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PerfeCTa MultiPlex qPCR ToughMix

Advanced 1-tube SuperMix optimized to support highly multiplexed DNA amplifications in miniaturized reaction volumes and withstand a broad spectrum of PCR inhibitors
Features & Benefits
  • 1-tube SuperMix minimizes pipetting steps, simplifying reaction assembly and improving accuracy
  • 5x concentrated reagent provides more flexibility with dilute DNA samples sensitivity
  • ToughMix reagent technology neutralizes a broad spectrum of PCR inhibitors common in plant and animal tissues, environmental sources, clinical specimens and complex food matrices
  • Superior assay sensitivity and specificity with ultrapure AccuStart II enzyme technology – maximum-yielding Taq DNA polymerase mutant controlled by stringent, multi-epitope antibody hotstart
  • Supports efficient vortex mixing with proprietary anti-foaming additives

 

PerfeCTa Multiplex 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 Multiplex qPCR ToughMix

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Kit Size:  250 x 25 μL rxns (1 x 1.25 mL)
Part Number:  95147-250
Price:  $570.00
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Kit Size:  1000 x 25 μL rxns (4 x 1.25 mL)
Part Number:  95147-01K
Price:  $2,150.00
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Kit Size:  5000 x 25 μL rxns (1 x 25 mL)
Part Number:  95147-05K
Price:  $9,409.00
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PerfeCTa Multiplex qPCR ToughMix ROX

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Kit Size:  250 x 25 μL rxns (1 x 1.25 mL)
Part Number:  95148-250
Price:  $571.00
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Kit Size:  1000 x 25 μL rxns (4 x 1.25 mL)
Part Number:  95148-01K
Price:  $2,150.00
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Kit Size:  5000 x 25 μL rxns (1 x 25 mL)
Part Number:  95148-05K
Price:  $9,412.00
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PerfeCTa Multiplex qPCR ToughMix Low ROX

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Kit Size:  250 x 25 μL rxns (1 x 1.25 mL)
Part Number:  95149-250
Price:  $571.00
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Kit Size:  1000 x 25 μL rxns (4 x 1.25 mL)
Part Number:  95149-01K
Price:  $2,150.00
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Kit Size:  5000 x 25 μL rxns (1 x 25 mL)
Part Number:  95149-05K
Price:  $9,409.00
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Description

Suppression of low copy amplicons by high copy reference targets during multiplex co-amplification skews the apparent representation and quantification of low copy target sequences. PerfeCTa MultiPlex qPCR ToughMix transcends these limitations by enabling sensitive, broad linear dynamic detection range with co-amplification of four abundant (106) targets. PerfeCTa MultiPlex qPCR ToughMix enables multiplex qPCR assay performance comparable to single-plex qPCR probe assays without the need to rigorously titrate primer concentration. PerfeCTa MultiPlex qPCR ToughMix is a 5x concentrated, ready to-use reaction cocktail for real-time quantitative PCR (qPCR) with ToughMix reagent technology that neutralizes a broad spectrum of PCR inhibitors that compromise assay performance with crude extracts, clinical specimens, plant, soil environmental or complex food matrices. A key component of the kit is an ultrapure, highly-processive thermostable DNA polymerase that is free of detectable E. coli DNA. PerfeCTa MultiPlex qPCR ToughMix is ideal for demanding qPCR applications such as bacterial pathogen detection where residual host DNA in typical recombinant enzyme preparations can limit assay sensitivity and obscure detection of low copy samples.

Featured Publication

A Multiplexed Real-time PCR Assay to Detect SCID and SMN1 Homozygous Exon 7 Deletion, and A Droplet Digital PCR Assay to Assess SMN2 Copy Numbers, in Newborn screening for Spinal Muscular Atrophy

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Details

Details

Contents

Single-tube, 5X concentrated reagent containing:

  • Reaction buffer with optimized concentrations of molecular-grade MgCl2, dATP, dCTP, dGTP, dTTP.
  • AccuStart II Taq DNA Polymerase.
  • 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 MultiPlex qPCR ToughMix

"The Perfecta Multiplex qPCR ToughMix greatly simplified my qPCR assay by removing DNA isolation steps and complexity of passive reference dyes. I will definitely be using this product in the future."

Bethany B. | Biotech company

Details

Contents

Single-tube, 5X concentrated reagent containing:

  • Reaction buffer with optimized concentrations of molecular-grade MgCl2, dATP, dCTP, dGTP, dTTP.
  • AccuStart II Taq DNA Polymerase.
  • 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

Resources

Flyers

Product Manuals

Application Notes

CofA (PSFs)

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SDSs

Publications

Rainbow smelt (Osmerus mordax) influence on walleye (Sander vitreus) recruitment decline: mtDNA evidence supporting the predation hypothesis
Jesse M. Lepak - 2023
Abstract
Rainbow smelt (Osmerus mordax) have been introduced widely but are associated with declines in walleye (Sander vitreus) recruitment. A primary hypothesis for these declines is that O. mordax consume larval S. vitreus. We confirmed overlapping spatial–temporal distributions of larval S. vitreus and O. mordax in our study system and used mtDNA analyses to determine if O. mordax stomach contents contained S. vitreus. Approximately 20% of O. mordax composite stomach samples were considered positive for S. vitreus consumption. These findings support the predation hypothesis and have S. vitreus management/stocking implications.
Molecular Detection of Cyclospora cayetanensis in Two Main Types of Farm Soil Using Real-Time PCR Assays and Method Modification for Commercial Potting Mix
Joseph Arida - 2023
Abstract
Cyclospora cayetanensis is a foodborne protozoan parasite that causes outbreaks of diarrheal illness (cyclosporiasis) with clear seasonality worldwide. In the environment, C. cayetanensis oocysts are very robust, and contact with contaminated soil may serve as an important vehicle in the transmission of this organism, and it is considered a risk factor for this infection. The present study evaluated a flotation concentration method, previously shown to provide the best detection results when compared with DNA isolation directly from soil samples, in two main types of farm soil, silt loam soil and sandy clay loam, as well as in commercial potting mix samples inoculated with different numbers of C. cayetanensis oocysts. The flotation method was able to detect as few as 10 oocysts in 10 g of either type of farm soil without modifications, but needed an extra wash and samples of reduced size for the processing of the commercial potting mix to be able to detect 20 oocysts/5 g. A recently modified real-time PCR method for the detection of C. cayetanensis based on a mitochondrial gene target was also evaluated using selected samples of each type of soil. This comparative study confirmed that the concentration of oocysts in soil samples by flotation in high-density sucrose solutions is a sensitive method that can detect low numbers of oocysts in different types of soil.
A high-throughput newborn screening approach for SCID, SMA, and SCD combining multiplex qPCR and tandem mass spectrometry
Rafael Tesorero - 2023
Abstract
Early diagnosis of severe combined immunodeficiency (SCID), spinal muscular atrophy (SMA), and sickle cell disease (SCD) improves health outcomes by providing a specific treatment before the onset of symptoms. A high-throughput nucleic acid-based method in newborn screening (NBS) has been shown to be fast and cost-effective in the early detection of these diseases. Screening for SCD has been included in Germany’s NBS Program since Fall 2021 and typically requires high-throughput NBS laboratories to adopt analytical platforms that are demanding in terms of instrumentation and personnel. Thus, we developed a combined approach applying a multiplexed quantitative real-time PCR (qPCR) assay for simultaneous SCID, SMA, and 1st-tier SCD screening, followed by a tandem mass spectrometry (MS/MS) assay for 2nd-tier SCD screening. DNA is extracted from a 3.2-mm dried blood spot from which we simultaneously quantify T-cell receptor excision circles for SCID screening, identify the homozygous SMN1 exon 7 deletion for SMA screening, and determine the integrity of the DNA extraction through the quantification of a housekeeping gene. In our two-tier SCD screening strategy, our multiplex qPCR identifies samples carrying the HBB: c.20A>T allele that is coding for sickle cell hemoglobin (HbS). Subsequently, the 2nd tier MS/MS assay is used to distinguish heterozygous HbS/A carriers from samples of patients with homozygous or compound heterozygous SCD. Between July 2021 and March 2022, 96,015 samples were screened by applying the newly implemented assay. The screening revealed two positive SCID cases, while 14 newborns with SMA were detected. Concurrently, the qPCR assay registered HbS in 431 samples which were submitted to 2nd-tier SCD screening, resulting in 17 HbS/S, five HbS/C, and two HbS/β thalassemia patients. The results of our quadruplex qPCR assay demonstrate a cost-effective and fast approach for a combined screening of three diseases that benefit from nucleic-acid based methods in high-throughput NBS laboratories.
Development and Single Laboratory Evaluation of a Refined and specific Real-time PCR Detection Method, Using Mitochondrial Primers (Mit1C), for the Detection of Cyclospora cayetanensis in Produce
Kannan V. Balan - 2023
Abstract
Regulatory methods for detection of the foodborne protozoan parasite Cyclospora cayetanensis must be specific and sensitive. To that end, we designed and evaluated (in a single laboratory validation) a novel and improved primer/probe combination (Mit1C) for real-time PCR detection of C. cayetanensis in produce. The newly developed primer/probe combination targets a conserved region of the mitochondrial genome of C. cayetanensis that varies in other closely related organisms. The primer/probe combination was evaluated both in silico and using several real-time PCR kits and polymerases against an inclusivity/exclusivity panel comprised of a variety of C. cayetanensis oocysts, as well as DNA from other related Cyclospora spp. and closely related parasites. The new primer/probe combination amplified only C. cayetanensis, thus demonstrating specificity. Sensitivity was evaluated by artificially contaminating cilantro, raspberries, and romaine lettuce with variable numbers (200 and 5) of C. cayetanensis oocysts. As few as 5 oocysts were detected in 75%, 67.7%, and 50% of the spiked produce samples (cilantro, raspberries, and romaine lettuce), respectively, all uninoculated samples and no-template real-time PCR controls were negative. The improved primer/probe combination should prove an effective analytical tool for the specific detection of C. cayetanensis in produce.
Development and single laboratory evaluation of a refined and specific real-time PCR detection method, using mitochondrial primers (Mit1C), for the detection of Cyclospora cayetanensis in produce
Kannan Balan - 2023
Abstract
Regulatory methods for detection of the foodborne protozoan parasite Cyclospora cayetanensis must be specific and sensitive. To that end, we designed and evaluated (in a single laboratory validation) a novel and improved primer/probe combination (Mit1C) for real-time PCR detection of C. cayetanensis in produce. The newly developed primer/probe combination targets a conserved region of the mitochondrial genome of C. cayetanensis that varies in other closely related organisms. The primer/probe combination was evaluated both in silico and using several real-time PCR kits and polymerases against an inclusivity/exclusivity panel comprised of a variety of C. cayetanensis oocysts, as well as DNA from other related Cyclospora spp. and closely related parasites. The new primer/probe combination amplified only C. cayetanensis, thus demonstrating specificity. Sensitivity was evaluated by artificially contaminating cilantro, raspberries, and romaine lettuce with variable numbers (200 and 5) of C. cayetanensis oocysts. As few as 5 oocysts were detected in 75%, 67.7%, and 50% of the spiked produce samples (cilantro, raspberries, and romaine lettuce), respectively. All uninoculated samples and no-template real-time PCR controls were negative. The improved primer/probe combination should prove an effective analytical tool for the specific detection of C. cayetanensis in produce.
Absolute quantitative detection of genetically modified soybean MON87708×MON89788 with stacked traits by digital polymerase chain reaction
Junyi Xu - 2022
Abstract
The main advantage of digital PCR (dPCR) is that it facilitates absolute quantification of the target without reference to the standard/calibration curve. Crystal droplet dPCR has a three-color staining detection function, which enables multiplex PCR reaction. In this study, this technique was used to establish triple dPCR detection for the genetically modified soybean MON87708×MON89788 with stacked traits. Specific absolute quantitative detection was accomplished for the genomic DNA extracted from the homogenized seeds of GM stack MON87708×MON89788 soybean. Our results can serve as a reference for the absolute quantitative detection of stacked events of genetically modified crops.
Establishment of a Cynomolgus Macaque Model of Human T-Cell Leukemia Virus Type 1 (HTLV-1) Infection by Direct Inoculation of Adult T-Cell Leukemia Patient-Derived Cell Lines for HTLV-1 Infection
Emiko Urano - 2022
Abstract
Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). However, the precise mechanisms leading to HTLV-1 chronic infection and the onset of the diseases have remained unclear, and effective vaccines for inhibiting the infection and the progression of pathogenesis have therefore not been developed. The use of a nonhuman primate (NHP) model is thought to be important for revealing the mechanisms of the progressive status and for the development of prevention procedures. In this study, we developed a cynomolgus macaque (CM) model of HTLV-1 infection by direct intravenous inoculation of HTLV-1-producing cells derived from ATL patients. The cell line used for infection, ATL-040, was selected as the most infectious one in our cell line library. CMs inoculated intravenously with 1 108 ATL-040 cells per animal became persistently infected with HTLV-1, as shown by the HTLV-1 provirus load (PVL) in peripheral blood mononuclear cells and HTLV-1-specific antibodies (2/2 animals). One CM inoculated intravenously with 1 107 ATL-040 cells did not have detectable PVLs despite the fact that anti-HTLV-1 antibodies were maintained for more than 2 years. Furthermore, immunological approaches, including CD81 T cell depletion prior to infection (3/3 animals) and intrathecal inoculation (3/3 animals), led to increased proviral loads in the cynomolgus monkeys. The present method and the cynomolgus monkey model of HTLV-1 infection will be beneficial for immunological and virological studies on HTLV-1 aiming at the development of anti-HTLV-1 prophylactic vaccines and therapy drugs. IMPORTANCE HTLV-1 was discovered in the 1980s as the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, the precise mechanisms leading to HTLV-1 chronic infection and the onset of the diseases still remain unidentified. Thus, no effective vaccines to inhibit the infection and the progressive of pathogenesis have been developed. The use of appropriate animal models is essential for understanding HTLV-1 infection and pathogenesis. In order to establish a new nonhuman primate model for studies on HTLV-1 infection, cynomolgus monkeys were infected with HTLV-1 under a variety of experimental conditions. Our method, using a cell line generated from an ATL patient as a source of HTLV-1, was able to establish HTLV-1 infection in monkeys with a 100% success rate. This cynomolgus macaque model of HTLV-1 infection will contribute to the elucidation of HTLV-1 infection and its associated disease development.
Efficiency-corrected PCR quantification for identification of prevalence and load of respiratory disease-causing agents in feedlot cattle
RJ Barnewall - 2022
Abstract
Bovine respiratory disease (BRD) is the most prevalent disease in feedlot cattle worldwide with Bovine alphaherpesvirus 1 (BoAHV1), Histophilus somni, Mannheimia haemolytica, Mycoplasma bovis, Pasteurella multocida and Trueperella pyogenes accepted to be common etiological agents associated with BRD. Although these agents are common in the upper and lower airways in clinical BRD cases, some also exist as normal flora suggesting their presence in the upper airways alone is not necessarily informative with respect to disease status or risk. To determine the relationship between presence, load and disease status, we investigated the relationship between load in the upper airways at induction and active BRD cases in feedlot cattle using efficiency-corrected PCR quantification. By this approach, we were able to accurately determine the prevalence and load of the key BRD agents in the upper respiratory tract showing that cattle in the hospital pen had a higher prevalence, and load, of these agents both singly and in combination compared to cattle sampled at feedlot induction. A combination of agents was the most accurate indicator of BRD risk with cattle with four or more agents detected in the upper airway more likely to be undergoing treatment for BRD than non-BRD ailments. In addition, M. bovis was rarely detected at feedlot induction but was identified at high prevalence in cattle in the hospital pen. These findings present a potential new technological approach for the investigation, analysis and identification of BRD-associated viral and bacterial agents for Australian feedlot systems as well as for BRD disease management and treatment
A telomere-to-telomere gap-free reference genome of watermelon and its mutation library provide important resources for gene discovery and breeding
Yun Deng - 2022
Abstract
Watermelon, Citrullus lanatus, is the world’s third largest fruity crop. Reference genomes with gaps and narrow genetic base hinder functional genomics and genetic improvement of watermelon. Here, we report the assembly of a telomere-to-telomere (T2T) gap-free genome of the elite watermelon inbred G42 by incorporating the high-coverage and accurate long-read sequence data with multiple assembly strategies. All 11 chromosomes have been assembled into single contig pseudomolecules without gap, representing the highest completeness and assembly quality till now. The G42 reference genome contained a total length of 369,321,829 bp and 24,205 predicted protein-coding genes, with all 22 telomeres and 11 centromeres characterized. Over 200,000 M1 seeds from inbred G42 were generated using pollen EMS mutagenesis. In a sampling pool, 48 monogenic phenotypic mutations, selected from 223 M1 and 78 M2 mutants with morphological changes, were confirmed. The average density of mutation is 1 SNP/1.69 Mb and 1 indel/4.55 Mb per M1 plant, and 1 SNP/1.08 Mb and 1 indel/6.25 Mb per M2 plant. Taking advantage of the gap-free G42 genome, 8,039 mutations from the 32 plants sampled from M1 and M2 families were identified with 100% accuracy, whereas only 25% of the randomly selected mutations identified using 97103v2 reference genome could be confirmed. Using this library and the gap-free genome, two genes responsible for elongated fruit shape and male sterility (ClMS1) were identified, both being caused by a single base change from G to A. The validated gap-free genome and its EMS mutation library provide invaluable resources for functional genomics and genetic improvement of watermelon.
Molecular characterization of Xanthomonas species isolated from Araceae and the development of a triplex TaqMan assay for detection of Xanthomonas phaseoli pv. dieffenbachiae
Jan van der Wolf - 2022
Abstract
In total 58 Xanthomonas strains isolated from Araceae worldwide, together with 13 other phylogenetically-related Xanthomonas strains, were characterized using multilocus sequence analysis based on concatenated sequences of seven single copy orthologous genes, extracted from whole genome sequences. The analysis revealed a monophyletic clade of 48 strains, 44 isolated from Anthurium, identified as X. phaseoli pv. dieffenbachiae (Xpd) confirmed by nucleotide identity analysis. The other strains from aroids were identified as Xanthomonas euvesicatoria (2 strains), X citri (5 strains) and Xanthomonas sacchari (3 strains). Two TaqMan assays were designed for specific detection of Xpd, one targeting sequences of a hypothetical protein and one targeting a type I restriction endonuclease subunit S. The two assays showed similar reaction kinetics and were merged with an assay comprising an amplification and extraction control into a triplex assay. The assay was able to detect minimally 100 copies of a target sequence delivered as a gBlock, 100 fg of genomic DNA and 104 cells per mL in an Anthurium leaf extract.
Variable Legionella Response to Building Occupancy Patterns and Precautionary Flushing
William J. Rhoads - 2022
Abstract
When stay-at-home orders were issued to slow the spread of COVID-19, building occupancy (and water demand) was drastically decreased in many buildings. There was concern that widespread low water demand may cause unprecedented Legionella occurrence and Legionnaires’ disease incidence. In lieu of evidenced-based guidance, many people flushed their water systems as a preventative measure, using highly variable practices. Here, we present field-scale research from a building before, during, and after periods of low occupancy, and controlled stagnation experiments. We document no change, a > 4-log increase, and a > 1.5-log decrease of L. pneumophila during 3- to 7-week periods of low water demand. L. pneumophila increased by > 1-log after precautionary flushing prior to reoccupancy, which was repeated in controlled boiler flushing experiments. These results demonstrate that the impact of low water demand (colloquially called stagnation) is not as straight forward as is generally assumed, and that some flushing practices have potential unintended consequences. In particular, stagnation must be considered in context with other Legionella growth factors like temperature and flow profiles. Boiler flushing practices that dramatically increase the flow rate and rapidly deplete boiler temperature may mobilize Legionella present in biofilms and sediment.
Challenging the “gold standard” of colony-forming units - Validation of a multiplex real-time PCR for quantification of viable Campylobacter spp. in meat rinses
Kerstin Stingl - 2022
Abstract
Campylobacter jejuni is the leading bacterial food-borne pathogen in Europe. Despite the accepted limits of cultural detection of the fastidious bacterium, the “gold standard” in food microbiology is still the determination of colony-forming units (CFU). As an alternative, a live/dead differentiating qPCR has been established, using propidium monoazide (PMA) as DNA-intercalating crosslink agent for inactivating DNA from dead, membrane-compromised cells. The PMA treatment was combined with the addition of an internal sample process control (ISPC), i.e. a known number of dead C. sputorum cells to the samples. The ISPC enables i), monitoring the effective reduction of dead cell signal by the light-activated DNA-intercalating dye PMA, and ii), compensation for potential DNA losses during processing. Here, we optimized the method for routine application and performed a full validation of the method according to ISO 16140-2:2016(E) for the quantification of live thermophilic Campylobacter spp. in meat rinses against the classical enumeration method ISO 10272-2:2017. In order to render the method applicable and cost-effective for practical application, the ISPC was lyophilized to be distributable to routine laboratories. In addition, a triplex qPCR was established to simultaneously quantify thermophilic Campylobacter, the ISPC and an internal amplification control (IAC). Its performance was statistically similar to the two duplex qPCRs up to a contamination level of 4.7 log10 Campylobacter per ml of meat rinse. The limit of quantification (LOQ) of the alternative method was around 20 genomic equivalents per PCR reaction, i.e. 2.3 log10 live Campylobacter per ml of sample. The alternative method passed a relative trueness study, confirming the robustness against different meat rinses, and displayed sufficient accuracy within the limits set in ISO 16140-2:2016(E). Finally, the method was validated in an interlaboratory ring trial, confirming that the alternative method was fit for purpose with a tendency of improved repeatability and reproducibility compared to the reference method for CFU determination. Campylobacter served as a model organism, challenging CFU as “gold standard” and could help in guidance to the general acceptance of live/dead differentiating qPCR methods for the detection of food-borne pathogens.
QUANTITATIVE ASSESSMENT OF METHODS FOR BACTERIAL AND VIRAL PURIFICATION AND CONCENTRATION
Nina Sara Fraticelli-Guzmán - 2021
Abstract
The COVID-19 global pandemic has led to the exploration and implementation of rapid tests for viral load identification and thus, control of the spread. For this, there are varying approaches with the gold standard being quantitative Polymerase Chain Reaction [1]and a potential more rapid alternate approach being the use of an antibody coated biosensor such as a field effect transistor (FET). Analogously, bacterial pathogens such as anthrax need sensing and quantification as well. Therefore, methods for pathogen collection and detection for both viruses and bacteria are needed. Regardless of the analysis technique, it is ideal for samples to contain little to zero non-target particles or contaminants that might interfere with the detection approach. As such, traditional ways of purifying and concentrating samples prior to any type of analysis involve both chemical, physical, physiochemical, or biological approaches such as filtration, centrifugation, affinity chromatography, immunomagnetic separation, etc. Of these, we will focus on the physical approach, filtration, due to its simplicity, low cost, varying options, and ability to process large and small sample volumes. In this work, we investigate how well the method works for virus or bacteria in the presence of high interferent concentrations that could potentially be present in saliva samples or other hydrosol samples taken from an environment to be tested. The research presented here characterizes the applicability of syringe filters and a tangential flow filtration device for the purification and concentration of bacteria and virus samples, respectively. Furthermore, automation of such systems was explored. Varying syringe filter pore sizes yielded different recoveries of bacteria for purifying and concentrating the sample. Furthermore, varying volumes were analyzed for xviii optimal recovery and concentration of the target. We also developed a fully automated method for double filter filtration to enable hands-free purification and concentration in 5.5 minutes for 5 mL of input volume with a 42 ± 13-fold enrichment improvement (n = 3). Furthermore, the purification and concentration of virus using a manually operated tangential flow filtration device was also explored and yielded modest concentration increases of around 2× with an enrichment improvement of up to 1,916 ± 1,839-fold (n = 3) under one configuration. By characterizing and automating these readily available items, we can enhance the detection of samples by decreasing labor time and processing complexity required for the purification and concentration of the target pathogens.
Improving Soil Quality and Potato Productivity with Manure and High-Residue Cover Crops in Eastern Canada
Judith Nyiraneza - 2021
Abstract
Under intensive low residue agricultural systems, such as those involving potato (Solanum tuberosum L.)-based systems, stagnant crop yields and declining soil health and environmental quality are common issues. This study evaluated the effects of pen-pack cow (Bos Taurus) manure application (20 Mg·ha−1) and cover crops on nitrate dynamics and soil N supply capacity, subsequent potato yield, selected soil properties, and soil-borne disease. Eight cover crops were tested and included grasses, legumes, or a mixture of legumes and grasses, with red clover (Trifolium pratense L.) used as a control. Forage pearl millet (Pennisetum glaucum L.) was associated with highest dry matter. On average, red clover had 88% higher total N accumulation than the treatments mixing grasses and legumes, and the former was associated with higher soil nitrate in fall before residue incorporation and overwinter, but this was not translated into increased potato yields. Pearl millet and sorghum sudangrass (Sorghum bicolor × sorghum bicolor var. Sudanese) were associated with lower soil nitrate in comparison to red clover while being associated with higher total potato yield and lower numerical value of root-lesion nematodes (Pratylenchus penetrans), although this was not statistically significant at 5% probability level. Manure incorporation increased total and marketable yield by 28% and 26%, respectively, and increased soil N supply capacity by an average of 44%. Carbon dioxide released after a short incubation as a proxy of soil microbial respiration increased by an average of 27% with manure application. Our study quantified the positive effect of manure application and high-residue cover crops on soil quality and potato yield for the province of Prince Edward Island.
Detection of Ralstonia pseudosolanacearum in drain water based on concentration, enrichment and the use of a duplex TaqMan PCR test
N. Sedighian - 2020
Abstract
The Ralstonia solanacearum species complex (RSSC) can cause bacterial wilt in a wide variety of plant species, including a number of ornamental glasshouse crops. Recently in Europe, ornamental rose plants for the production of cut flowers and propagation materials have been strongly affected by Ralstonia pseudosolanacearum, phylotype I, biovar 3. To test for the presence of the pathogen in the glasshouse, sampling of water from a drainage gutter or well may be an efficient strategy since it is known that RSSC can be released from infected root systems in the water. A protocol was developed to detect low densities of R. pseudosolanacearum in drain water collected from rose growers. Drain water was filtered through a bacterial filter, the filtrate was collected and target bacteria enriched for 48 h in Semi-selective Medium South Africa (SMSA) broth supplemented with sterilized tomato plant extracts. DNA extracted from the enrichment broth was analysed using a TaqMan test in a duplex format, based on specific egl sequences of RSSC and the use of an extraction and amplification control. The optimized protocol had a detection level of ≤1–10 colony forming units of R. pseudosolanacearum in drain water.
Sensitive, quantitative detection of Besnoitia darlingi and related parasites in intermediate hosts and to assess felids as definitive hosts for known and as-yet undescribed related parasite species
Gereon Schares - 2020
Abstract
Besnoitia darlingi, B. neotomofelis and B. oryctofelisi are closely related coccidian parasites with cats as definitive hosts. While B. darlingi uses opossums as intermediate hosts, B. neotomofelis and B. oryctofelisi have been described in Southern Plains woodrats (Neotoma micropus) from the USA and in domestic rabbits from Argentina, respectively. A comparison of the Internal Transcribed Spacer-1 (ITS-1) region of the ribosomal DNA (rDNA) of these Besnoitia spp. showed only a few differences. The present study aimed at developing a real-time PCR to detect B. darlingi, B. neotomofelis and B. oryctofelisi in tissues of intermediate and in faeces of definitive hosts in order to support studies of these organisms’ epidemiology and pathogenesis. The established PCR was based on primer regions distinct from the ITS-1 sequences of ungulate Besnoitia spp. and made use of a Besnoitia universal probe. To monitor inhibition, a heterologous internal control was established based on the enhanced green fluorescent protein gene. The real-time PCR reacted with B. darlingi, B. neotomofelis and B. oryctofelisi, while the novel PCR did not recognize ungulate Besnoitia spp. (B. besnoiti, B. bennetti, B. tarandi). DNA of Apicomplexa ascribed to other Besnoitia-related genera, including other gut parasites of cats (Cryptosporidium parvum, Giardia duodenalis, Tritrichomonas foetus), was not recognized. The real-time PCR had an analytic sensitivity of less than 1 tachyzoite per reaction. In feline faeces spiked with B. darlingi oocysts, the limit of detection was a DNA amount equivalent to 1 oocyst per PCR reaction. In B. darlingi infected ɣ-interferon knock-out mice, the lung was identified as the predilection organ. In conclusion, this real-time PCR should advance further studies on these parasites and may inspire research on related species, not only in the Americas, but also in other parts of the world.
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