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.

the reporting criteria, such as outpatients in the community and hospitalized patients who were suspected to have SARS-CoV-2 infection, as well as people who came to Taiwan from abroad. Clinical specimens were then transported to the laboratories of the SARS-CoV-2 laboratory network for virus detection by real-time RT-PCR. 12 Original specimens that tested positive in each local laboratory were sent back to the Taiwan CDC for further viral genetic characterization and virus isolation.

| Dual-target conventional and POC real-time RT-PCR tests for SARS-CoV-2 detection
Conventional real-time RT-PCR for SARS-CoV-2 detection was defined as the workflow in which viral nucleic acid extraction and detection are performed separately. The dual-target method that was evaluated in this study was performed as previously reported [16][17][18] and served as the reference method. Two of the SARS-CoV-2 genomic segments, including the envelope (E) and nu- The reaction mixtures were incubated at 50°C for 10 min, followed by 95°C for 30 s. The reaction mixtures were then subjected to 45 cycles of 95°C for 5 s, 53°C for 15 s, and 60°C for 15 s. Specimens that tested positive for at least one of the two assays were interpreted as positive.
The POC real-time RT-PCR tests evaluated in this study were defined as those that combined viral nucleic acid extraction and detection in a closed system, and the Xpert Xpress SARS-CoV-2 (Cepheid) and cobas Liat SARS-CoV-2 & Influenza A/B (Roche) tests were selected for evaluation. Each POC test was performed according to the manufacturer's instructions. Xpert Xpress detects the gene sequences of the viral E and N proteins of SARS-CoV-2. Cobas Liat detects the viral ORF1a/b and N genes. Interpretation of the test results of each POC real-time RT-PCR also followed the manufacturer's instructions.

| Generation of the semiquantitative standard curve of the conventional N2 assay for estimation of viral RNA loads in clinical specimens
To determine the viral RNA loads in each SARS-CoV-2-positive clinical specimen taken for evaluation, their cycles of threshold (Ct values) determined by the conventional N2 assay were used for proper estimation. For this purpose, the Amplirun Total SARS-CoV-2 Control (Swab) (Vircell) was purchased and served as a reference material to construct the semiquantitative standard curve. Vials of lyophilized inactivated SARS-CoV-2 particles were reconstituted in molecular biology grade water, resulting in a final concentration of 35,000 viral RNA copies/ml according to the manufacturer's instructions.
Afterward, 2-fold serially diluted standards with known amounts of viral RNA ranging from approximately 3500-109.375 copies/ml were prepared and subjected to nucleic acid extraction followed by the N2 assay to determine the respective Ct values. A standard curve of the N2 assay was constructed by incorporating the Ct values of each diluted standard tested in 18 replicates.

| Preparation of artificial clinical specimen pools
In this study, a 5-specimen pooled testing strategy for SARS-CoV-2 nucleic acid detection by using conventional or POC real-time RT-

| Statistical analysis
Statistical analyses were performed using a one-tailed t test. Data were considered to be statistically significant at a p < 0.05.

| Performance of dual-target conventional realtime RT-PCR for the detection of SARS-CoV-2 in the 5-specimen pooled sample
To evaluate the performance of the dual-target conventional realtime RT-PCR for the detection of SARS-CoV-2 through the pooling strategy, we first determined the semiquantitative standard curve of the E and N2 assays (Figure 1), and the latter was used to estimate the viral RNA loads in a given specimen. A total of the 50 artificially spiked 5-specimen pooled samples, each of which con-  Table 1. It showed that dual-target conventional real-time RT-PCR can correctly detect SARS-CoV-2 viral RNA through the 5-specimen pooled testing strategy among all the enrolled positive specimens with viral RNA load levels ranging from 3.48 × 10 5 to 3.44 × 10 3 copies/ml before pooling, achieving a positive percent agreement (PPA) of 100% compared to individual testing. For the 5-specimen pooled samples with low viral loads (1.59 × 10 3 -3.42 × 10 2 copies/ml) before pooling, 1 out of the 16 had a negative result, and the PPA was 93.75%.
The specimen that was missed had Ct values of 34.92 and 37.97 in the E and N2 assays, respectively, when tested individually.
Comparing the Ct values of the 5-specimen pooled samples to those of the respective positive specimens that were tested individually showed that the average Ct values obtained through the pooling strategy significantly increased by 1.7 (p < 0.0001) and 2.0 (p < 0.0001) in the E and N2 assays, respectively (Figure 2A), mirroring the dilution effect caused by the pooled testing.

| Performance of POC real-time RT-PCR tests for SARS-CoV-2 detection from the individual and 5-specimen pooled samples
To understand the clinical applicability of POC real-time RT-PCR tests to detect SARS-CoV-2, another two panels of 50 F I G U R E 1 Standard curves for dual-target conventional realtime 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 (log 2 copies/ml) and the cycles of threshold (Ct) are indicated on the X-and Y-axes, respectively nasopharyngeal/oropharyngeal specimens and the respective RT-PCR tests evaluated in this study could both be finished within 60 min, we proposed that they are beneficial to overcome these data reporting issues. Another recommendation could be to implement pooled testing in low virus prevalence scenarios to minimize the requirement for a second round of individual testing. In summary, we demonstrate that the use of a rapid and sensitive real-time RT-PCR platform will maximize testing capacity and resource conservation through a pooled testing strategy with a group size of 5.
Limitations of this study include the small numbers of specimens and diagnostic platforms that were enrolled and tested which may bias the interpretation of the evaluation data. Future works by incorporating more representative clinical specimens are suggested to obtain a better scenario for application of pooled testing strategy in SARS-CoV-2 nucleic acid detection.

| CON CLUS ION
We demonstrated that the pooling strategy is practical for routine SARS-CoV-2 diagnosis by using such real-time RT-PCR tests without significant loss of detection sensitivity, even for specimens with low RNA loads. Furthermore, the applicability of POC real-time RT-PCR platforms in either individual or pooled testing strategies was particularly highlighted since they can produce comparable data to that of the conventional test in a shortened time period. Our data serve as proof of concept supporting the use of various real-time RT-PCR platforms in the routine detection of SARS-CoV-2 from pooled specimens and the feasibility of enforcing POC real-time RT-PCR tests in local laboratories for efficient and rapid COVID-19 diagnosis.

ACK N OWLED G M ENTS
This study was supported by grants from the Centers for Disease Control and the Ministry of Health and Welfare, Taiwan (MOHW110-CDC-C-315-124302).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analyzed during this study are included in this published article.