SalivaSTAT: Direct-PCR and Pooling of Saliva Samples Collected in Healthcare and Community Setting for SARS-CoV-2 Mass Surveillance
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Abstract
Objectives: Limitations of widespread current COVID-19 diagnostic testing exist in both the pre-analytical and analytical stages. To alleviate these limitations, we developed a universal saliva processing protocol (SalivaSTAT) that would enable an extraction-free RT-PCR test using commercially available RT-PCR kits. Methods: We optimized saliva collection devices, heat-shock treatment, and homogenization. Saliva samples (879) previously tested using the FDA-EUA method were reevaluated with the optimized SalivaSTAT protocol using two widely available commercial RT-PCR kits. A five-sample pooling strategy was evaluated as per FDA guidelines. Results: Saliva collection (done without any media) showed performance comparable to that of the FDA-EUA method. The SalivaSTAT protocol was optimized by incubating saliva samples at 95 °C for 30-min and homogenization, followed by RT-PCR assay. The clinical sample evaluation of 630 saliva samples using the SalivaSTAT protocol with PerkinElmer (600-samples) and CDC (30-samples) RT-PCR assay achieved positive (PPA) and negative percent agreements (NPAs) of 95.0% and 100%, respectively. The LoD was established as ~60–180 copies/mL by absolute quantification. Furthermore, a five-sample-pooling evaluation using 250 saliva samples achieved a PPA and NPA of 92% and 100%, respectively. Conclusion: We have optimized an extraction-free RT-PCR assay for saliva samples that demonstrates comparable performance to FDA-EUA assay (Extraction and RT-PCR).
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SciScore for 10.1101/2020.11.23.20236901: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
No key resources detected.
Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:However, several limitations exist in the current methodology at both pre-analytical and analytical stages. In th pre-analytical stage, NPS are associated with exposure risk to healthcare workers, high cost, invasive …
SciScore for 10.1101/2020.11.23.20236901: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
No key resources detected.
Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:However, several limitations exist in the current methodology at both pre-analytical and analytical stages. In th pre-analytical stage, NPS are associated with exposure risk to healthcare workers, high cost, invasive collection, and supply-chain constraints [6-8]. Further, the RNA extraction step in the analytical stage is the most significant rate-limiting step in this protocol because of wide range of reasons that include, the requirement for competent testing personnel, cost of reagents/ kits, equipment, and turnaround time. To overcome the pre-analytical and analytical limitations of current COVID-19 testing methods, saliva samples and extraction-free RT-PCR assays have recently been explored. Significant efforts have been made to develop an extraction-free RT-PCR assay using NPS swabs, and several groups have optimized dry swabs, transport media, heat inactivation, different RT-PCR reaction chemistries, and RT-PCR methods [19-22], but minimal information has emerged on extraction-free RT-PCR assay using saliva samples. To our knowledge, only one report ha evaluated the performance of extraction-free RT-PCR assay using saliva samples, and this assay was limited to an asymptomatic population, used early morning saliva, and yielded low sensitivity [23]. Hence, the goal of this study was to optimize both pre-analytical and analytical variables by developing a universal saliva processing protocol that would enable an extraction free RT-PCR test using any of the commercially a...
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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