Assessing the effectiveness of sample and RNA extract pool testing for the detection of SARS-CoV-2 RNA by real time RT-PCR

Introduction: Pooled testing is a cost-effective approach to increasing testing capacity during pandemics. This study analysed the effectiveness of pooling of samples and RNA extracts for the detection of SARS-CoV-2 by real time RT-PCR.
Methods: Twenty SARS-CoV-2 positive samples with Ct value of 25–35 and 60 known negative samples based on initial PCR results were used for this study. The samples were used to prepare 2-, 4- and 8-fold pooled samples prior to extraction. The RNA extracts of a further 10 PCR positive samples were pooled to prepare 2-, 4- and 8-fold RNA extract pools. The Ct values of neat samples and pooled samples were compared using the paired t-test with a 95% confidence interval. The same was done for the neat RNA extracts and the extract pools.
Results: The detection capacity was considerably lower when the pool size was increased from 2- to 8-fold in sample pooling, whereas pools of RNA extracts showed 100% detection from 2- to 8-fold dilution. The increase in Ct value of 2-, 4- and 8-fold sample dilutions were 1.69 ± 0.78, 3.84 ± 1.47 and 8.98 ± 2.25, respectively (P < 0.0001). However, there was a small rise in the Ct value of 2-, 4- and 8-fold extract pools (1.01 ± 0.38, 2.18 ± 0.82 and 3.05 ± 0.77, respectively).
Conclusions: Large scale screening of asymptomatic individuals for SARS-CoV-2 can be maximised with optimal use of resources by 2- or 4-fold pooling of samples or 4- or 8-fold pooling of RNA extracts without significantly compromising the detection capacity.