBP of zoonotic or veterinary importance. Leishmania infantum was found in 18% of dogs, reaching a prevalence as high as 38% in urbanized areas of Sarajevo. These data highlight substantially higher levels of L. infantum prevalence when compared to that previously reported using conventional methods using the same samples. Additionally, this high-throughput microfluidic qPCR assay was able to detect pathogens rarely or never reported in canines in BiH, including Anaplasma phagocytophilum (3%), Anaplasma platys (0.2%), haemotropic Mycoplasma (1%) and Hepatozoon canis (26%). Our report of the endemicity of important zoonotic pathogens and those of clinical significance to dogs emphasizes the need for urgent implementation of surveillance and control for VBPs in BiH, targeting both animal and human infections within the country.
Human Air-Liquid-Interface Organotypic Airway Cultures Express Significantly More ACE2 Receptor Protein and Are More Susceptible to HCoV-NL63 Infection than Monolayer Cultures of Primary Respiratory Epithelial Cells
Gino Castillo - 2022
Abstract
Human coronavirus NL63 (HCoV-NL63) is commonly associated with mild respiratory tract infections in infants, being that the respiratory epithelial cells are the main target for infection and initial replication of this virus. Standard immortalized cells are highly permissive to HCoV-NL63, and they are routinely used for isolation and propagation of the virus from clinical specimens. However, these cell lines are not the natural cell target of the virus and lack sufficient complexity to mimic the natural infection process in vivo. This study comparatively evaluated the differences on the susceptibility to HCoV-NL63 infection and virus replication efficiency of submerged monolayer cultures of LLC-MK2 and primary human respiratory epithelial cells (HRECs) and organotypic airway cultures of respiratory cells (ALI-HRECs). Productive viral infection and growth kinetics were assessed by morphologic examination of cytopathic effects, immunofluorescence, reverse transcription quantitative real-time PCR, and flow cytometry. Results from this study showed higher susceptibility to HCoV-NL63 infection and replication in LLC-MK2 cells followed by ALI-HRECs, with very low susceptibility and no significant virus replication in HRECs. This susceptibility was associated with the expression levels of angiontensin-converting enzyme 2 (ACE2) receptor protein in LLC-MK2, ALI-HRECs, and HRECs, respectively. Remarkably, organotypic ALI-HREC cultures expressed significantly more ACE2 receptor protein and were more susceptible to HCoV-NL63 infection than monolayer cultures of HREC. The ACE2 receptor is, therefore, a critical factor for susceptibility to HCoV-NL63 infection and replication, as is the type of culture used during infection studies. IMPORTANCE HCoV-NL63 is widespread globally, accounting for a significant number of respiratory infections in children and adults. HCoV-NL63 gains entrance into respiratory epithelial cells via the ACE2 receptor, the same cell receptor used by severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Thus, HCoV-NL63 has been suggested as safe surrogate for studying disease mechanisms and therapeutic interventions against SARSlike CoVs, while working under BSL-2 conditions. The present study not only showed the critical role of ACE2 for effective HCoV-NL63 infection and replication, but also shed light on the need of more refined and complex in vitro organotypic models that recapitulate the proxy of air-liquid respiratory epithelia cell composition, structure, and functionality. These cultures have broaden virological studies toward improving our understanding of how coronaviruses cause disease and transmission not just within humans but also in animal populations.
Monitoring of Campylobacter jejuni in a chicken infection model by measuring specific volatile organic compounds and by qPCR
Julia Hankel - 2022
Abstract
Campylobacter is one of the leading bacterial foodborne pathogens worldwide. Poultry is the host species with this pathogen with the highest clinical impact. Flocks become colonised with Campylobacter, which leads to contamination of product entering the food-chain. Rapid and reliable Campylobacter detection methods could support controls to minimize the risks of contamination within the food-chain, which would easier enable the implementation of a logistical slaughter schedule or other control options. The present study evaluates current and emerging C. jejuni detection technologies on air samples in a unique study set-up of pre-defined C. jejuni prevalences. Both non-invasive detection technologies on air samples by subsequent measuring of volatile organic compounds (VOCs) or by qPCR detected the C. jejuni presence and could additionally distinguish between the number of present C. jejuni-positive birds in the study set-up. Nevertheless, electrostatic air samplers diagnosed fewer birds as C. jejuni-positive compared to the cultivation-based method. By measuring the VOCs, it was possible to detect the presence of two positive birds in the room. This apparent high sensitivity still needs to be verified in field studies. Techniques, such as these promising methods, that can facilitate C. jejuni surveillance in poultry flocks are desirable to reduce the risk of infection for humans.
A Mobile Laboratory Enables Fecal Pollution Source Tracking in Catchments Using Onsite qPCR Assays
Rixia Zan - 2022
Abstract
Onsite molecular diagnostics can revolutionize fecal pollution source tracking. We aimed to validate a method for onsite qPCR assays with a miniature speaker-sized Q qPCR instrument and other portable equipment items. We showed that marker genes for total bacteria (16S) and E. coli (rodA) in 100 mL of river water measured with this method agreed within ±0.3 log10 units with results obtained when using conventional laboratory equipment items. We then deployed the portable method in a mobile laboratory (‘lab in a van’) and quantified HF183 marker genes for human host associated Bacteroides in river water within 3 h of sampling. We also used the mobile laboratory to investigate urban river water and effluents from two storm drains and a retention pond and collected comprehensive microbial and physicochemical water quality data. We found significantly higher HF183 gene levels in the older storm drain compared to the river water (6.03 ± 0.04 vs. 4.23 ± 0.03 log10 gene copies per 100 mL), and a principal component analysis revealed that storm drain effluent retention in a pond beneficially altered water characteristics, making them more like those of the receiving river. In conclusion, onsite qPCR assays can be performed with portable equipment items to quickly test water.
Occurrence of Naegleria fowleri and faecal indicators in sediments from Lake Pontchartrain, Louisiana
Shalina A. Shahin - 2022
Abstract
The occurrence of amoeba, Naegleria fowleri, in sediment samples from Lake Pontchartrain in Louisiana was investigated. This amoeba is pathogenic and can cause primary amoebic meningoencephalitis. In this study, quantitative polymerase chain reaction methods were used to test for the prevalence of Naegleria fowleri, HF183, and E. coli. N. fowleri was detected in 51.25% of our sediment samples. Illumina sequencing of sediment samples revealed ten different phyla, with Cyanobacteria being the most predominant at sites that generally presented with the highest median N. fowleri concentrations. N. fowleri was however strongly negatively correlated with HF183 (r = −0.859, p < 0.001). Whenever sediment E. coli concentrations were below 1.54 Log GC/g, there was only a 37.5% chance that N. fowleri would be detected in the same sample. When sediment E. coli concentrations exceeded 2.77 Log GC/g, the chances of detecting N. fowleri in the same sample increased to 90%, potentially suggesting predatory activity by the amoeba. The effect of temperature was observed to be different in relation to observed N. fowleri concentrations and detection rates. Although sediment samples collected during periods of higher temperatures had significantly lower mean N. fowleri concentrations (2.7 Log GC/g) compared to those collected at lower temperatures (3.7 Log GC/g, t(39) = 4.167, p < 0.001), higher N. fowleri detection rates in the overall samples were observed at higher temperatures (>19.1 °C) than at lower temperatures (<19.1 °C).
Non-lethal sampling for the detection of Renibacterium salmoninarum by qPCR for diagnosis of bacterial kidney dsisease
Eva Jansson - 2022
Abstract
Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum (Rs), can be transmitted both horizontally and vertically and there is no available cure or prophylaxis. The control of BKD requires continuous surveillance, which is challenging in aquaculture as well as in programs for conservation and restoration of salmonid fish strains. BKD is a notifiable disease in Sweden and is monitored through the mandatory health control program using a polyclonal ELISA for detection of the Rs p57 protein in kidney. Fish must be killed for sampling, an obvious disadvantage especially regarding valuable broodfish. The present study shows that gill-/cloacal swabs collected in vivo for real-time PCR (qPCRgc), allow a sensitive and specific detection of Rs. The sensitivity of qPCRgc was estimated to 97.8% (credible interval (ci) 93.8%–100%) compared to 98.3% (ci 92.7%–100%) and 48.8% (ci 38.8%–58.8%) of kidney samples for qPCR (qPCRk) and ELISA (ELISAk) respectively, by use of the Bayesian Latent Class Analysis (BLCA). Since the goal of the program is eradication of BKD the most sensitive test is preferrable. Using qPCRgc instead of ELISAk will result in a lower false negative rate and can be useful for surveillance in aquaculture and in breeding programs with valuable fish. However, a higher false positive rate warrants confirmatory lethal testing before a previously Rs negative farm is subject to restrictions.
Profiling Multiomic Biomarkers using Particle Detection Counters and Spectral-FLIM Microscopy
Tam Vu - 2021
Abstract
Profiling multiomic biomarkers in bulk and in situ provides critical information which enables basic research and clinical applications. Unfortunately, most existing profiling methods are limited due to either low multiplexing, sensitivity, costs, or assay complexity. This thesis aims to develop two core technologies that address 1) bulk profiling issues with sensitivity and low throughput as well as 2) in situ profiling issues with low multiplexing capabilities, costs, and limited throughput. To address the first issue, this work introduces a novel liquid biopsy approach that utilizes a platform technology called Integrated Comprehensive Droplet Digital Detection (IC3D). This integrated approach combines microfluidic droplet partitioning technology, fluorescent multiplexed PCR chemistry, and our own unique and rapid particle counting technology to deliver ultrasensitive and ultrafast detection of colorectal cancer-specific genomic biomarkers from minimally processed blood samples. xv To address the second issue, this work introduces a new spatial multi-omics technology termed Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA) that integrates a) in situ labeling of molecular markers (e.g. mRNA, proteins) in cells or tissues with combinatorial fluorescence spectral and lifetime encoded probes, and b) spectra and timeresolved fluorescence imaging and analysis to enable rapid, high-throughput, and cost-effective spatial profiling of multi-omics biomarkers. By utilizing both time and intensity domains for labeling and imaging, this technology seeks to discriminate a vast repertoire of lifetime and spectral components simultaneously within the same pixel or image of a sample to enable highly increased multiplexing capabilities with standard optical systems. Overall, these two technologies represent simple, versatile, and scalable tools which enable more rapid, sensitive, and/or multiplexed protein/transcriptomic analysis.
Design of Bacterial Strain-Specific qPCR Assays Using NGS Data and Publicly Available Resources and Its Application to Track Biocontrol Strains
Iker Hernandez - 2020
Abstract
Biological control is emerging as a feasible alternative to chemical pesticides in agriculture. Measuring the microbial biocontrol agent (mBCA) populations in the environment is essential for an accurate environmental and health risk assessment and for optimizing the usage of an mBCA-based plant protection product. We hereby show a workflow to obtain a large number of qPCR markers suitable for robust strain-specific quantification. The workflow starts from whole genome sequencing data and consists of four stages: (i) identifying the strain-specific sequences, (ii) designing specific primer/probe sets for qPCR, and (iii) empirically verifying the performance of the assays. The first two stages involve exclusively computer work, but they are intended for researchers with little or no bioinformatic background: Only a knowledge of the BLAST suite tools and work with spreadsheets are required; a familiarity with the Galaxy environment and next-generation sequencing concepts are strongly advised. All bioinformatic work can be implemented using publicly available resources and a regular desktop computer (no matter the operating system) connected to the Internet. The workflow was tested with five bacterial strains from four different genera under development as mBCAs and yielded thousands of candidate markers and a triplex qPCR assay for each candidate mBCA. The qPCR assays were successfully tested in soils of different natures, water from different sources, and with samples from different plant tissues. The mBCA detection limits and population dynamics in the different matrices are similar to those in qPCR assays designed by other means. In summary, a new accessible, cost-effective, and robust workflow to obtain a large number of strain-specific qPCR markers is presented.
Detection of freshwater mussels (Unionidae) using environmental DNA in riverine systems
Louis Gasparini - 2020
Abstract
Environmental DNA (eDNA) methods are being developed for use in conservation biology to improve upon conventional species survey techniques. Validation of eDNA methods in different environmental contexts is required if they are to be widely adopted. One potential application of eDNA methods is for the detection of freshwater mussels (Bivalvia: Unionidae), which are among the most imperiled species in North America. Conventional unionid survey methods are highly invasive and can be difficult to conduct due to issues with morphological identification and their cryptic use of habitat. eDNA methods can potentially provide a non‐invasive, extremely specific, and highly sensitive alternative. Here, we examine the effectiveness of eDNA methods at detecting an imperiled unionid, the wavy‐rayed lampmussel (Lampsilis fasciola), in lotic systems with moderate discharge. We developed a novel qPCR assay for the detection of L. fasciola eDNA, which included a custom internal positive control to check for PCR inhibition. We used different experimental densities of caged L. fasciola specimens as a point source of eDNA within two rivers of the Grand River watershed in Southern Ontario. Sampling occurred at set distances downstream of the cage using purpose‐built sampling equipment. Detection was obtained at the cage (i.e., point of eDNA shedding) but not downstream at distances ≥10 m during stream discharges of approximately 1,632–2,332 L/s. The results indicate that eDNA is diluted rapidly in rivers with moderate discharge and that high‐resolution spatial sampling efforts may be necessary to obtain meaningful eDNA‐based distribution data of unionids, and other sessile organisms, present at low density in lotic systems.
T-cell Receptor Excision Circles in Newborns with Heart Defects
Kiran A. Gul - 2020
Abstract
In the fetus, the cardiac neural crest gives rise to both the thymus and the conotruncus of the heart. In newborn screening for severe T-cell lymphopenia neonates with congenital heart defects may be detected. In this study, we investigated the occurrence of T-cell lymphopenia in neonates with or without 22q11.2 deletion syndrome (del) suffering from heart defects. This retrospective cohort study included 125 patients with heart defects. T-cell receptor excision circles (TRECs), a measure for T-cell lymphopenia, were quantified by RT-PCR using stored newborn screening blood spots. Three patient groups were compared: non-conotruncal defects (n = 57), conotruncal defects (n = 42), and 22q11.2 del with conotruncal defects (n = 26). Significantly lower TREC values were detected in patients with 22q11.2 del and conotruncal heart defects compared to those with non-syndromic conotruncal (p < 0.001) and non-conotruncal (p < 0.001) defects. In contrast, no significant difference was found between patients with non-syndromic conotruncal and non-conotruncal heart defects (p = 0.152). Low TREC levels were obtained in neonates treated with heart surgery/intervention within 2 weeks after birth and in those with a fatal outcome (p = 0.02) independent of patient group. A correlation was found between low TREC numbers and oxygen saturation, SpO2 below 95% (p = 0.017). The SpO2 was significantly lower in the non-syndromic conotruncal group compared to non-conotruncal (p < 0.001) and 22q11.2 del group (p = 0.015). No correlation was found between low neonatal TRECs and infections needing hospitalization later in life (p = 0.135). Patients with 22q11.2 del and conotruncal defects have significantly lower TREC levels compared to patients with heart defects without this syndrome.
MAPK-induced miR-29 restrains melanoma progression by targeting MAFG
Olga Vera, Ilah Bok - 2020
Abstract
The tumor suppressive miR-29 family of microRNAs is encoded by two clusters, miR-29b1∼a and miR-29b2∼c, which are regulated by oncogenic and tumor suppressive stimuli, including p53. Here we investigated whether MAPK hyperactivation-induced oncogenic stress regulates miR-29 abundance and how this signaling axis impacts melanoma development. Using mouse embryonic fibroblasts and human melanocytes, we found that oncogenic MAPK signaling stimulates p53-independent and p53-dependent transcription of pri-miR-29b1∼a and pri-miR-29b2∼c, respectively. Expression analyses revealed that while pri-miR-29a∼b1 remains elevated, pri-miR-29b2∼c levels decrease during melanoma progression. Using a rapid mouse modeling platform, we showed that inactivation of miR-29 in vivo accelerates the development of frank melanomas and decreases overall survival. We identified MAFG as a relevant miR-29 target that has oncogenic potential in melanocytes and is required for growth of melanoma cells. Our findings suggest that MAPK-driven miR-29 induction constitutes a tumor suppressive barrier by targeting MAFG, which is overcome by attenuation of miR-29b2∼c expression.
Rickettsia felis identified in two fatal cases of acute meningoencephalitis
Arthur H. P. Mawuntu - 2020
Abstract
Background Rickettsia felis has recently emerged worldwide as a cause of human illness. Typically causing mild, undifferentiated fever, it has been implicated in several cases of non-fatal neurological disease in Mexico and Sweden. Its distribution and pathogenicity in Southeast Asia is poorly understood. Methodology/Principal findings We retroactively tested cerebrospinal fluid (CSF) or sera from 64 adult patients admitted to hospital in North Sulawesi, Indonesia with acute neurological disease. Rickettsia felis DNA was identified in the CSF of two fatal cases of meningoencephalitis using multi-locus sequence typing semi-nested PCR followed by Sanger sequencing. DNA from both cases had 100% sequence homologies to the R. felis reference strain URRWXCal2 for the 17-kDa and ompB genes, and 99.91% to gltA. Conclusion/Significance The identification of R. felis in the CSF of two fatal cases of meningoencephalitis in Indonesia suggests the distribution and pathogenicity of this emerging vector-borne bacteria might be greater than generally recognized. Typically Rickettsia are susceptible to the tetracyclines and greater knowledge of R. felis endemicity in Indonesia should lead to better management of some acute neurological cases.
The retinoic acid receptor (RAR) α-specific agonist Am80 (tamibarotene) and other RAR agonists potently inhibit hepatitis B virus transcription from cccDNA
Shirin Nkongolo - 2019
Abstract
Chronic infection with the human Hepatitis B virus (HBV) is a major global health problem. Hepatitis D virus (HDV) is a satellite of HBV that uses HBV envelope proteins for cell egress and entry. Using infection systems encoding the HBV/HDV receptor human sodium taurocholate co-transporting polypeptide (NTCP), we screened 1181 FDA-approved drugs applying markers for interference for HBV and HDV infection. As one primary hit we identified Acitretin, a retinoid, as an inhibitor of HBV replication and HDV release. Based on this, other retinoic acid receptor (RAR) agonists with different specificities were found to interfere with HBV replication, verifying that the retinoic acid receptor pathway regulates replication. Of the eight agonists investigated, RARα-specific agonist Am80 (tamibarotene) was most active. Am80 reduced secretion of HBeAg and HBsAg with IC50s < 10 nM in differentiated HepaRG-NTCP cells. Similar effects were observed in primary human hepatocytes. In HepG2-NTCP cells, profound Am80-mediated inhibition required prolonged treatment of up to 35 days. Am80 treatment of cells with an established HBV cccDNA pool resulted in a reduction of secreted HBsAg and HBeAg, which correlated with reduced intracellular viral RNA levels, but not cccDNA copy numbers. The effect lasted for >12 days after removal of the drug. HBV genotypes B, D, and E were equally inhibited. By contrast, Am80 did not affect HBV replication in transfected cells or HepG2.2.15 cells, which carry an integrated HBV genome. In conclusion, our results indicate a persistent inhibition of HBV transcription by Am80, which might be used for drug repositioning.
Differentiation between wild boar and domestic pig in food by targeting two gene loci by real-time PCR
Maria Kaltenbrunner - 2019
Abstract
Studies indicate that many meat products are not authentic, most frequently because the meat species differ from those given on the food labels. At present, DNA based methods play the most important role in meat species authentication. Discrimination of wild boar and domestic pig meat in food is challenging because it is differentiation on the subspecies level. We developed and validated two singleplex real-time PCR assays targeting SNP rs81416363 on chromosome 9 and a duplex real-time PCR assay targeting SNP g.299084751 C > T in the NR6A1 gene located on chromosome 1. The singleplex real-time PCR assays led to some ambiguous results for Mangalica and Krškopolje pig breeds and wild boar individuals from Germany, the duplex real-time PCR assay particularly for the Turopolje pig breed. We demonstrate that the probability of misclassification can be substantially reduced if the results of both the singleplex real-time PCR assays and the duplex real-time PCR assay are taken into consideration. 86 (91.5%) of a total of 94 individuals, comprising 64 domestic pigs (14 different breeds and 6 cross-breeds) and 30 wild boars (from Austria, Germany, Romania, USA and Estonia), were classified correctly.
Vector competence, vectorial capacity of Nyssorhynchus darlingi and the basic reproduction number of Plasmodium vivax in agricultural settlements in the Amazonian Region of Brazil
Maria Anice M. Sallum - 2019
Abstract
Background Brazilian malaria control programmes successfully reduced the incidence and mortality rates from 2005 to 2016. Since 2017, increased malaria has been reported across the Amazon. Few field studies focus on the primary malaria vector in high to moderate endemic areas, Nyssorhynchus darlingi, as the key entomological component of malaria risk, and on the metrics of Plasmodium vivax propagation in Amazonian rural communities. Methods Human landing catch collections were carried out in 36 houses of 26 communities in five municipalities in the Brazilian states of Acre, Amazonas and Rondônia states, with API (> 30). In addition, data on the number of locally acquired symptomatic infections were employed in mathematical modelling analyses carried out to determine Ny. darlingi vector competence and vectorial capacity to P. vivax; and to calculate the basic reproduction number for P. vivax. Results Entomological indices and malaria metrics ranged among localities: prevalence of P. vivax infection in Ny. darlingi, from 0.243% in Mâncio Lima, Acre to 3.96% in Machadinho D’Oeste, Rondônia; daily human-biting rate per person from 23 ± 1.18 in Cruzeiro do Sul, Acre, to 66 ± 2.41 in Lábrea, Amazonas; vector competence from 0.00456 in São Gabriel da Cachoeira, Amazonas to 0.04764 in Mâncio Lima, Acre; vectorial capacity from 0.0836 in Mâncio Lima, to 1.5 in Machadinho D’Oeste. The estimated R0 for P. vivax (PvR0) was 3.3 in Mâncio Lima, 7.0 in Lábrea, 16.8 in Cruzeiro do Sul, 55.5 in São Gabriel da Cachoeira, and 58.7 in Machadinho D’Oeste. Correlation between P. vivax prevalence in Ny. darlingi and vector competence was non-linear whereas association between prevalence of P. vivax in mosquitoes, vectorial capacity and R0 was linear and positive. Conclusions In spite of low vector competence of Ny. darlingi to P. vivax, parasite propagation in the human population is enhanced by the high human-biting rate, and relatively high vectorial capacity. The high PvR0 values suggest hyperendemicity in Machadinho D’Oeste and São Gabriel da Cachoeira at levels similar to those found for P. falciparum in sub-Saharan Africa regions. Mass screening for parasite reservoirs, effective anti-malarial drugs and vector control interventions will be necessary to shrinking transmission in Amazonian rural communities, Brazil.
Real‐time PCR assays for rapid detection of Zeugodacus cucumis and Bactrocera jarvisi (Diptera: Tephritidae) for quarantine application
Dongmei Li - 2019
Abstract
Zeugodacus cucumis and Bactrocera jarvisi are pests of fruit and vegetable crops and cause damage to horticulture industries. Immature stages of these two fruit fly species have been intercepted in New Zealand a number of times. Identification to species was not possible using morphological characters; thus, it is important to develop an assay for their species‐level identification. Here, the real‐time PCR assays for rapid identification of Z. cucumis and B. jarvisi were developed and validated. The PCR protocols demonstrated their specificity by amplifying the two target species successfully, with no cross‐reactions observed in the tested tephritid species. The in silico test of the primer and probe binding sites of the two assays also demonstrated the assays’ specificity by no mismatches present in the binding regions of the target species, but 1–4 mismatches in the binding regions of the non‐target fruit fly species. The thresholds of detection for the two assays are as low as 10 copies/µl of the target DNA, indicating that the assays have a very high sensitivity. The application of the real‐time PCR assays has greatly assisted in routine pest identifications at the New Zealand border and surveillance programme. Therefore, the assays have the potential to be used by diagnostic agencies and research organizations worldwide.
Digital Loop-Mediated Isothermal Amplification on a Commercial Membrane
Xingyu Lin - 2019
Abstract
In this work, we report digital loop-mediated isothermal amplification (LAMP) or reverse-transcription LAMP (RT-LAMP) on a commercial membrane, without the need for complex chip fabrication or use of specialized equipment. Due to the pore size distribution, the theoretical error for digital LAMP on these membranes was analyzed, using a combination of Random Distribution Model and Multivolume Theory. A facile peel-off process was developed for effective droplet formation on the commercial track-etched polycarbonate (PCTE) membrane. Each pore functions as an individual nanoreactor for single DNA amplification. Absolute quantification of bacteria genomic DNA was realized with a dynamic range from 11 to 1.1 × 105 copies/μL. One-step digital RT-LAMP was also successfully performed on the membrane for the quantification of MS2 virus in wastewater. With the introduction of new probes, the positive pores can be easily distinguished from negative ones with 100 times difference in fluorescence intensities. Finally, the cost of a disposable membrane is less than $0.10/piece, which, to the best of our knowledge, is the most inexpensive way to perform digital LAMP. The membrane system offers opportunities for point-of-care users or common laboratories to perform digital quantification, single cell analysis, or other bioassays in an inexpensive, flexible, and simplified way.
A Brief Review of Non-Avian Reptile Environmental DNA (eDNA), with a Case Study of Painted Turtle (Chrysemys picta) eDNA Under Field Conditions
Clare I. M. Adams - 2019
Abstract
Environmental DNA (eDNA) is an increasingly used non-invasive molecular tool for detecting species presence and monitoring populations. In this article, we review the current state of non-avian reptile eDNA work in aquatic systems, and present a field experiment on detecting the presence of painted turtle (Chrysemys picta) eDNA. Thus far, turtle and snake eDNA studies have shown mixed results in detecting the presence of these animals under field conditions. However, some instances of low detection rates and non-detection occur for these non-avian reptiles, especially for squamates. We explored non-avian reptile eDNA quantification by sampling four lentic ponds with different densities (0 kg/ha, 6 kg/ha, 9 kg/ha, and 13 kg/ha) of painted turtles over three months to detect differences in eDNA using a qPCR assay amplifying the COI gene of the mtDNA genome. Only one sample of the highest-density pond amplified eDNA for a positive detection. Yet, estimates of eDNA concentration from pond eDNA were rank-order correlated with turtle density. We present the “shedding hypothesis”—the possibility that animals with hard, keratinized integument do not shed as much DNA as mucus-covered organisms—as a potential challenge for eDNA studies. Despite challenges with eDNA inhibition and availability in water samples, we remain hopeful that eDNA can be used to detect freshwater turtles in the field. We provide key recommendations for biologists wishing to use eDNA methods for detecting non-avian reptiles.
Quantification of Hepatitis B Virus Covalently Closed Circular DNA in Infected Cell Culture Models by Quantitative PCR
Bingqian Qu - 2019
Abstract
Persistence of the human hepatitis B virus (HBV) requires the maintenance of covalently closed circular (ccc)DNA, the episomal genome reservoir in nuclei of infected hepatocytes. cccDNA elimination is a major aim in future curative therapies currently under development. In cell culture based in vitro studies, both hybridization- and amplification-based assays are currently used for cccDNA quantification. Southern blot, the current gold standard, is time-consuming and not practical for a large number of samples. PCR-based methods show limited specificity when excessive HBV replicative intermediates are present. We have recently developed a real-time quantitative PCR protocol, in which total cellular DNA plus all forms of viral DNA are extracted by silica column. Subsequent incubation with T5 exonuclease efficiently removes cellular DNA and all non-cccDNA forms of viral DNA while cccDNA remains intact and can reliably be quantified by PCR. This method has been used for measuring kinetics of cccDNA accumulation in several in vitro infection models and the effect of antivirals on cccDNA. It allowed detection of cccDNA in non-human cells (primary macaque and swine hepatocytes, etc.) reconstituted with the HBV receptor, human sodium taurocholate cotransporting polypeptide (NTCP). Here we present a detailed protocol of this method, including a work flowchart, schematic diagram and illustrations on how to calculate “cccDNA copies per (infected) cell”.
Linking the resistome and plasmidome to the microbiome
Thibault Stalder - 2019
Abstract
The rapid spread of antibiotic resistance among bacterial pathogens is a serious human health threat. While a range of environments have been identified as reservoirs of antibiotic resistance genes (ARGs), we lack understanding of the origins of these ARGs and their spread from environment to clinic. This is partly due to our inability to identify the natural bacterial hosts of ARGs and the mobile genetic elements that mediate this spread, such as plasmids and integrons. Here we demonstrate that the in vivo proximity-ligation method Hi-C can reconstruct a known plasmid-host association from a wastewater community, and identify the in situ host range of ARGs, plasmids, and integrons by physically linking them to their host chromosomes. Hi-C detected both previously known and novel associations between ARGs, mobile genetic elements and host genomes, thus validating this method. We showed that IncQ plasmids and class 1 integrons had the broadest host range in this wastewater, and identified bacteria belonging to Moraxellaceae, Bacteroides, and Prevotella, and especially Aeromonadaceae as the most likely reservoirs of ARGs in this community. A better identification of the natural carriers of ARGs will aid the development of strategies to limit resistance spread to pathogens.
Development of event-specific qPCR detection methods for genetically modified alfalfa events J101, J163 and KK179
Patrick Guertler - 2019
Abstract
Genetically modified alfalfa is authorized for cultivation in several countries since 2005. On the other hand, cultivation in or export to the European Union is not allowed and thus neither certified reference material nor official event-specific detection methods are available. Therefore, based on patent sequence information, eventspecific real-time PCR detection methods targeting the junction sequence of the alfalfa genome and the transgenic insert of the respective events J101, J163 and KK179 were developed. Newly developed plasmids were used as reference material for assay optimization and in-house validation. Plasmid standards were quantified using digital droplet PCR and LOD95%, PCR efficiency, robustness and specificity of the assays were determined using real-time PCR. A LOD95% of 10 copies per PCR reaction was observed and PCR efficiencies of 95–97 % were achieved. Different real-time PCR instruments and PCR conditions were applied to test for robustness of the assays using DNA at a concentration of 30 copies per μL for each gm alfalfa event. All replicates were positive independent of the instrument or the PCR condition. DNA from certified reference material of different genetically modified crops as well as reference materials of the three events was used to experimentally test for specificity. No unspecific amplification signal was observed for any of the assays. Validation results were in line with the “Minimum Performance Requirements for Analytical Methods of GMO Testing” of the European Network of GMO Laboratories. Furthermore, an inter-laboratory comparison study was conducted to show the transferability and applicability of the methods and to verify the assay performance parameters.
A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis
Henk J. Wisselink - 2019
Abstract
Background: Several species-specific PCR assays, based on a variety of target genes are currently used in the diagnosis of Mycoplasma bovis infections in cattle herds with respiratory diseases and/or mastitis. With this diversity of methods, and the development of new methods and formats, regular performance comparisons are required to ascertain diagnostic quality. The present study compares PCR methods that are currently used in six national veterinary institutes across Europe. Three different sample panels were compiled and analysed to assess the analytical specificity, analytical sensitivity and comparability of the different PCR methods. The results were also compared, when appropriate, to those obtained through isolation by culture. The sensitivity and comparability panels were composed of samples from bronchoalveolar fluids of veal calves, artificially contaminated or naturally infected, and hence the comparison of the different methods included the whole workflow from DNA extraction to PCR analysis. Results: The participating laboratories used i) five different DNA extraction methods, ii) seven different real-time and/or end-point PCRs targeting four different genes and iii) six different real-time PCR platforms. Only one commercial kit was assessed; all other PCR assays were in-house tests adapted from published methods. The analytical specificity of the different PCR methods was comparable except for one laboratory where Mycoplasma agalactiae was tested positive. Frequently, weak-positive results with Ct values between 37 and 40 were obtained for non-target Mycoplasma strains. The limit of detection (LOD) varied from 10 to 103 CFU/ml to 103 and 106 CFU/ ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down to 102 CFU/ml. Although Ct values showed considerable variation with naturally infected samples, both between laboratories and tests, the final result interpretation of the samples (positive versus negative) was essentially the same between the different laboratories. Conclusion: With a few exceptions, all methods used routinely in the participating laboratories showed comparable performance, which assures the quality of diagnosis, despite the multiplicity of the methods.
Effect of Thermotherapy on the Acquisition of Candidatus Liberibacter Asiaticus by the Asian Citrus Psyllid (Hemiptera: Liviidae)
Alicia J. Kelley - 2019
Abstract
The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is the most detrimental insect pest of citrus crops due to its role as a vector of Candidatus Liberibacter asiaticus (Las), the bacterial causal agent of huanglongbing, also known as citrus greening disease. Trees infected with Las decline rapidly and fruit production decreases until eventual tree death. Few treatment options for infected trees are available for disease management. A technique called “thermotherapy” is under development to reduce bacterial titers in infected trees; however, the effect of these treatments on the transmission cycle of Las is not known. Field and laboratory assays were conducted to determine whether thermotherapy treatment reduced Las acquisition by D. citri. Trees in the field were treated with a mobile heat treatment system. Potted trees in the laboratory were treated in a steam chamber. We monitored acquisition rates in D. citri following thermal treatment of Las-positive Citrus sinensis (L.) (Rutaceae). Psyllid acquisition and Las titer in thermotherapy-treated trees were compared with untreated Las-positive and untreated Las-negative trees. Our results confirmed the efficacy of whole-tree thermotherapy on Las in potted citrus trees. In contrast, thermotherapy did not significantly reduce plant Las titers or acquisition of Las by D. citri under field conditions. These results suggest that further development of field application methods is needed to determine the utility of thermotherapy as a tool for huanglongbing management.
Using discrete and online ATP measurements to evaluate regrowthpotential following ozonation and (non)biological drinking watertreatment
Glen Andrew de Vera - 2019
Abstract
Water utilities must control microbial regrowth in the distribution system to protect public health. In thisstudy, an adenosine triphosphate (ATP)-based biomass production potential test using indigenous bac-terial communities were used to evaluate regrowth potential following ozonation with either bio-filtration (BF) or sustained chlorination (SCl2). Two full-scale water treatment plants with differentupstream processes (i.e., WTP-BF: ozonation, coagulation/flocculation, biofiltration, UV irradiation,chlorination; and WTP-SCl2: ozonation, chlorination, coagulation/flocculation,filtration, chlorination)were compared. Characterization of indigenous bacteria using 16S rRNA gene sequencing, qPCR, andcellular ATP (cATP) showed microbial diversity changes across treatment, biomass sloughing from bio-filters (effluent cATP¼30±1 ng/L), and disinfection by chlorine (cATP<1 ng/L). For both WTPs, 14-daycumulative biomass production (CBPt¼Ptt¼0ATPðtÞDt) was highest for ozonated water samples(CBP14¼1.2103e3.0103d ngATP/L). CBP further increased with increasing ozone dose due to pro-duction of more biodegradable carbon. Growth promotion by carbon was confirmed from the con-sumption of ozonation byproducts (carboxylic acids, aldehydes) and the increase in CBP (9.5102e2.9103d ngATP/L) after addition of 50e300mgC/L acetate. Ozone followed by sustained chlorination(WTP-SCl2) effectively controlled biomass growth across the treatment process (CBP14<10 d ngATP/L). Incontrast, ozone followed by biofiltration (WTP-BF) reduced regrowth potential by 30% (biofilter influentCBP14¼1.3103d ngATP/L; biofilter effluent CBP14¼9.3102d ngATP/L). After adding chlorine to thebiofilter effluent, CBP14was reduced to<10 d ngATP/L. Lastly, online ATP measurements confirmed thediscrete measurements and improved identification of the cATP peak and growth phases of indigenousbacteria.
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