新型冠状病毒N_sgRNA荧光定量PCR检测方法的建立  

Establishment of real-time fluorescence quantitative PCR method for detecting the N subgenome of SARS-CoV-2

作　　者：韩桃利 张帜 赵嘉欣 卢盼 焦洋[1] 赵剑虹[1] 高艳[1] 张士尧[1] 刘宽宽 刘玉洁 樊茹 李文静[4] 孙灵利[1] Han Taoli;Zhang Zhi;Zhao Jiaxin;Lu Pan;Jiao Yang;Zhao Jianhong;Gao Yan;Zhang Shiyao;Liu Kuankuan;Liu Yujie;Fan Ru;Li Wenjing;Sun Lingli(Department of Microbiology,Beijing Chaoyang District Centre for Disease Control and Prevention,Beijing 100021,China;Beijing Fangshan District Center for Disease Control and Prevention,Beijing 100021,China;Yanjing Medical College,Capital Medical University,Beijing 101300,China;Department of Environment and Health,Beijing Chaoyang District Centre for Disease Control and Prevention,Beijing 100021,China)

机构地区：[1]北京市朝阳区疾病预防控制中心微生物检验科,北京100021 [2]北京市房山区疾病预防控制中心,北京100021 [3]首都医科大学燕京医学院,北京101300 [4]北京市朝阳区疾病预防控制中心环境与卫生科,北京100021

出　　处：《中华实验和临床病毒学杂志》2025年第1期96-101,共6页Chinese Journal of Experimental and Clinical Virology

基　　金：国家重点研发计划项目(2022YFE0102400);食物中毒诊断溯源技术北京市重点实验室开放基金课题(BZ0063-202403);首都卫生发展科研专项(首发2022-1G-4231);北京市朝阳区科技计划(CYSF2206)。

摘　　要：目的基于SARS-CoV-2的N_sgRNA建立实时荧光定量PCR检测方并进行初步应用。方法基于Wuhan-Hu-1/2019_MN908947和亚基因组RNA(subgenomic RNA,sgRNA)形成机制设计合成N_sgRNA重组质粒及其特异性引物探针。将重组质粒做为模板,根据实验结果的Ct值、荧光强度、扩增曲线等筛选最佳反应条件和反应体系,并采用评价指标对优化后的SARS-CoV-2病毒N_sgRNA荧光定量PCR检测方法进行灵敏性、重复性和特异性评估。同时,将该方法初步应用于172份临床样本和256份城市生活污水的检测探索该方法实际应用的可能性。结果初步建立了检测新冠病毒N_sgRNA的qRT-PCR方法,该方法检测下限为20 copies/mL,批内重复(0.002%~0.767%)和批间重复(0.016%~0.752%)实验变异系数均小于1%,特异性实验仅有N_sgRNA重组质粒检出,建立的方法灵敏度较高,特异性较强,重复性好。临床样本HK.3、EG.5.1和JN.1及其亚分支与其流行时期对应的城市污水样本的Ratio_(sgRNA/gRNA)均具有统计学差异(P<0.05);同时期临床样本和污水城市样本中Ratio_(sgRNA/gRNA)中位数具有较强的相关性(相关系数r=1.000,P=0.010),提示污水样本与人群临床样本中Ratio_(sgRNA/gRNA)趋势一致。结论本研究建立了一种检测SARS-CoV-2的N_sgRNA实时荧光定量PCR方法,该方法具有灵敏度较高,特异性较强,重复性好的特点,可用于检测SARS-CoV-2感染性。ObjectiveTo establish a fluorescent quantitative RT-PCR assay based on N_sgRNA of SARS-CoV-2 and preliminarily apply it on real samples.MethodsRecombinant plasmid,specific primers and probes of N_sgRNA were designed and synthesized based on Wuhan-Hu-1/2019_MN908947 and synthesis mechanism of subgenomic RNA(sgRNA).Using recombinant plasmid as amplification templates,the optimal reaction conditions and reaction system were screened according to the Ct value,fluorescence intensity,and shape of amplification curve and was evaluated for sensitivity,reproducibility,and specificity.Meanwhile,the possibility of practical application of the method was explored by testing 172 clinical samples and 256 municipal wastewater samples.ResultsA qRT-PCR assay for N_sgRNA in SARS-CoV-2 was initially established.The detection limit of the assay was 20 copies/mL,and the variation coefficients of in-batch(0.002%~0.767%)and batch to batch repetition(0.016%~0.752%)were less than 1%.Only N_sgRNA recombinant plasmid was detected in the specificity assay.So the method is more highly sensitive,specific and reproducible.The Ratio_(sgRNA/gRNA)of clinical samples HK.3,EG.5.1,JN.1 and their sub-lineages and their corresponding urban sewage samples in epidemic period were significantly different(P<0.05).There is a strong correlation between the median of Ratio_(sgRNA/gRNA)in clinical samples and sewage samples in the same period(correlation coefficient r=1.000,P=0.010).ConclusionsIn this study,a qRT-PCR method for detecting N_sgRNA of SARS-CoV-2 was established and the method has the characteristics of higher sensitivity,stronger specificity and better repeatability,and it can be used to detect SARS-CoV-2 infectivity.

关 键 词：新型冠状病毒 亚基因组 实时荧光定量PCR 

分 类 号：R73[医药卫生—肿瘤]