复方PCR用于分支杆菌菌种鉴定的初步研究

摘要/Abstract

摘要： 目的　寻找新的、快捷简便的分支杆菌菌种鉴定技术,进一步提高临床结核病诊断的水平。方法将3套分支杆菌基因组DNA的不同标记引物同时放入一个体系中,并在同一退火温度下进行PCR扩增,通过对扩增产物的不同带型进行分析,对已知标准株和部分临床株进行菌种鉴定。结果复方PCR方法可将结核复合群中的结核分支杆菌、牛结核分支杆菌与非结核分支杆菌一次性地区分开,特异性达到100%,灵敏度为10ng/反应。结论复方PCR技术是一种有效而又简捷的分支杆菌诊断方法,可用于临床上分结核与非结核分支杆菌或分支杆菌与非分支杆菌的初步区分。

关键词: 分支杆菌, 菌种鉴定, 聚合酶链反应

Abstract: Objective To find a new rapid and easy method of species identification of Mycobacteria for improving diagnosis level on tube rculosis.Methods Use three sets primers for mycobacteria togeth er to amplify the genome DNA of Mycobacteria including reference and some cl inical isolates.Results This method can distinguish M.tuberculosis complex from Nontuberculous Mycobacteria or does M.tuberculosis from M.bovis in a single step.Its specificity was 100%,and its sensitivity i s 10ng /response. Conclusion It showed that multiprimer PCR is a effective specie s identification method for Mycobacteria.So it can be used for initial diffe rentiational diagnosis between M.tuberculosis complex and Nontuberculous Mycobacteria or Mycobacteria and non-mycobacteria.

Key words: Mycobacterium, Species identification, PCR

王仲元,匡铁吉,金关甫,陈红兵,李净,安慧茹,王涛,. 复方PCR用于分支杆菌菌种鉴定的初步研究[J]. 中国防痨杂志, 2003, 25(6): 391-393.

WANG Zhong-yuan,KUANG Tie-ji,JIN Guan-fu,et al.. Species identification of Mycobacteria with multiprimer PCR[J]. Chinese Journal of Antituberculosis, 2003, 25(6): 391-393.

参考文献

1.Borremans M L，Witt D,G. Volckaert, et al. Cloning, sequence de-termination ,and expression of a 32-kilodalton-protein gene of My-cobacterium tuberculosis [J]. Infect Immun, 1989,57:3123～30.
2.Parra C A, Londono L P, Portillo P D,et al. Isolation,characteriza-tion, and molecular cloning of a specific Mycobacterium tuberculosis antigen:identification of a species-specific sequence [J]. Infect Im-mun, 1991,59:3411～17.
3.Thierry D A,Brisson-Noel, V Vincent-Levy Frebault,et al. Charac-terization of a Mycobacterium tuberculosis insertion sequence,IS6110, and its application in diagnosis [J].J Clin Microbiol,1990,28:2668～73.
4.Noordhock G T,Kolk AH J,Bjune G,et al.Sensitivity and specific-ity of PCR for detection of Mycobacterium tuberculosis：a blind comparison study among seven laboratories[J].J Clin Microbiol,1994,32:277～84.
5.Tevere V J, Hewitt P L, Dare A,et al. Detection of Mycobacterium tuberculosis by PCR amplification with Pan-Mycobacterium prim-ers and hybridization to a M. Tuberculosis specific probe [J].J Clin M icrobiol，1996,34:918～23.
6 Forbes B A, Hicks K E S. Direct detection of Mycobacterium tuber-culosis in respiratory specimens in a clinical laboratory by polymera-sa chain reaction [J].J Clin Microbiol, 1993,31:1688～94.
7.Das S C,Paramasivan N，Lowrie D B,et al. IS6110 restriction frag-ment length polymorphism typing of Mycobacterium tuberculosis from patients with pulmonary tuberculosis in Madras. South India [J].Tubercle Lung Dis,1995,76:550～54.
8.Sahadevan R，Narayanan S,Paramasivan C N, et al. Restriction fragment length polymorphism typing of clinical isolates of Myco-bacterium tuberculosis from patients with pulmonary tuberculosis in Madras, India, by use of Direct-Repeat Probe [J].J Clin Micro-biol, 1995,33:3037～39.

[1]	李爱芳, 崔晓利, 康磊, 雷静, 党丽云, 杨翰. 荧光PCR探针熔解曲线法检测结核分枝杆菌耐药性的价值[J]. 中国防痨杂志, 2020, 42(9): 998-1001.
[2]	张静, 陈曦, 王彬, 付雷, 陆宇, 陈效友. 改良单叠氮丙啶-荧光定量PCR法的建立及其检测抗结核药物活性的价值[J]. 中国防痨杂志, 2020, 42(5): 472-480.
[3]	于佳佳, 张旭霞, 张雨晴, 任卫聪, 姚丛, 李传友, 刘毅, 唐神结. PCR扩增技术联合CRISPR-Cas13a系统对MTB DNA检测方法的初步研究[J]. 中国防痨杂志, 2020, 42(12): 1280-1288.
[4]	吕纯芳,吴健虹,卢留珠,徐阳凤,刘盛元. 国产实时荧光定量PCR试剂与GeneXpert MTB/RIF检测结核分枝杆菌的对比分析[J]. 中国防痨杂志, 2020, 42(1): 60-65.
[5]	曹志华,赵悦竹,胡双双. 荧光 PCR 探针熔解曲线技术检测结核分枝杆菌对五种抗结核药物耐药性的研究[J]. 中国防痨杂志, 2019, 41(2): 156-161.
[6]	吴慧娜,孙付胜,刘清文,戎中录,张纪东,高阳,李永福,常斐. 荧光PCR探针熔解曲线法检测结核分枝杆菌复合群对利福平和异烟肼耐药性的价值[J]. 中国防痨杂志, 2019, 41(1): 74-79.
[7]	程丽平,张晓岩,沙巍. 痰标本PCR-反向斑点杂交法对疑似非结核分枝杆菌肺病的诊断价值[J]. 中国防痨杂志, 2018, 40(8): 834-839.
[8]	董宇杰,张莉,王宇轩,周立娟,曲杨,张晨,张海青,车南颖. 免疫组织化学及PCR技术在淋巴结结核病理诊断中的应用价值[J]. 中国防痨杂志, 2018, 40(4): 348-352.
[9]	郭俊红,武春燕,董正伟,吴伟,张莉萍,李辉,黄焰. CT引导下肺穿刺活检标本行PCR检测诊断肺结核的临床价值[J]. 中国防痨杂志, 2018, 40(11): 1159-1163.
[10]	程丽平，肖和平. 三种检测方法联合使用对结核性胸膜炎的诊断价值[J]. 中国防痨杂志, 2016, 38(11): 956-962.
[11]	张洁，邢青，王甦民，易俊莉，杨新宇，丁北川，苏建荣. 传统培养法与PCR-荧光探针法鉴定分枝杆菌的对比研究[J]. 中国防痨杂志, 2015, 37(3): 291-294.
[12]	周伟东，魏光喜，王军，赵明伟. 颈部淋巴结核96例诊断分析[J]. 中国防痨杂志, 2014, 36(10): 888-892.
[13]	董伟杰，秦世炳，赵立平，付育红，黄海荣. 骨关节结核各类标本进行结核分枝杆菌培养与PCR检测的阳性率结果分析[J]. 中国防痨杂志, 2014, 36(1): 46-48.
[14]	袁小亮张天托朱家馨郑文争雷建平涂少华罗一钧刘惟优. 江西省结核分枝杆菌耐二线抗结核药的分子学特征[J]. 中国防痨杂志, 2013, 35(9): 649-654.
[15]	王桂荣付育红梁倩尚媛媛姜广路赵立平于霞陈素婷黄海荣. 应用多重PCR方法快速鉴定结核分枝杆菌复合群与非结核分枝杆菌的研究[J]. 中国防痨杂志, 2013, 35(9): 686-689.