Evaluation of Rapid Multiplex Reverse Transcription-Quantitative Polymerase Chain Reaction Assays for SARS-CoV-2 Detection in Individual and Pooled Samples

Abstract

Although coronavirus disease 2019 (COVID-19) is no longer a Public Health Emergency of International Concern (PHEIC), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has had a vast impact to date. Hence, continuous management is required, given the uncertainty caused by the potential evolution of SARS-CoV-2. Reverse transcription-quantitative PCR (RT-qPCR) diagnosis has been fundamental in overcoming this issue. In this study, the performances of two rapid RT-qPCR assays (Real-Q Direct SARS-CoV-2 Detection Kit and Allplex™ SARS-CoV-2 fast PCR Assay) with short PCR times were comparatively evaluated using a STANDARD M nCoV Real-Time Detection Kit (STANDARD M, conventional RT-qPCR assay). All kits showed a limit of detection values (10²-10³ copies/reaction). The evaluation showed that the two rapid assay tests had ≥97.89% sensitivity and ≥99.51% specificity (κ = 0.98) for individual samples and ≥97.32% sensitivity and ≥97.67% specificity for pooled samples compared to STANDARD M. These results indicate that the two rapid RT-qPCR kits, which showed significant time reduction in performance, are as effective as a conventional RT-qPCR assay. They are likely to increase not only the number of tests that can be performed but also the efficiency of sustainable management of COVID-19 in the long term.

Keywords: COVID-19; SARS-CoV-2; infection management; rapid RT-qPCR.

Figure 1

Correlation between the Ct values and SARS-CoV-2 B.1.351 RNA at eight concentrations, as analyzed using linear regression based on LoD experiments. Graphs prepared using the results of the STANDARD M kit for (A) E and (B) RdRP; the Real-Q Direct kit for (C) E and (D) RdRP; and the Allplex™ fast kit for (E) E, (F) RdRP, and (G) N. Percentage efficiency was calculated from the slope using the formula E (%) = 100 × (−1 + 10^−1/slope). Circles represent the results of each test, and 24 replicates were used per concentration. The dotted line represents the LoD value. E, envelope gene; RdRP, RNA-dependent RNA polymerase gene; N, nucleocapsid gene; Ct, Cycle threshold; and LoD, limit of detection.

Figure 2

Bland–Altman plot analysis of the differences in Ct values for the same target genes, as tested with the STANDARD M and rapid RT-qPCR kits (Real-Q Direct kit for (A) E and (B) RdRP; Allplex™ fast kit for (C) E and (D) RdRP). The x-axis represents the average Ct value for the STANDARD M and rapid RT-qPCR kits. The y-axis shows the difference in the Ct value between the STANDARD M and two rapid RT-qPCR kits. E, envelope gene; RdRP, RNA-dependent RNA polymerase gene; Ct, cycle threshold; and SD, standard deviation.

Figure 3

Comparison of Ct values between individual and pooled samples, as tested with the (A) STANDARD M, (B) Real-Q Direct, and (C) Allplex™ fast kits. Samples that were negative are marked with gray dots beyond the dotted line. The x-axis represents the average Ct value of individual samples, and the y-axis represents the average Ct value of pooled samples. Ct, cycle threshold.