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

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 range 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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study did not receive any funding. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by the Institutional Review Committee (IRC) of KAHS (IRC approval reference no 077/078/03) Jumla, Nepal. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present work are contained in the manuscript.