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Background: The coronavirus disease (COVID-19) pandemic has revealed the global public health importance of robust diagnostic testing. To overcome the challenge of nucleic acid (NA) extraction and testing kit availability efficient method is urgently needed. Objectives: To establish an efficient, time and resource-saving and cost-effective methods, and to propose an ad hoc pooling approach for mass screening of SARS-CoV-2 Methods: Direct clinical sample and NA pooling approach was used for the standard reverse transcriptase polymerase chain reaction (RT-PCR) test of the SARS CoV-2 targeting the envelop (E) and open reading frame (ORF1ab) genomic region of the virus. In this approach, experimental pools were created using SARS CoV-2 positive clinical samples spiked with up to 9 negative samples prior to NA extraction step to have a final extraction volume of 200L (maximum dilution factor of 10). Viral NA was also subsequently extracted from each pool and tested using the SARS CoV-2 RT-PCR assay. Results: We found that a single positive sample can be amplified and detected in pools of up to 7 samples depending on the ct value of the original sample, corresponding to high, medium, and low SARS CoV-2 viral copies/reaction. However, to minimize false negativity of the assay with pooling strategies and with unknown false negativity rate of the assay under validation, we recommend poling of 4 in 1 using the standard protocols of the assay, reagents and equipment. The predictive algorithm indicated a pooling ratio of 4 in 1 was expected to retain accuracy of the test irrespective of the ct value (relative RNA copy number) of the sample spiked and result in a 237% increase in testing efficiency. Conclusions: The approaches showed its concept in easily customized and resource-saving manner and would allow expanding of current screening capacities and enable the expansion of detection in the community.
Collection : COVID-19 SARS-CoV-2 preprints from medRxiv and bioRxiv
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