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Extractionless nucleic acid detection: a high capacity solution to COVID-19 testing.

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.

SUBMITTER: Baksh S

PROVIDER: S-EPMC8222080 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Extractionless nucleic acid detection: a high capacity solution to COVID-19 testing.

Baksh Shairaz S Volodko Natalia N Soucie Merle M Geier Sheena Brandon SB Diep Anthony A Rozak Kallie K Chan Tak Yin TY Mustapha Jelili J Lai Raymond R Estey Mathew M Verity Bob B Lee Mao-Cheng MC

Diagnostic microbiology and infectious disease 20210617 2

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, respective ...[more]

PMID: 34274751

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Project description:BackgroundFrom 20 July to 26 August 2021, local outbreaks of COVID-19 occurred in Nanjing City and Yangzhou City (Jiangsu Province, China). We analyzed the characteristics of these outbreaks in an effort to develop specific and effective intervention strategies.MethodsPublicly available data on the characteristics of the COVID-19 outbreaks in Jiangsu Province were collected. Logistic regression was used to assess the association of age and sex with clinical severity. Analysis of onset dates, generation time distributions, and locations were used to estimate the mean transmission distance. A branching process model was used to evaluate different management strategies.ResultsFrom 20 July to 26 August 2021, 820 patients were diagnosed with COVID-19 in Jiangsu Province, with 235 patients (28.7%) from Nanjing, 570 (69.5%) from Yangzhou, and 15 (1.8%) from other cities. Overall, 57.9% of the patients were female, 13.7% were under 20 years-old, and 58.3% had moderate disease status. The mean transmission distance was 4.12 km, and closed-loop management of the area within 2.23 km of cases seemed sufficient to control an outbreak. The model predicted that the cumulative cases in Yangzhou would increase from 311 to 642 if the interval between rounds of nucleic acid amplification testing (NAAT) increased from 1 to 6 days. It also predicted there would be 44.7% more patients if the NAAT started 10 days (rather than 0 days) after diagnosis of the first case. The proportion of cases detected by NAAT would increase from 11.16 to 44.12% when the rounds of NAAT increased from 1 to 7 within 17 days. When the effective vaccine coverage was 50%, the outbreak would be controlled even when using the most relaxed non-pharmaceutical interventions.ConclusionsThe model predicted that a timely closed-loop management of a 2.23 km area around positive COVID-19 cases was sufficient to control the outbreak. Prompt serial NAAT is likely to contain an outbreak quickly, and our model results indicated that three rounds of NAAT sufficiently controlled local transmission. Trial registration We did not involve clinical trial.

Dataset Information

Extractionless nucleic acid detection: a high capacity solution to COVID-19 testing.

Publications

Extractionless nucleic acid detection: a high capacity solution to COVID-19 testing.

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