Effect of virus inactivation on weak positive results of nucleic acid test for 2019 novel coronavirus

Abstract

Objective To investigate the effect of virus inactivation on weak positive result of 2019 Novel Coronavirus (2019-nCoV) nucleic acid test. Method A retrospective study was conducted on the nasopharyngeal swabs of three patients with positive PCR nucleic acid test for Novel Coronavirus at different concentrations in the Second affiliated Hospital of Zhejiang University Medical College from January to February 2020.The virus in nasopharyngeal swab specimens were inactivated by water bath at 56 ℃ for 30 min, dry bath at 56 ℃ for 60 min and dry bath at 60 ℃ for 30 min respectively. After treatment, the sample RNA were extracted and then detected by three new commercial quantitative real-time polymerase chain reaction reagent kits for 2019-nCoV.Cycle threshold(Ct)value was used to evaluate the effect of virus inactivation on nucleic acid detection of 2019-nCoV. Results There was no significant difference between the groups before and after inactivation. Ct values of ORF1ab gene before inactivation were 23.28±0.28, 25.25±0.25, 28.93±0.44, 32.06±0.47, 35.20±0.38, 32.89±0.38, 36.24±0.23, 33.30±0.46, and those after inactivation were, group 1:23.60±0.20, 27.29±0.30, 31.83±0.51, 37.41±0.46, group 2:24.25±0.34, 27.18±0.42, 31.84±0.61, 34.99±1.01, 34.89±0.45,group 3:23.37±0.17, 26.89±0.52, 32.05±0.50.Ct value of N gene before inactivation were24.38±0.09, 26.64±0.11, 30.35±0.12, 33.29±0.33, 36.93±0.11, 34.50±0.12, 35.63±0.12, those after inactivation were, group 1:24.66 ±0.11, 28.52±0.14, 32.71±0.14, 37.00±0.13.group 2:25.41 ±0.10, 28.79±0.15, 33.29±0.28. group 3:23.37±0.11, 28.68±0.11, 33.54±0.13, 37.18±0.23(ORF1ab gene: t =-1.416; N gene: t=-1.379, P> 0.05). There was no significant difference amongthe three inactivation groups, the specific Ct values are shown above(ORF1ab gene: t =-0.460; N gene: t =-0.132, P>0.05). However, the Ct values of the inactivated groups (1,2,3) and the non-inactivated group at different dilution times were different (10 ×:Ct value of ORF1abwas 25.25±0.25 in the non-inactivated group, and 27.29±0.30, 27.18±0.42 and 26.89±0.52 in the inactivated group1,2 and 3.t (ORF1ab) = -7.327, P 0.05) and among the three reagents(reagent 1:7/11, 4/11, 3/11, 3/11, reagent 2:8/11, 4/11, 3/11, 3/11, reagent 3:5/11, 3/11, 3/11, 2/11)(χ2=1.199, P>0.05). Conclusion The virus inactivation can degrade the nucleic acid of the 2019nCoV, resulting in the decrease of the Ct value and the false negative results of the low-concentration specimens. Key words: Coronavirus; Virus inactivation; Nucleic acids; False negative reactions; Real-time polymerase chain reaction