Dataset Information

Development and implementation of a simple and rapid extraction-free saliva SARS-CoV-2 RT-LAMP workflow for workplace surveillance.

ABSTRACT: Effective management of the COVID-19 pandemic requires widespread and frequent testing of the population for SARS-CoV-2 infection. Saliva has emerged as an attractive alternative to nasopharyngeal samples for surveillance testing as it does not require specialized personnel or materials for its collection and can be easily provided by the patient. We have developed a simple, fast, and sensitive saliva-based testing workflow that requires minimal sample treatment and equipment. After sample inactivation, RNA is quickly released and stabilized in an optimized buffer, followed by reverse transcription loop-mediated isothermal amplification (RT-LAMP) and detection of positive samples using a colorimetric and/or fluorescent readout. The workflow was optimized using 1,670 negative samples collected from 172 different individuals over the course of 6 months. Each sample was spiked with 50 copies/μL of inactivated SARS-CoV-2 virus to monitor the efficiency of viral detection. Using pre-defined clinical samples, the test was determined to be 100% specific and 97% sensitive, with a limit of detection of 39 copies/mL. The method was successfully implemented in a CLIA laboratory setting for workplace surveillance and reporting. From April 2021-February 2022, more than 30,000 self-collected samples from 755 individuals were tested and 85 employees tested positive mainly during December and January, consistent with high infection rates in Massachusetts and nationwide.

SUBMITTER: Li Z

PROVIDER: S-EPMC9135294 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Development and implementation of a simple and rapid extraction-free saliva SARS-CoV-2 RT-LAMP workflow for workplace surveillance.

Li Zhiru Z Bruce Jacqueline L JL Cohen Barry B Cunningham Caileigh V CV Jack William E WE Kunin Katell K Langhorst Bradley W BW Miller Jacob J Moncion Reynes A RA Poole Catherine B CB Premsrirut Prem K PK Ren Guoping G Roberts Richard J RJ Tanner Nathan A NA Zhang Yinhua Y Carlow Clotilde K S CKS

PloS one 20220526 5

Effective management of the COVID-19 pandemic requires widespread and frequent testing of the population for SARS-CoV-2 infection. Saliva has emerged as an attractive alternative to nasopharyngeal samples for surveillance testing as it does not require specialized personnel or materials for its collection and can be easily provided by the patient. We have developed a simple, fast, and sensitive saliva-based testing workflow that requires minimal sample treatment and equipment. After sample inact ...[more]

PMID: 35617204

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Project description:The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic demanded rapid diagnosis to isolate new COVID-19 cases and prevent disease transmission. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) rapidly became the gold standard for diagnosis. However, due to the high cost and delay of the results, other types of diagnosis were implemented, such as COVID-19 Ag Rapid Tests and Reverse Transcription Technique followed by Loop-Mediated isothermal Amplification (RT-LAMP). In this work, we validated the use of RT-LAMP in saliva samples rather than nasopharyngeal swabs, as the collection is more comfortable. First, we selected 5 primer sets based on the limit of detection for SARS-CoV-2 RNA, then validated their sensitivity and specificity in patient samples. A total of 117 samples were analyzed by fluorometric RT-LAMP and compared with qRT-PCR results. Our results show that the use of a high-sensitive primer ORF1-a, together with a low-sensitive primer set Gene E (time to threshold of 22.9 and 36.4 minutes, respectively, using 200 copies of viral RNA), achieved sensitivity in purified RNA from saliva samples of 95.2% (95% CI 76.1‒99.8) with 90.5% specificity (95% CI 69.6‒98.8) (n = 42).As RNA purification increases the turnaround time, we tested the outcome of RT-LAMP utilizing raw saliva samples without purification. The test achieved a sensitivity of 81.8% (95% CI 59.7‒94.8) and a specificity of 90.9% (95% CI 70.8‒98.8). As a result, the accuracy of 92.9% (95% CI 80.5‒98.5) in purified RNA-saliva samples was lowered to an acceptable level of 86.4% (95% CI 72.6‒94.8) in raw saliva. Although mass vaccination has been implemented, new strains and low vaccination progress helped to spread COVID-19. This study shows that it is feasible to track new COVID-19 cases in a large population with the use of raw saliva as sample in RT-LAMP assay which yields accurate results and offers a less invasive test.

Dataset Information

Development and implementation of a simple and rapid extraction-free saliva SARS-CoV-2 RT-LAMP workflow for workplace surveillance.

Publications

Development and implementation of a simple and rapid extraction-free saliva SARS-CoV-2 RT-LAMP workflow for workplace surveillance.

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