Lab practices that improve coronavirus disease 2019 detection accuracy using real-time PCR.

ABSTRACT: The number of coronavirus disease 2019 (COVID-19) cases significantly increased with the emergence of multiple variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This has led to an ongoing effort focused on developing the diagnostic detection tests. Among the currently available tests, real-time reverse transcriptase PCR (RT-PCR) has been considered as the 'golden method' for the detection of SARS-COV-2. However, a significant number of inaccurate (false-negative/false-positive) results have been reported in spite of this method's reliability and effectiveness. These unreliable results may arise because of various issues encountered throughout the entire testing process starting with the sampling phase, going through the PCR process, and ending with the result analysis. This article aims to shed light on the errors that occur during the COVID-19 testing process and suggest ways to overcome them effectively. Accurate testing could be optimized by following the correct swabbing technique, using adequate RT-PCR kits and controls, setting clear lab guidelines, and properly interpreting the results.

A rapid and low-cost protocol for the detection of B.1.1.7 lineage of SARS-CoV-2 by using SYBR Green-based RT-qPCR.

BACKGROUND: The new SARS-CoV-2 variant VOC (202012/01), identified recently in the United Kingdom (UK), exhibits a higher transmissibility rate compared to other variants, and a reproductive number 0.4 higher. In the UK, scientists were able to identify the increase of this new variant through the rise of false negative results for the spike (S) target using a three-target RT-PCR assay (TaqPath kit). METHODS: To control and study the current coronavirus pandemic, it is important to develop a rapid and low-cost molecular test to identify the aforementioned variant. In this work, we designed primer sets specific to the VOC (202012/01) to be used by SYBR Green-based RT-PCR. These primers were specifically designed to confirm the deletion mutations Δ69/Δ70 in the spike and the Δ106/Δ107/Δ108 in the NSP6 gene. We studied 20 samples from positive patients, detected by using the Applied Biosystems TaqPath RT-PCR COVID-19 kit (Thermo Fisher Scientific, Waltham, USA) that included the ORF1ab, S, and N gene targets. 16 samples displayed an S-negative profile (negative for S target and positive for N and ORF1ab targets) and four samples with S, N and ORF1ab positive profile. RESULTS: Our results emphasized that all S-negative samples harbored the mutations Δ69/Δ70 and Δ106/Δ107/Δ108. This protocol could be used as a second test to confirm the diagnosis in patients who were already positive to COVID-19 but showed false negative results for S-gene. CONCLUSIONS: This technique may allow to identify patients carrying the VOC (202012/01) or a closely related variant, in case of shortage in sequencing.