Analytical Validation of a COVID-19 qRT-PCR Detection Assay Using a 384-well Format and Three Extraction Methods
diagnostics molecular biology
Authors: Andrew C. Nelson et al.
Link to paper: https://doi.org/10.1101/2020.04.02.022186
Journal/ Pre-Print: bioRxiv
Key Words: qRT-PCR, SARS-CoV-2 detection,
1. Validation of a 384-well format qRT-PCR employing a RUO Promega one-step mastermix for the detection of SARS-CoV-2.
2. Validation of the Macherey-Nagel Nucelospin Virus extraction kit as suitable alterative to the QIAmp Viral RNA kit (Qiagen) and EasyMag NucliSENS kit (biomérieux) for effective viral RNA extraction.
3. Reduction of qRT-PCR reaction volume from 20 to 10 μL still provides detection sensitivity down to 5 viral genome copies/ μL.
One of the preferred methods for COVID-19 diagnosis is by qRT-PCR, that requires use of RNA extraction kits with external lysis buffers capable of deactivating SARS-CoV-2. However, demand for approved RNA extraction kits and associated reagents are in short supply. Nelson et al. developed a qRT-PCR SARS-CoV-2 detection method employing a 384-well format validating its analytic accuracy, sensitivity and specificity. Additionally, they demonstrated that Macherey-Nagel Nucelospin Virus extraction kit has a suitable performance in comparison to the two CDC-approved kits, which are able to detect down to 5 viral genome copies per microliter (cp/μL).
IMPACT FOR SARS-COV2/COVID19 RESEARCH EFFORTS
· Develop additional diagnostic tools for SARS-CoV2/COVID19. (This alternative 384-well format qRT-PCR diagnosis assay reduces consumption of limited reagents).
· In vitro study
STRENGTHS AND LIMITATIONS OF THE PAPER
Novelty: Validation of a qRT-PCR method carried out with a 384-well format and a reaction volume of 10 μL. Viral RNA extraction can be performed by Macherey-Nagel Nucelospin Virus extraction kit.
Standing in the field: For SARS-CoV-2 this appears to be the first of its kind. However, another proposed alternative strategy is group testing. Group testing has long been established as a way of detecting syphilis in amongst US American troops. Group testing has also been demonstrated recently to be an efficient way of reducing reagent usage as it allows for the pooling of samples. Sinnott-Armstrong et al 2020 establish this strategy for the SARS-CoV-2 virus. This could be used to test individuals on mass in asymptomatic, mild disease and low risk groups, prior to follow up tests (Sinnott-Armstrong et al 2020, medRxiv preprint).
Appropriate statistics: Yes- authors calculate the confidence interval for the analytical accuracy of the assay counting every unique extraction of a clinical sample or of a synthetic positive extraction control once. They also only count each biologically unique nucleic acid source once. This enables the authors to have analytical sensitivity (95% CI: 0.9244-1) and 100% analytical specificity (95% CI: 0.8957-1).
Viral model used: SARS-CoV-2 (residual clinical biospecimens and synthetic standard controls)
Translatability: The purposed method highlights new assay components and approaches for COVID-19 diagnosis possibly helping clinical needs avoiding supply chain shortages
Main limitations: Some CT values are very high down (>36) when only 5 cp/ μL were used per sample. However, the authors establish a 97% accuracy at 1X to 2X of the limit of detection (5-10 cp/μL)