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  • Reverse Transcription
    Quantitative RT-qPCR
    Extractionless Nucleic Acid Detection: A High Capacity Solution to COVID-19 Testing
    Shairaz Baksh, Science Direct - 2021
    Abstract
    We describe an extractionless real-time reverse transcriptase-PCR (rRT-PCR) protocol for SARS‐CoV‐2 nucleic acid detection using heat as an accurate cost-effective high-capacity solution to COVID‐19 testing. We present the effect of temperature, transport media, rRT-PCR mastermixes and gene assays on SARS‐CoV‐2 gene amplification and limits of detection. Utilizing our heated methodology, our limits of detection were 12.5 and 1 genome copy/reaction for singleplex E‐ and N1‐gene assays, respectively, and 1 genome copy/reaction by utilizing an E/N1 or Orf1ab/N1 multiplex assay combination. Using this approach, we detected up to 98% of COVID‐19 positive patient samples analyzed in our various cohorts including a significant percentage of weak positives. Importantly, this extractionless approach will allow for > 2‐fold increase in testing capacity with existing instruments, circumvent the additional need for expensive extraction devices, provide the sensitivity needed for COVID‐19 detection and significantly reduce the turn‐around time of reporting COVID‐19 test results.
    Comparative effects of viral-transport- medium heat inactivation upon downstream SARS-CoV- 2 detection in patient samples
    Jamie L. Thompson, Journal of Medical Microbiology - 2021
    Abstract
    Introduction. The COVID-19 pandemic, which began in 2020 is testing economic resilience and surge capacity of healthcare providers worldwide. At the time of writing, positive detection of the SARS-CoV-2 virus remains the only method for diagnosing COVID-19 infection. Rapid upscaling of national SARS-CoV-2 genome testing presented challenges: (1) Unpredictable supply chains of reagents and kits for virus inactivation, RNA extraction and PCR-detection of viral genomes. (2) Rapid time to result of <24 h is required in order to facilitate timely infection control measures.Hypothesis. Extraction-free sample processing would impact commercially available SARS-CoV-2 genome detection methods.Aim. We evaluated whether alternative commercially available kits provided sensitivity and accuracy of SARS-CoV-2 genome detection comparable to those used by regional National Healthcare Services (NHS).Methodology. We tested several detection methods and tested whether detection was altered by heat inactivation, an approach for rapid one-step viral inactivation and RNA extraction without chemicals or kits.Results. Using purified RNA, we found the CerTest VIASURE kit to be comparable to the Altona RealStar system currently in use, and further showed that both diagnostic kits performed similarly in the BioRad CFX96 and Roche LightCycler 480 II machines. Additionally, both kits were comparable to a third alternative using a combination of Quantabio qScript one-step Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) mix and Centre for Disease Control and Prevention (CDC)-accredited N1 and N2 primer/probes when looking specifically at borderline samples. Importantly, when using the kits in an extraction-free protocol, following heat inactivation, we saw differing results, with the combined Quantabio-CDC assay showing superior accuracy and sensitivity. In particular, detection using the CDC N2 probe following the extraction-free protocol was highly correlated to results generated with the same probe following RNA extraction and reported clinically (n=127; R2=0.9259).Conclusion. Our results demonstrate that sample treatment can greatly affect the downstream performance of SARS-CoV-2 diagnostic kits, with varying impact depending on the kit. We also showed that one-step heat-inactivation methods could reduce time from swab receipt to outcome of test result. Combined, these findings present alternatives to the protocols in use and can serve to alleviate any arising supply-chain issues at different points in the workflow, whilst accelerating testing, and reducing cost and environmental impact.
    Development and validation of a real-time RT-PCR test for screening pepper and tomato seed lots for the presence of pospiviroids
    Marleen Botermans, BioRxiv - 2020
    Abstract
    Potato spindle tuber viroid and other pospiviroids can cause serious diseases in potato and tomato crops. Consequently, pospiviroids are regulated in several countries. Since seed transmission is considered as a pathway for the introduction and spread of pospiviroids, some countries demand for the testing of seed lots of solanaceous crops for the presence of pospiviroids. A real-time RT-PCR test, named PospiSense, was developed for testing pepper (Capsicum annuum) and tomato (Solanum lycopersicum) seeds for seven pospiviroid species known to occur naturally in these crops. The test consists of two multiplex reactions running in parallel, PospiSense 1 and PospiSense 2, that target Citrus exocortis viroid (CEVd), Columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), tomato chlorotic dwarf viroid (TCDVd) and tomato planta macho viroid (TPMVd, including the former Mexican papita viroid). Dahlia latent viroid (DLVd) is used as an internal isolation control. Validation of the test showed that for both pepper and tomato seeds the current requirements of a routine screening test are fulfilled, i.e. the ability to detect one infested seed in a sample of c.1000 seeds for each of these seven pospiviroids. Additionally, the Pospisense test performed well in an inter-laboratory comparison, which included two routine seed-testing laboratories, and as such provides a relatively easy alternative to the currently used tests.
    The effect of increased inoculum on oral rotavirus vaccine take amonginfants in Dhaka, Bangladesh: A double-blind, parallel group,randomized, controlled trial
    Benjamin Lee, Vaccine - 2020
    Abstract
    Background:Oral, live-attenuated rotavirus vaccines suffer from impaired immunogenicity and efficacyin low-income countries. Increasing the inoculum of vaccine might improve vaccine response, but thisapproach has been inadequately explored in low-income countries.Methods:We performed a double-blind, parallel group, randomized controlled trial from June 2017through June 2018 in the urban Mirpur slum of Dhaka, Bangladesh to compare vaccine take (primary out-come) among healthy infants randomized to receive either the standard dose or double the standard doseof oral Rotarix (GlaxoSmithKline) vaccine at 6 and 10 weeks of life. Infants with congenital malforma-tions, birth or enrollment weight <2000 gm, known immunocompromising condition, enrollment inanother vaccine trial, or other household member enrolled in the study were excluded. Infants were ran-domized using random permuted blocks. Vaccine take was defined as detection of post-vaccination fecalvaccine shedding by real-time reverse transcription polymerase chain reaction with sequence confirma-tion or plasma rotavirus-specific immunoglobulin A (RV-IgA) seroconversion 4 weeks following the sec-ond dose.Results:220 infants were enrolled and randomized (110 per group). 97 standard-dose and 92 high-doseinfants completed the study per-protocol. For the primary outcome, no significant difference wasobserved between groups: vaccine take occurred in 62 (67%) high-dose infants versus 69 (71%)standard-dose infants (RR 0.92, 95% CI 0.67–1.24). However, in post-hoc analysis, children with con-firmed vaccine replication had significantly increased RV-IgA responses, independent of the intervention.No significant adverse events related to study participation were detected.Conclusions:Administration of double the standard dose of an oral, live-attenuated rotavirus vaccine(Rotarix) did not improve vaccine take among infants in urban Dhaka, Bangladesh. However, improvedimmunogenicity in children with vaccine replication irrespective of initial inoculum provides further evi-dence for the need to promote in-host replication and improved gut health to improve oral vaccineresponse in low-income settings.
    The effect of increased inoculum on oral rotavirus vaccine take among infants in Dhaka, Bangladesh: A double-blind, parallel group, randomized, controlled trial
    Benjamin Lee, Vaccine - 2020
    Abstract
    Background Oral, live-attenuated rotavirus vaccines suffer from impaired immunogenicity and efficacy in low-income countries. Increasing the inoculum of vaccine might improve vaccine response, but this approach has been inadequately explored in low-income countries. Methods We performed a double-blind, parallel group, randomized controlled trial from June 2017 through June 2018 in the urban Mirpur slum of Dhaka, Bangladesh to compare vaccine take (primary outcome) among healthy infants randomized to receive either the standard dose or double the standard dose of oral Rotarix (GlaxoSmithKline) vaccine at 6 and 10 weeks of life. Infants with congenital malformations, birth or enrollment weight <2000 gm, known immunocompromising condition, enrollment in another vaccine trial, or other household member enrolled in the study were excluded. Infants were randomized using random permuted blocks. Vaccine take was defined as detection of post-vaccination fecal vaccine shedding by real-time reverse transcription polymerase chain reaction with sequence confirmation or plasma rotavirus-specific immunoglobulin A (RV-IgA) seroconversion 4 weeks following the second dose. Results 220 infants were enrolled and randomized (110 per group). 97 standard-dose and 92 high-dose infants completed the study per-protocol. For the primary outcome, no significant difference was observed between groups: vaccine take occurred in 62 (67%) high-dose infants versus 69 (71%) standard-dose infants (RR 0.92, 95% CI 0.67–1.24). However, in post-hoc analysis, children with confirmed vaccine replication had significantly increased RV-IgA responses, independent of the intervention. No significant adverse events related to study participation were detected. Conclusions Administration of double the standard dose of an oral, live-attenuated rotavirus vaccine (Rotarix) did not improve vaccine take among infants in urban Dhaka, Bangladesh. However, improved immunogenicity in children with vaccine replication irrespective of initial inoculum provides further evidence for the need to promote in-host replication and improved gut health to improve oral vaccine response in low-income settings. ClinicalTrials.gov: NCT02992197.
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