新型冠状病毒核酸检测过程中灵敏度损失的定量分析  

Quantitative analysis of the loss of sensitivity in the nucleic acid detection of SARS-CoV-2

作　　者：董宏杰 张俊梅[1,2] 王宏伟 王茂凤[1] 张坤迪 张冯瑜 王帅 胡玮[1] 谢时灵[2] 谷立川 DONG Hongjie;ZHANG Junmei;WANG Hongwei;WANG Maofeng;ZHANG Kundi;ZHANG Fengyu;WANG Shuai;HU Wei;XIE Shiling;GU Lichuan(State Key Laboratory of Microbial Technology,Shandong University,Qingdao 266000,Shandong,China;Shandong Shtars Biological Industry Co.,Ltd.,Jinan 250000,Shandong,China)

机构地区：[1]山东大学微生物技术国家重点实验室,山东青岛266000 [2]山东仕达思生物产业有限公司,山东济南250000

出　　处：《山东大学学报（医学版）》2021年第1期1-7,共7页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金(31970043)。

摘　　要：目的定量分析新型冠状病毒核酸检测过程中的灵敏度损失,为改进检测流程、解决假阴性问题提供支持。方法以N基因假病毒为样本,对核酸提取方法、保存液pH、保存液体积、提取核酸所用的样本保存液体积中可能影响诊断灵敏度的各种因素进行系统的定量分析和优化,并对优化的检测方法与目前常规检测法进行比较。结果用柱法提取核酸,使用酸性样本保存液可提高检测灵敏度;随着保存液体积增大,假病毒释放得更充分,当保存液体积达到3 mL时假病毒接近完全洗脱;分别取200μL和3 mL保存液样本提取核酸,检测灵敏度相差10倍以上;全部保存液样本用离心柱法提取核酸且PCR体系中加入12μL模板时,检测灵敏度达70 copies/mL;取200μL样本提取核酸且PCR体系中加入4μL模板时,灵敏度为700 copies/mL。结论将新型冠状病毒标本存放于偏酸性样本保存液中,利用尽量多的样本保存液,用离心柱法提取核酸,并在PCR体系中加入最大量的模板,该操作法可较目前常规检测法将最终检测灵敏度提高10倍以上。Objective To conduct the quantitative analysis of SARS-CoV-2 nucleic acid detection sensitivity loss, and to provide support for improving the detection process and solving the false negative problem. Methods The N gene pseudovirus was taken as the sample, the factors that might affect the diagnostic sensitivity in nucleic acid extraction method, pH of preservation solution, volume of preservation solution used for nucleic acid extraction were analyzed and optimized systematically, and the optimized detection method was compared with the current conventional detection method. Results The detection sensitivity could be improved by extraction of nucleic acid by centrifugal column method and using acid sample preservation solution. With the increase of the volume of the preservation solution, the pseudovirus was released more fully. When the volume of the preservation solution reached 3 mL, the pseudovirus was almost completely eluted. The detection sensitivity exhibited more than ten times the difference when 200 μL and 3 mL preservation solution were used to extract nucleic acid, respectively. When the whole preservation samples was extracted by centrifugal column method, and 12 μL template was added into the PCR system, the detection sensitivity could reach 70 copies/mL;when 200 μL preservation sample was taken to extract the nucleic acid, and 4 μL template was added into the PCR system, the detection sensitivity was 700 copies/mL. Conclusion The novel coronavirus specimens are stored in the slightly acidic sample, and the nucleic acid is extracted by centrifugation column method using as many preservation solution samples as possible, and the largest number of nucleic acid templates are added into the PCR system. The sensitivity of the nucleic acid detection method is 10 times higher than that of the conventional detection method.

关 键 词：新型冠状病毒 核酸检测 逆转录-聚合酶链反应 假阴性 

分 类 号：R574[医药卫生—消化系统]