SARS-CoV-2核酸提取前样本混匀后不同静置时间对假阴性结果影响的初步探讨

Preliminary study on the effect of different standing time on false negative results after mixing samples before extraction of SARS-CoV-2 nucleic acid

目的探讨新型冠状病毒在治疗初期和治疗后期的核酸检测情况,分析假阴性结果出现的实验因素。方法所有样本来源于1例COVID-19确诊病例患者在住院治疗期间,入院后治疗的前6 d设为治疗初期,这期间送检的6个鼻咽拭子编号为1、2、3、4、5、6,设为A组。在出院前一周的治疗设为治疗后期,这期间送检的6个鼻咽拭子编号为7、8、9、10、11、12,设为B组。将A组和B组的12个样本充分混匀后,每个样本以每管200 ul的液量分装到四个不同的1.5 mL EP管中,第一管即刻加样提取,第二管静置5 min加样提取,第三管静置15 min加样提取,第四管静置30 min加样提取,RT-PCR扩增。用Pearson积矩相关分析比较A组的N基因和ORF1ab基因Ct值的相关系数,并用MedCalc软件绘制Bland-Altman曲线进行一致性分析。用Excel 2016比较B组的结果。结果治疗初期A组N基因Ct值0 min与其余3种静置时间两两比较,结果具有较好的一致性;而ORF1ab基因Ct值0 min与30 min一致性较差。治疗后期B组中同一个样本的N基因和ORF1ab基因在加样提取前因静置时间不同而出现不同的结果,甚至出现了假阴性结果。结论样本静置时间对扩增检测结果有影响,治疗后期和疑似患者的样本应充分震荡并静置30 min后再进行提取和扩增实验。

Objective To investigate the nucleic acid detection of novel coronavirus in the initial stage and post-period of COVID-19 treatment and analyze the experimental factors of false negative results.Method All samples were from a patient with a confirmed case of COVID-19 during hospitalization.The first 6 days of treatment after admission were set as the initial stage of treatment.The 6 nasopharyngeal swabs sent during this period were numbered 1,2,3,4,5,6,and were set as group A.The treatment of the week before discharge was set as the later stage of treatment.During this period,6 nasopharyngeal swabs numbered 7,8,9,10,11 and 12 were sent for inspection,which were set as group B.After fully mixing 12 samples of group A and group B,each sample was divided into four different 1.5 ml EP tubes with 200 uL liquid volume per tube.The first tube was immediately added and extracted,the second tube was placed for 5 min and extracted,the third tube was placed for 15 min and extracted,and the fourth tube was placed for 30 min and extracted,RT-PCR amplification.Pearson product moment correlation analysis was used to compare the correlation coefficient between Ct values of N gene and ORF1 ab gene in group A,and Bland-Altman curve was drawn by MedCalc software for consistency analysis.Excel 2016 was used to compare the results of group B.Results The Ct value of N gene with 0 min in group A.Group A with the initial stage of treatment was compared with the other three standing time,the results had good consistency,while the Ct value of ORF1ab gene 0 min and 30 min had poor consistency.The N gene and ORF1ab gene of the same sample in group B in post-period of treatment had different results due to the different standing time before the sample extraction,and even had false negative results.Conclusion The standing time of the sample has an effect on the amplification test results.Especially the samples in the post stage of treatment and suspected cases should be fully shaken and standing for 30 minutes before proceeding with the extraction and amplification experiment.