Evaluation of RT-PCR pooling test for the detection of SARS-CoV-2 in low-resource setting

Abstract

Background: Specimen pooling is an efficient method when there is limited accessibility or scarcity of test kits and reagents for nucleic acid extraction and molecular detection. We evaluated the ability of the standard real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test for detecting a single positive sample of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within a pool of negative samples and to find out the maximum pool dilution limit up to which a single positive sample can be detected. Methods: RNA extracts from nasopharyngeal and oropharyngeal samples were pooled for the detection of the SARS-CoV-2 virus by RT-PCR. Positive samples were serially diluted in negative samples pools with dilutions ranging from 1/2 to 1/64 to estimate the optimal pool size. The viral transport medium (VTM) of three positive samples was also evaluated for optimal pool size determination. Results: A single positive sample with a Cycle threshold (Ct) value ranging from 16-23 (high viral load) can be detected in dilution pools upto1/64 for both genes. In pooling before RNA extraction, a positive sample with a low Ct value (13) and intermediate Ct value (25) was detected till 1/32 dilution pool but a positive sample with a high Ct value (32) was not detected further 1/4 dilution. Besides, two positive VTM samples were detected in pools of sizes 5, 8, and 10. Conclusions: This study concluded that sample testing by pooling is reliable if done properly and can help increase testing capacity in a low-resource setting.