Impact of sample pooling strategy on 2019-nCoV RNA detection results

ABSTRACT

Objective: To evaluate the impact of sample pooling strategy on 2019-nCoV RNA detection results. Methods: Ten negative swabs were stored in 6 ml virus transport medium, mixed thoroughly and diluted 12 and 110. Inactivated 2019-nCoV culture medium was added to simulate pooling samples 10 pooling samples, 5 pooling samples and 1 swab sample. Extraction and amplification were made using three nucleic acid extraction reagents a, b, and c with different extraction methods and systems, as well as five 2019-nCoV detection reagents A-E with various template loading volumes and sensitivities respectively. Results: For the same sample, the Ct values of extracted templates a were 2.10±0.47 and 3.46±0.62 earlier than extracted templates b and c. For samples with identical amplifying, the Ct valves of N and ORF1ab gene of A reagent were 1.16±0.48 and 2.36±0.54 earlier than that of reagent B. Adding nucleic acid of 10 negative swabs to the amplification system lagged the Ct values of reagent A by about 1.36±0.32 Ct, while Ct values of reagent B were not affected. Extracted by regent a, a lag of 1.66±0.39 Ct on average was observed in C, D, and E reagents in detecting pooling samples of ten swabs as compared with one swab sample. When extracting 400 copies/ml pooling samples of ten swabs by reagent a, N gene could be detected by reagents C and E, but not by reagent D. Conclusion: Large amount of extraneous DNA is introduced by sample pooling, which could interfere the effiency of extraction and amplification. Strategies of using extraction reagents with large loading volume and high effiency, together with amplification reagents with large template volume and low limit of detection are helpful for ensuring detection sensitivity of pooling samples, and greatly reducing the risk of false negative results.