Dataset Information

Evaluation of Alternative Transport Media for RT-qPCR-Based SARS-CoV-2 Testing.

ABSTRACT: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), is still rapidly spreading as of March 2022. An accurate and rapid molecular diagnosis is essential to determine the exact number of confirmed cases. Currently, the viral transport medium (VTM) required for testing is in short supply due to a sharp increase in the laboratory tests performed, and alternative VTMs are needed to alleviate the shortage. Guanidine thiocyanate-based media reportedly inactivate SARS-CoV-2 and are compatible with quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays, but the compatibility and the viral detection capacity have not been fully validated. To evaluate the guanidine thiocyanate-based Gene Transport Medium (GeneTM) as an alternative VTM, we prepared 39 SARS-CoV-2-positive and 7 SARS-CoV-2-negative samples in GeneTM, eNAT™, and phosphate-buffered saline (PBS). The cycle threshold (Ct) values of three SARS-CoV-2 targets (the S, RdRP, and N genes) were analyzed using RT-qPCR testing. The comparison of Ct values from the positive samples showed a high correlation (R ²= 0.95-0.96) between GeneTM and eNAT™, indicating a comparable viral detection capacity. The delta Ct values of the SARS-CoV-2 genes in each transport medium were maintained for 14 days at cold (4°C) or room (25°C) temperatures, suggesting viral samples were stably preserved in the transport media for 14 days. Together, GeneTM is a potential alternative VTM with comparable RT-qPCR performance and stability to those of standard media.

SUBMITTER: Baek YH

PROVIDER: S-EPMC9385321 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Evaluation of Alternative Transport Media for RT-qPCR-Based SARS-CoV-2 Testing.

Baek Young Hyun YH Park Min Young MY Lim Ho Jae HJ Jung Hye Soo HS Yang Jae-Hyun JH Sohn Yong-Hak YH Lee Sun-Hwa SH Park Jung Eun JE Yang Yong-Jin YJ

International journal of analytical chemistry 20220810

Severe acute respiratory syndrome coronavirus 2 <b>(</b>SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), is still rapidly spreading as of March 2022. An accurate and rapid molecular diagnosis is essential to determine the exact number of confirmed cases. Currently, the viral transport medium (VTM) required for testing is in short supply due to a sharp increase in the laboratory tests performed, and alternative VTMs are needed to alleviate the shortage. Guanidine thiocyanate-based m ...[more]

PMID: 35992557

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Project description:BackgroundThe extent to which children and adolescents contribute to SARS-CoV-2 transmission remains not fully understood. Novel high-capacity testing methods may provide real-time epidemiological data in educational settings helping to establish a rational approach to prevent and minimize SARS-CoV-2 transmission. We investigated whether pooling of samples for SARS-CoV-2 detection by RT-qPCR is a sensitive and feasible high-capacity diagnostic strategy for surveillance of SARS-CoV-2 infections in schools.MethodsIn this study, students and school staff of 14 educational facilities in Germany were tested sequentially between November 9 and December 23, 2020, two or three times per week for at least three consecutive weeks. Participants were randomized for evaluation of two different age adjusted swab sampling methods (oropharyngeal swabs or buccal swabs compared to saliva swabs using a 'lolli method'). Swabs were collected and pooled for SARS-CoV-2 RT-qPCR. Individuals of positive pooled tests were retested by RT-qPCR the same or the following day. Positive individuals were quarantined while the SARS-CoV-2 negative individuals remained in class with continued pooled RT-qPCR surveillance. The study is registered with the German Clinical Trials register (registration number: DRKS00023911).Findings5,537 individuals were eligible and 3970 participants were enroled and included in the analysis. In students, a total of 21,978 swabs were taken and combined in 2218 pooled RT-qPCR tests. We detected 41 positive pooled tests (1·8%) leading to 36 SARS-CoV-2 cases among students which could be identified by individual re-testing. The cumulative 3-week incidence for primary schools was 564/100,000 (6/1064, additionally 1 infection detected in week 4) and 1249/100,000 (29/2322) for secondary schools. In secondary schools, there was no difference in the number of SARS-CoV-2 positive students identified from pooled oropharyngeal swabs compared to those identified from pooled saliva samples (lolli method) (14 vs. 15 cases; 1·3% vs. 1·3%; OR 1.1; 95%-CI 0·5-2·5). A single secondary school accounted for 17 of 36 cases (47%) indicating a high burden of asymptomatic prevalent SARS-CoV-2 cases in the respective school and community.InterpretationIn educational settings, SARS-CoV-2 screening by RT-qPCR-based pooled testing with easily obtainable saliva samples is a feasible method to detect incident cases and observe transmission dynamics.FundingFederal Ministry of education and research (BMBF; Project B-FAST in "NaFoUniMedCovid19"; registration number: 01KX2021).

Project description:Given the challenges that SARS-CoV-2 variants have caused in terms of rapid spread and reduced vaccine efficacy, a rapid and cost-effective assay that can detect new and emerging variants is greatly needed worldwide. We have successfully applied the xenonucleic acid-based molecular-clamping technology to develop a multiplex reverse-transcription quantitative real-time PCR assay for SARS-CoV-2 multivariant detection. The assay was used to test 649 nasopharyngeal swab samples that were collected for clinical diagnosis or surveillance. The assay was able to correctly identify all 36 Delta variant samples as it accurately detected the D614G, T478K, and L452R mutations. In addition, the assay was able to correctly identify all 34 Omicron samples by detecting the K417N, T478K, N501Y, and D614G mutations. This technique reliably detects a variety of variants and has an analytical sensitivity of 100 copies/mL. In conclusion, this novel assay can serve as a rapid and cost-effective tool to facilitate large-scale detection of SARS-CoV-2 variants.ImportanceWe have developed a multiplex reverse-transcription quantitative real-time PCR (RT-qPCR) testing platform for the rapid detection of SARS-CoV-2 variants using the xenonucleic acid (XNA)-based molecular-clamping technology. The XNA-based RT-qPCR assay can achieve high sensitivity with a limit of detection of about 100 copies/mL for variant detection which is much better than the next-generation sequencing (NGS) assay. Its turnaround time is about 4 hours with lower cost and a lot of Clinical Laboratory Improvement Amendments (CLIA) labs own the instrument and meet skillset requirements. This assay provides a rapid, reliable, and cost-effective testing platform for rapid detection and monitoring of known and emerging SARS-CoV-2 variants. This testing platform can be adopted by laboratories that perform routine SARS-CoV-2 PCR testing, providing a rapid and cost-effective method in lieu of NGS-based assays, for detecting, differentiating, and monitoring SARS-CoV-2 variants. This assay is easily scalable to any new variant(s) should it emerge.

Dataset Information

Evaluation of Alternative Transport Media for RT-qPCR-Based SARS-CoV-2 Testing.

Publications

Evaluation of Alternative Transport Media for RT-qPCR-Based SARS-CoV-2 Testing.

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