Abstract: Objective To determine the reasonable sample sizes of diluted mixed sample test and n-in-1 test in the detection of SARS-CoV-2, so as to provide a more efficient scheme for nucleic acid screening of large samples. Methods A method was designed to extract nucleic acid from the preservation solution of large volume samples using a syringe purification column. In both diluted mixed sample test and n-in-1 test, this method was used in the extraction of nucleic acid from the total preservation solution and from 200 μL solution. The test results were compared. Results In diluted mixed sample test, for a single positive sample, the Ct values of ORF1ab gene and N gene were 3.583 and 2.904 lower from the total preservation solution than from the 200 μL solution; when one positive sample was mixed with 9 negative samples and 19 negative samples, there was no statistical difference in Ct values between the two sample sizes(P>0.05); when the number of mixed samples was more than 20, the Ct values increased significantly. In the n-in-1 test, the Ct values of nucleic acid extracted from total preservation solution was significantly lower than that from 200 μL solution(the current scheme). Conclusion In mixed sample detection, using a syringe purification column to extract nucleic acid from the total preservation solution can increase the maximum number of sample size from 20 to 50. With this strategy, the Ct values of nucleic acid test can be significantly reduced, so as to improve the detection rate and reduce the number of false-negative cases in n-in-1 test.

Key words: Severe Acute Respiratory Syndrome Coronavirus 2, Diluted mixed sample test, n-in-1 test, Syringe purification column, RT-PCR