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. 2021 Jul:293:114149.
doi: 10.1016/j.jviromet.2021.114149. Epub 2021 Apr 8.

Enhanced throughput of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time RT-PCR panel by assay multiplexing and specimen pooling

Xiaoyan Lu  1 , Senthilkumar K Sakthivel  2 , Lijuan Wang  3 , Brian Lynch  2 , Sheila M Dollard  4
Affiliations

Enhanced throughput of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time RT-PCR panel by assay multiplexing and specimen pooling

Xiaoyan Lu et al. J Virol Methods. 2021 Jul.

Abstract

A multiplex real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was developed based on the same primer and probe sequences of an existing U.S. CDC Emergency Use authorized test panel, targeting SARS-CoV-2 N1, N2 and human RNase P genes in singleplex. Both singleplex and multiplex assays demonstrated linear dynamic ranges of 8 orders of magnitude and analytical limits of detection of 5 RNA transcript copies/reaction. Both assays showed 100 % agreement with 364 previously characterized clinical specimens (146 positive and 218 negative) for detection of SARS-CoV-2 RNA. To further increase testing throughput, 40 positive and 20 negative four-specimen pools were tested by the multiplex assay and showed 97.75 % and 100 % congruence with individual specimen tests, respectively. rRT-PCR assay multiplexing and sample pooling, individually or in combination, can substantially increase throughput of SARS-CoV-2 testing.

Keywords: COVID-19; Coronavirus; Multiplex; Pooling; Real-time RT-PCR; SARS-CoV-2.

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Conflict of interest statement

The authors report no declarations of interest.

Figures

Fig. 1
Fig. 1
Standard curves of serial 10-fold dilutions ranging from 5 to 5 × 107 copies/reaction of the nucleocapsid synthetic RNA transcripts tested in five replicates by the multiplex SARS-CoV-2 rRT-PCR. Plot inserts show calculated linear correlation coefficients (R2) and amplification efficiencies (Eff.) for N1 and N2 targets.
Fig. 2
Fig. 2
A) Comparison of the SARS-CoV-2 multiplex Real-Time RT-PCR (rRT-PCR) assay with the singleplex assays in the CDC 2019-nCoV rRT-PCR panel with 146 SARS-CoV-2 positive clinical specimens. Linear regression lines fitted to cycle threshold (Ct) data with regression equations and coefficients of determination (R²) insets. B) Bland-Altman plot analysis: ΔCt (Ct value difference between the SARS-CoV-2 rRT-PCR multiplex assay and the singleplex assays in the 2019-nCoV rRT-PCR panel with 146 positive specimens) vs average Ct values obtained from the SARS-CoV-2 multiplex rRT-PCR and the 2019-nCoV rRT-PCR panel. Solid lines represent the mean ΔCt and dashed lines represent the upper and lower limits of agreement (mean ΔCT ± 1.96 standard deviation of ΔCt).
Fig. 3
Fig. 3
A) Comparison of cycle threshold (Ct) values of 39 positive 4-specimen pools containing one SARS-CoV-2 positive sample with three negative specimens vs individual positive specimens tested with the SARS-CoV-2 multiplex real-time RT-PCR (rRT-PCR) assay. Linear regression lines fitted to Ct values with regression equations and coefficients of determination (R²) insets. B) Bland-Altman plot analysis: ΔCt (Ct value difference between 39 positive 4-specimen pools each containing one SARS-CoV-2 positive sample and 3 negative specimens and individual positive specimens) vs average Ct values of individual and pooled specimens obtained from the SARS-CoV-2 multiplex rRT-PCR assay. Solid lines represent the mean ΔCt and dashed lines represent the upper and lower limits of agreement (mean ΔCT ± 1.96 standard deviation of ΔCt).
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