Comparative Analysis of Pooling Methods for SARS-CoV-2 Real-time Polymerase Chain Reaction Testing

Abstract

Abstract Background: Wide spread coronavirus 2019 (COVID-19) infections in 2020 and 2021 and fewer real-time polymerase chain reaction (PCR) laboratories placed extraordinary demand on molecular diagnostics. To accommodate demand for increased testing volumes, the Indian Council of Medical Research had initially allowed pooling of five samples. This study aimed to conduct a comparative analysis of three sample pooling methods for SARS-CoV-2 testing to assess their effectiveness, sensitivity, and specificity. Methods: In this cross-sectional study, samples intended for SARS-CoV-2 testing were initially tested individually and then combined in a 1:4 positive-to-negative ratio. A total of 90 pools, comprising 450 samples, were analyzed using three distinct pooling methods: swab pooling, viral transport medium (VTM) pooling, and RNA pooling, all targeting the detection of the RdRp gene. PCR testing was conducted for all 90 pools comprising 450 samples in a uniform manner. Results: Swab pooling and RNA pooling methods correctly identified RdRp gene in all samples. VTM pooling method failed to identify RdRp gene in 4 samples. Analysis of cycle threshold (CT) value showed, loss of mean CT value of 2.80 ± 0.86 in swab pooling method with Z score of 6.03, VTM pooling method showed loss of mean CT value of 1.96 ± 0.76 with Z score of 3.22 and RNA pooling method showed loss of mean CT value of 1.73 ± 0.58 with Z score of 4.29 in. P value was statistically significant (<0.001) in all three methods. Conclusion: This study concludes that the RNA pooling method is superior, with minimal sensitivity loss in CT values. Swab pooling is cost-effective for negative pools but impractical for positive ones, while the VTM pooling method offers greater cost-effectiveness compared to RNA pooling.