RT-qPCR half-reaction optimization for the detection of SARS-CoV-2

Abstract

INTRODUCTION: The main laboratory test for the diagnosis of coronavirus disease 2019 (COVID-19) is the reverse transcription real-time polymerase chain reaction (RT-qPCR). However, RT-qPCR is expensive because of the number of tests required. This study aimed to evaluate an alternative to the RT-qPCR approach for the detection of sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is half of the total volume currently recommended by the US Centers for Disease Control and Prevention. METHODS: The analytical limit of detection (LoD) and the reaction efficiency using half volumes of the RT-qPCR assay were evaluated for the N1 and N2 regions using a synthetic control RNA. A panel of 76 SARS-CoV-2-positive and 26 SARS-CoV-2-negative clinical samples was evaluated to establish clinical sensitivity and specificity. RESULTS: The RT-qPCR assay efficiency was 105% for the half and standard reactions considering the N2 target and 84% (standard) and 101% (half) for N1. The RT-qPCR half-reaction LoD for N1 and N2 were 20 and 80 copies/µL, respectively. The clinical sensitivity and specificity were 100%. The half reaction presented a decrease of up to 5.5 cycle thresholds compared with standard RT-qPCR. CONCLUSIONS: The use of the RT-qPCR half-reaction proved feasible and economic for the detection of SARS-CoV-2 RNA.

SARS-CoV-2 Variants of Concern: Presumptive Identification via Sanger Sequencing Analysis of the Receptor Binding Domain (RBD) Region of the S Gene

“…Due to the large size of the fragment 75L/77R, a high viral load is required to provide reliable results from Sanger sequencing. Based on a comparative analysis, the LOD was determined as a minimal viral load of 500,000 copies/uL using an adapted RT-qPCR CDC protocol [ 14 ], corresponding to Ct values of up to 20, the same described in another study [ 22 ]. This viral load is necessary to generate a proper sequencing electropherogram that allows for an accurate analysis with no ambiguous base calls.…”

“…To determine the limit of detection, we evaluated a serial dilution (from 1:1 to 1:10,000) of SARS-CoV-2 positive samples in a RT-qPCR assay and used a standard curve to quantify the viral load, as previously described by Wink et al [ 14 ]. The same dilutions of SARS-CoV-2 positive specimens were submitted for RBD PCR.…”

Optimization of Da An Gene Kit for SARS-CoV-2 Detection in Real-Time RT-PCR

“…The results of the tests are expected to save the use of reagents for the detection of SARS-CoV-2. In the Wink et al, 2021 study it was argued that regardless of the reliable results of SARS-CoV-2 infection detection testing with reagent volume plus optimized samples, this test can save the use of available reagents.…”

Evaluation of filter paper to transport oro/nasopharyngeal samples to detect SARS-CoV-2 by RT-qPCR

“…Primers and probes were purchased from Integrated DNA Technologies (Coralville, IA, USA). The reaction conditions were used as previously described ( Wink et al, 2021 ) and the Superscript III (SSIII) one-step RT-qPCR system (Thermo Fisher Scientific Inc, California, USA) was used for RT-qPCR reactions. The master mix was composed of 5uL of 2X reaction buffer (0.4 mM of each dNTP and 6 mM MgSO 4 ); 0.2 µL of SuperScript™ III RT/Platinum™ Taq Mix; 0.2 µL of ROX (dilution 1:10); 0.75 µL of combined primers/probes mix of nCOV1 (N1 primer) or nCOV2 (N2 primer) or RP (2019-nCoV RUO Kit, IDT, Integrated DNA Technologies Inc, Iowa, USA) and 4 µL of extracted RNA.…”