双靶标实时荧光PCR检测技术用于牛结核病快速诊断的研究  

Study on the rapid diagnosis of bovine tuberculosis by dual-target real-time fluorescent PCR

作　　者：欧喜超[1] 赵冰[1] 滕冲 张思琦 许晔 邢睿达 裴少君 夏辉[1] 王胜芬[1] 范伟兴[5] 赵雁林[1] Ou Xichao;Zhao Bing;Teng Chong;Zhang Siqi;Xu Ye;Xing Ruida;Pei Shaojun;Xia Hui;Wang Shengfen;Fan Weixing;Zhao Yanlin(National Center for Tuberculosis Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Department of Tuberculosis,Beijing Dongcheng District Center for Disease Control,Beijing 100050,China;School of Life Sciences,Xiamen University,Xiamen 361102,China;School of Public Health,Peking University,Beijing 100191,China;Chinese Center for Animal Health and Epidemiology,National Animal Tuberculosis Reference Laboratory,Qingdao 266033,China)

机构地区：[1]中国疾病预防控制中心结核病预防控制中心,北京102206 [2]北京市东城区疾病预防控制中心结核科,北京100050 [3]厦门大学生命科学学院,厦门361102 [4]北京大学公共卫生学院,北京100191 [5]中国动物卫生与流行病学中心国家动物结核病参考实验室,青岛266033

出　　处：《中国防痨杂志》2023年第11期1052-1057,共6页Chinese Journal of Antituberculosis

基　　金：国家重点研发计划(2022YFC2305204);西藏自治区重点研发计划(XZ202201ZY0007N-01)。

摘　　要：目的:针对结核分枝杆菌复合群(Mycobacterium tuberculosis complex,MTBC)IS6110和IS1081基因,开发双靶标实时荧光PCR检测技术并评价其在剖检奶牛病料中进行MTBC快速检测的可行性,为分子检测技术在牛结核病早期诊断领域的推广应用提供基础数据支持。方法:以IS6110和IS1081为特异靶基因建立MTBC实时荧光PCR检测体系;通过制备并检测含有不同浓度牛分枝杆菌菌悬液的牛肺组织,确定双靶标实时荧光PCR检测体系的检出限;使用21株常见非结核分枝杆菌标准株的DNA作为扩增模板,确定双靶标实时荧光PCR检测MTBC的特异度;对剖检奶牛组织病料进行MGIT 960液体培养、Xpert MTB/RIF(简称“Xpert”)、Xpert MTB/RIF Ultra(简称“Xpert Ultra”)和双靶标实时荧光PCR检测,以液体培养结果为参照标准,分析双靶标实时荧光PCR检测在剖检奶牛病料中检测MTBC的效能。结果:双靶标实时荧光PCR检测21株非结核分枝杆菌菌株的DNA,均未检测到扩增曲线,MTBC检测特异度为100.0%(21/21)。组织病料液体培养、Xpert、Xpert Ultra和双靶标荧光PCR检测阳性率分别为64.0%(32/50)、56.0%(28/50)、78.0%(39/50)和76.0%(38/50)。双靶标荧光PCR检测阳性率明显高于Xpert检测阳性率,差异有统计学意义(χ^(2)=4.456,P=0.035),双靶标荧光PCR检测阳性率与Xpert Ultra检测阳性率差异无统计学意义(χ^(2)=0.056,P=0.812)。以液体培养结果为参照标准,双靶标荧光PCR检测MTBC的敏感度和特异度分别为90.3%(28/31)和40.0%(6/15);Xpert Ultra检测MTBC的敏感度和特异度分别为96.8%(30/31)和40.0%(6/15)。结论:双靶标荧光PCR检测技术可用于牛结核病的早期快速诊断,有助于控制人畜共患结核病的流行和传播。Objective:A dual-target real-time fluorescent PCR technique was developed for the detection of IS6110 and IS1081 genes of Mycobacterium tuberculosis complex(MTBC),to evaluate the feasibility of MTBC rapid detection in cow samples,and to provide basic data support for the application of molecular detection technology in the early diagnosis of bovine tuberculosis.Methods:MTBC real-time fluorescence PCR system was established with IS6110 and IS1081 as specific target genes.The limit of detection for two-target real-time fluorescent PCR system was determined by preparing and detecting bovine lung tissues containing different concentrations of Mycobacterium bovis suspensions.Using DNA of 21 common standard nontuberculous mycobacteria(NTM)strains as amplification template,the specificity of dual-target real-time fluorescent PCR detection of MTBC was determined.MGIT 960 liquid culture,Xpert MTB/RIF(Xpert),Xpert MTB/RIF Ultra(Xpert Ultra)and dual-target real-time fluorescent PCR were used to detect the tissue samples.Using liquid culture results as a reference standard,the efficacy of dual target real-time fluorescence PCR detection in detecting MTBC in dissected cow diseased materials was analyzed.Results:According to the results of dual-target real-time fluorescent PCR detecting the DNA of 21 NTM strains,no amplification curve was detected,the detection specificity of MTBC was 100.0%(21/21).The positive rates of liquid culture,Xpert,Xpert Ultra,and dual-target real-time fluorescent PCR detection were 64.0%(32/50),56.0%(28/50),78.0%(39/50),and 76.0%(38/50),respectively.The positive rate of dual-target real-time fluorescent PCR detection was significantly higher than that of Xpert detection(χ^(2)=4.456,P=0.035).There was no statistically significant difference between the positive rates of dual-target real-time fluorescent PCR detection and Xpert Ultra detection(χ^(2)=0.056,P=0.812).Based on the results of liquid culture experiments,the sensitivity and specificity of dual-target real-time fluorescent PCR for detecting

关 键 词：结核 牛 动物疾病 分子探针技术 诊断 

分 类 号：R52[医药卫生—内科学] R535[医药卫生—临床医学]