2019新型冠状病毒核酸检测试剂优化、验证及分析

Optimization, validation and analysis of a 2019-nCoV nucleic acid detection kit

目的优化敏感性及特异性更高的新型冠状病毒(2019-nCoV)核酸检测试剂盒,提高阳性检出率。并与市售试剂盒进行对比,对临床使用提出指导。方法收集2020年1月至3月在山东大学附属济南市中心医院等6家2019-nCoV核酸检测单位的确诊患者88例及阴性患者572例,共计660例。通过对2019-nCoV的全基因组进行序列分析,设计更加灵敏的引物、探针,优化buffer配比及扩增过程。采用交叉实验及内源性物质干扰实验评价其特异性。另外选择3家市售核酸检测试剂盒,对梯度稀释的阳性核酸样本进行对比验证实验。结果优化后的开放读码框1a/b(ORF1ab)、核衣壳蛋白(N)对于2019-nCoV的RNA靶点,比市售试剂盒具有更高的灵敏度。当核酸样本中1个病毒浓度为1个拷贝时即可检出,同时扩增曲线更加优越。相比较与世界卫生组织(WHO)或自行设计引物的试剂盒,ORF1ab的灵敏度提高了2倍(1∶10 vs 1∶5);而N基因的灵敏度比中国疾病预防控制中心(CDC)和自行设计引物的试剂盒提高了8倍(1∶80 vs 1∶10),比WHO试剂盒提高了2倍(1∶80 vs 1∶40)。临床验证实验显示,鼻咽拭子检测准确度均为100%,与其他感染部位相同或感染症状相似的其他病原体无交叉反应,试剂盒的分析特异性为100%,阳性符合率和阴性符合率均为100%。结论优化后的2019-nCoV核酸检测试剂盒具有更高的检测灵敏度和特异性,有助于解决核酸检测假阴性,提高阳性率,将对低病毒载量患者及出院患者的判定具有重要的意义。

Objective To optimize the sensitivity and specificity of a 2019-nCoV nucleic acid detection kit,so as to improve the positive detection rate and provide guidance for clinical use by comparison with different kits.Methods From January to March 2020,88 confirmed and 572 negative specimens of 2019-nCoV were recruited from 62019-nCOV nucleic acid testing centers including Jinan Central Hospital Affiliated to Shandong University.By sequence analysis of the whole genome of 2019-nCoV,a more sensitive primer and probe were designed to optimize the buffer ratio and amplification process.The sensitivity was evaluated with cross-reactive experiment and endogenous substances interference test.The optimized kit and 3 other commercial kits were compared with gradient dilution tests.Results The optimized ORF1 ab and N gene had higher sensitivity to the RNA targets of 2019-nCoV.When the concentration of virus was 1,the virus could be detected,and the amplification curve was better than the commercial kits.The sensitivity of ORF1 ab was increased by 2 times(1∶10 vs 1∶5)compared with WHO or other self-designed kits,and the sensitivity of N gene was increased by 8 times(1∶80 vs 1∶10)and 2 times(1∶80 vs 1∶40)compared with CDC and WHO kits respectively.The results of cross-reactive test and endogenous substances interference test showed that the accuracy of nasopharyngeal swabs was 100%,there was no cross-reaction with other pathogens with similar infection symptoms,and the accuracy and specificity of the kit were 100%.Conclusion The optimized 2019-nCoV nucleic acid detection kit has higher sensitivity and specificity,which is helpful to solve the false negative nucleic acid detection and increase the positive rate,and is significant to formulate discharge criteria of low viral load patients.