新型冠状病毒荧光定量PCR的Ct值与病毒分离的结果分析  

Analysis of SARS-CoV-2 Fluorescence Quantitative PCR Detection and Virus Isolation

作　　者：郑焕英[1] 张官婷 柯碧霞[1] 梁丹 李安安 洪文珊[3] 梁玉凤 王涛 黎薇[1] 彭晓放 柯昌文[1] 邓小玲[1] ZHENG Huanying;ZHANG Guanting;KE Bixia;LIANG Dan;LI Anan;HONG Wenshan;LIANG Yufeng;WANG Tao;LI Wei;PENG Xiaofang;KE Changwen;DENG Xiaoling(Guangdong Province Center for Disease Control and Prevention,Guangdong Workstation for Emerging Infectious Disease Control and Prevention,Guangzhou 511430,China;School of Public Health,Southern Medical University,Guangzhou 510515,China;Shantou University Medical College,Shantou 515041,China)

机构地区：[1]广东省疾病预防控制中心、广东省新发突发传染病防治工作站,广州511430 [2]南方医科大学、公共卫生学院,广州510515 [3]汕头大学医学院,汕头515041

出　　处：《病毒学报》2023年第1期199-205,共7页Chinese Journal of Virology

基　　金：国家重点研发计划(项目号:2021YFC0863300),题目:新型冠状病毒变异监测及功能影响分析;广州实验室应急攻关项目(项目号:EKPG21-27),题目:新型冠状病毒变异株的分离鉴定及其抗原性分析;广东省重点领域研发计划项目(项目号:2021A1111100008),题目:新冠肺炎确诊病例和无症状感染者系列临床样本的收集保藏与应用。

摘　　要：为探究利用SARS-CoV-2的ORF1ab基因和N基因作为靶标进行荧光定量检测的Ct值与SARS-CoV-2病毒分离阳性率之间的关系。本研究对广州入境的新型冠状病毒肺炎输入病例临床样本进行荧光定量PCR检测和用Vero细胞进行病毒分离,统计病毒分离率与荧光定量PCR检测Ct值之间的关系,再通过logistics回归模型用Ct值来预测病毒分离率;同时对收集到的临床样本进行三代测序及进化树的构建。结果显示,在160例新型冠状病毒肺炎输入病例临床样本中,分离到74株新型冠状病毒,分离阳性率为46.25%,分离到包括20株Alpha、9株Beta、2株Delta等VOC毒株和2株Epsilon、1株Eta等VOI毒株,其他变异株42株;病例样本荧光定量PCR检测ORF1ab基因Ct值为16.12~39.00,N基因的Ct值为17.82~39.41;病毒分离率与样本荧光定量PCR检测的ORF1ab基因和N基因的Ct值均呈负相关,在Ct值>31时,分离率仅9.10%(6/66),而当Ct值<23.1时,病毒分离阳性率可超过95%,分离到新冠病毒的病例样本荧光定量PCR检测的ORF1ab基因和N基因的最高Ct值分别为33.74和34.47。本研究结果提示,当SARS-CoV-2荧光定量PCR检测结果的Ct值越高时,毒株分离率越低,在Ct值>31时病毒分离阳性率显著降低。To explore the relationship between the cycle threshold(Ct) values of severe acute respiratory syndrome-coronavirus-2(SARS-CoV-2) quantitative PCR detection and the positive rate of SARS-CoV-2 virus isolation. Clinical samples of COVID-19 imported cases in Guangzhou,China were detected by quantitative PCR and virus isolation by Vero cells. The relationship between virus isolation rate and Ct value detected by quantitative PCR was analyzed, and the virus isolation rate was predicted by logistics regression model. At the same time, the collected clinical samples were sequenced with the third generations sequencing and evolutionary tree were constructied. From 160 clinical samples of imported cases of COVID-19, seventy-seven novel coronavirus strains were isolated. The isolation positive rate was 46.25%. VOC strains including 20 Alpha, 9Beta, 2 Delta and VOI strain 2 Epsilon and 1 Eta were isolated, together with forty-two other variants. The Ct values of ORF1ab gene measured by quantitative PCR ranged from 16.12 to 39.00, and the Ct values of the N gene ranged from 17.82 to 39.41. The virus isolation rate was negatively correlated with the Ct values of both the ORF1ab and N genes detected by quantitative PCR. At the Ct values greater than 31, the isolation rate is only 9.10%(6/66), while the Ct value is less than 23.1, the positive rate of virus isolation can exceed 95%.The highest Ct values for the ORF1ab and N genes detected by quantitative PCR were 33.74 and 34.47, respectively. The higher the Ct value of the SARS-CoV-2 quantitative PCR, the lower the strain isolation rate, it is difficult to isolate the strain from the sample at the Ct value greater than 31.

关 键 词：新型冠状病毒 病毒分离 荧光定量检测 

分 类 号：R373.1[医药卫生—病原生物学]