样本分析前质量控制对新型冠状病毒核酸检测结果的影响

Effect of quality control before sample analysis on results of 2019 novel coronavirus nucleic acid detection

目的评估新型冠状病毒(2019-nCoV)核酸检测阳性样本的运输保存温度以及保存时间对样本核酸结果稳定性的影响。方法对来自20例2019-nCoV核酸检测阳性患者鼻咽拭子的病毒核酸结果循环阈值(CT值)进行分析。将目标样本等量分装20份,在25℃、4℃、-20℃和-80℃温度下储存,并在1、2、3、7、14 d后测定。结果20份样本在不同温度下和保存不同时间后检测结果均为ORF-1ab基因和N基因双靶标阳性;由于RNA的不稳定性,样本在-20℃和-80℃储存的前7 d内和在4℃保存的48 h内相对稳定;样本在-20℃和-80℃储存7 d后、4℃储存48 h后和室温下储存24 h后核酸检测CT值呈上升趋势。在不同温度下,ORF-1ab基因在保存第7天时出现CV值均>5%;N基因在25℃、4℃和-20℃条件下保存至第7天时出现CV>5%;在忽略保存温度的前提下,样本保存时间为7 d时标本ORF-1ab基因和N基因CT值的CV较大,样本保存时间为14 d时20份标本中6份ORF-1ab基因的CV>5%,7份样本N基因CT值的CV>5%。结论样本的储存温度和储存时间是2019-nCoV核酸检测分析中潜在的分析前误差,由于RNA的不稳定性,当分析延迟时,尤其是处于较高的储存温度和经过较长的储存时间后,遇到弱阳性标本可能影响检测结果和临床诊断。样本采集后应在低温条件下保存运输并尽快检测,以减少因储存温度和时间而导致的分析前误差。

Objective To evaluate the effects of transportation storage temperature and storage time on the stability of sample nucleic acid results for 20 samples from patients with positive nucleic acid test for 2019 novel coronavirus(2019-nCoV).Methods The circulating threshold(CT value)of nasopharyngeal swabs from 20 patients who tested positive for 2019-nCoV nucleic acid were analyzed.Twenty aliquots of target samples were stored at 25℃,4℃,-20℃and-80℃,and concentrations were determined after 1,2,3,7 and 14 days.Results The 20 samples were positive for ORF-1ab gene and N gene dual target at different temperature and after different storage time.Due to the instability of RNA,samples were stored at-20℃and-80℃within the first 7 days and at 4℃within 48 hours.After the samples were stored at-20℃and-80℃for 7 days,at 4℃for 48 hours,and at room temperature for 24 hours,the CT value of nucleic acid detection showed an upward trend.At different temperature,the CV value of ORF-1ab gene was more than 5%on the 7th day of storage.The CV value of N gene was more than 5%at 25℃,4℃and-20℃on the 7th day of storage.Under the condition of ignoring the storage temperature,the CT values of ORF-1ab gene and N gene of the sample were higher when the sample was stored for 7 days.When the sample was stored for 14 days,the CV of ORF-1ab gene in 6 of 20 samples was more than 5%,and the CV of the CT value of N gene in 7 of 20 samples was more than 5%.Conclusions The storage temperature and time of the samples are potential pre-analytical errors in the detection and analysis of 2019-nCoV nucleic acid.Due to the instability of RNA,when the analysis is delayed,especially at higher storage temperature and longer storage time,weaker conditions are encountered.Positive patient specimens may affect test results and clinical diagnosis.Samples should be stored cryopreserved for transport and analyzed as soon as possible after collection to minimize pre-analytical errors due to storage temperature and time.