The Comparison of Three Real-Time PCR Kits for Sars-Cov-2 Diagnosis Reveals Discrepancies on the Identification of Positive Covid-19 Cases and Dispersion on the Values Obtained for the Detection of Sars-Cov-2 Variants

Abstract

The COVID-19 pandemic has generated a huge challenge and threat to public health throughout the world population. Reverse transcription associated with real-time Polymerase Chain Reaction (RT-qPCR) has been the gold-standard molecular tool for diagnosis and detection of the SARS-CoV-2. Currently, it is used as the main strategy for testing, traceability, and control of positive cases For this reason, the on-top high demand for reagents has produced stock-out on several occasions and the only alternative to keep population diagnosis has been the use of different RT-qPCR kits. Therefore, we evaluate the performance of three of the commercial RT-qPCR kits currently in use for SARS-CoV-2 diagnosis in Chile, consisting in: TaqMan 2019-nCoV Assay Kit v1 (Thermo). Real-Time Fluorescent RT-PCR Kit for Detecting SARS-CoV-2 (BGI), and LightCycler Multiplex RNA Virus Master (Roche). Results of quantification cycle (Cq) and relative fluorescence units (RFU) obtained from their RT-qPCR reactions revealed important discrepancies on the total RNA required for the identification of SARS-CoV-2 genes and diagnosis. Marked differences between kits in samples with 30>Cq value< 34 was observed. Samples with positive diagnoses for Covid-19 using the Thermo Fisher kit had different results when the same samples were evaluated with Roche and BGI kits. The displacement on the Cq value for SARS-CoV-2 identification between the three different RT-qPCR kits was also evident when the presence of single nucleotide variants was evaluated in the context of genomic surveillance. Taken together, this study emphasizes the special care adjusting RT-qPCR reaction conditions of the different kits must be taken by all the laboratories before carrying out the detection of SARS-CoV-2 genes from total RNA nasopharyngeal swab (NPS) samples.

First Identification of Reinfection by a Genetically Different Variant of SARS‐CoV‐2 in a Homeless Person from the Metropolitan Area of Santiago, Chile

“…In both samples, the RNase P amplification was quite similar (Cq = 16.82; Cq = 16.75, respectively) ( Figure 1(b) ). The detection limit for ORF1ab, as considered by our laboratory and described previously [ 14 ], confirmed the positive diagnosis for COVID-19 for both samples. The phylogenetic analysis showed that 0606-201 and 0308-063 samples were members of Nextstrain clades 20B and 20A, respectively ( Figure 1(c) ).…”

The Comparative Analysis of Two RT-qPCR Kits for Detecting SARS-CoV-2 Reveals a Higher Risk of False-Negative Diagnosis in Samples with High Quantification Cycles for Viral and Internal Genes

“…In diagnosis, it is a solid basis to validate the proper process of total RNA extraction and subsequent amplification of the SARS-CoV-2 genes of interest used (ORF1ab, N, S, E , and RdRp). Due to the nonamplification of an internal gene, the results may be invalidated [ 19 ].…”