Evaluation of conventional and point-of-care real-time RT-PCR tests for the detection of SARS-CoV-2 through a pooled testing strategy

Abstract

Background: The rapid identification and isolation of individuals infected with SARS-CoV-2 are fundamental countermeasures for the efficient control of the COVID-19 pandemic, which has affected millions of people around the world. Real-time RT-PCR is one of the most commonly applied reference methods for virus detection, and the use of pooled testing has been proposed as an effective way to increase the throughput of routine diagnostic tests. However, the clinical applicability of different types of real-time RT-PCR tests in a given group size remains inconclusive due to inconsistent regional disease prevalence and test demands.

Methods: In this study, the performance of one dual-target conventional and two point-of-care real-time RT-PCR tests in a 5-specimen pooled testing strategy for the detection of SARS-COV-2 was evaluated.

Results: We demonstrated the proof of concept that all of these real-time RT-PCR tests could feasibly detect SARS-CoV-2 from nasopharyngeal and oropharyngeal specimens that contain viral RNA loads in the range of 3.48 × 10⁵ to 3.42 × 10² copies/ml through pooled testing in a group size of 5 with overall positive percent agreement (pooling vs. individual testing) ranging from 100% to 93.75%. Furthermore, the two POC real-time RT-PCR tests exhibited comparable sensitivity to that of the dual-target conventional one when clinical specimens were tested individually.

Conclusion: Our findings support the feasibility of using real-time RT-PCR tests developed as a variety of platforms in routine laboratory detection of suspected COVID-19 cases through a pooled testing strategy that is beneficial to increasing the daily diagnostic capacity.

Keywords: SARS-CoV-2; point-of-care; pooled testing; real-time RT-PCR.

FIGURE 1

Standard curves for dual‐target conventional real‐time RT‐PCR. Serial twofold dilutions of commercially purchased inactivated SARS‐CoV‐2 particles with known RNA loads ranging from 3500 copies/ml to 109.375 copies/ml were used for RNA extraction followed by real‐time RT‐PCR analysis of (A) E and (B) N2 assays. Each dilution of the standard curve was analyzed by 18 replicates, and the respective mean value is illustrated by dotted and hollow circles, respectively, with standard deviation shown as error bars. The viral RNA loads (log₂ copies/ml) and the cycles of threshold (Ct) are indicated on the X‐ and Y‐axes, respectively

FIGURE 2

Comparison of Ct values of SARS‐CoV‐2 detection through individual and pooled testing strategies. Data of (A) dual‐target conventional real‐time RT–PCR, (B) Xpert Xpress, and (C) cobas Liat are presented as a scatter plot with mean values ± SD of the same testing strategy. The asterisks (**) and (***) indicate a significant difference (p < 0.01 and p < 0.0001, respectively) by a one‐tailed unpaired t test. The testing strategy and the resultant Ct values are indicated on the X‐ and Y‐axes, respectively