Please wait a minute...
浙江大学学报（医学版）
浙江大学学报（医学版）  2016, Vol. 45 Issue (1): 61-67    DOI: 10.3785/j.issn.1008-9292.2016.01.10
原著     
基于可变数目串联重复序列的痰液结核分枝杆菌检测方法建立及初步应用
苏敏1, 陈晋2, 白冰3, 黄云秀1, 魏兰1, 刘旻雁3, 陈婷梅1
1. 重庆医科大学检验系 教育部临床检验重点实验室, 重庆 400016;
2. 同济大学附属肺科医院检验科 上海市结核病临床研究中心重点实验室, 上海 200433;
3. 广西医科大学检验系, 广西 南宁 530021
Establishment and preliminary application of detection of Mycobacterium tuberculosis in sputum based on variable number tandem repeat
SU Min1, CHEN Jin2, BAI Bing3, HUANG Yunxiu1, WEI Lan1, LIU Minyan3, CHEN Tingmei1
1. Ministry of Education Key Laboratory of Medical Diagnostics, Chongqing Medical University, Chongqing 400016, China;
2. Department of Clinical Laboratory, Pulmonary Hospital, Tongji University, Shanghai 200433, China;
3. Medicine Laboratory Department, Guangxi Medical University, Nanning 530021, China
全文: PDF(868 KB)  
摘要: 

目的:结核分枝杆菌散在分布重复单位及可变数目串联重复序列(MIRU-VNTR)是一项应用于结核分枝杆菌分子分型的技术,本研究基于该技术旨在建立一种直接应用临床痰液标本检测结核分枝杆菌的实验室方法。方法:根据扩增条件及临床检测的实际需要,优化选择八个MIRU-VNTR位点(MTUB21、MTUB04、QUB18、QUB26、QUB11b、MIRU31、MIRU10和MIRU26)作为检测靶标,收集130例肺结核患者和200例非肺结核患者(其他肺部疾病)的痰液样本,分别以传统的罗氏培养法和临床诊断的方法为标准,用MIRU-VNTR分析和荧光定量聚合酶链反应(FQ-PCR)对上述样本进行检测。结果:以罗氏培养法为标准时,MIRU-VNTR的灵敏度为93.1%,特异度为97.7%;FQ-PCR检测的灵敏度为94.0%,特异度为96.7%,MIRU-VNTR分析与FQ-PCR检测结果准确度差异无统计学意义(χ2=0.352,P=0.569)。以临床诊断为标准时,MIRU-VNTR分析的灵敏度为86.9%,特异度为100%;FQ-PCR检测的灵敏度为87.7%,特异度为99.0%,两种方法检测结果准确度差异无统计学意义(χ2=0.030,P=0.862),且MIRU-VNTR分析中未发现假阳性结果。在MIRU-VNTR检测为阳性的结果中,98.2%(111/113)的样本分子编码互不相同,只有1.8%(2/113)分子编码一致(皆为5-4-6-8-5-5-3-8)。结论:应用临床痰液标本建立的基于MIRU-VNTR的结核分枝杆菌快速检测方法有着较好的应用价值。
关键词 ： 分枝杆菌, 结核/分离和提纯,  结核,肺/诊断,  痰液/微生物学,  分枝杆菌,结核/遗传学,  基因型,  串联重复序列    
Abstract：

Objective: To establish a laboratory method for detection of Mycobacterium tuberculosis in sputum based on variable number tandem repeat (VNTR). Methods: Mycobacterium tuberculosis was tested by VNTR and fluorescent quantitative reverse transcription polymerase chain reaction (FQ-PCR) in 130 sputum samples from patients with pulmonary tuberculosis and 200 specimens from patients with other lung diseases. According to the amplification conditions and clinical detection needs, MTUB21, MUTB04, QUB18, QUB26, QUB11b, MIRU31, MIRU10 and MIRU26 were selected as test targets. The results of VNTR and FQ-PCR were compared with Lowenstein-Jensen culture and clinical diagnosis, and analyzed by chi-square test. Results: With the results of L-J culture as the standard, the sensitivity and specificity of VNTR were 93.1% (108/116) and 97.7% (209/214), and those of FQ-PCR were 94.0% (109/116) and 96.7% (207/214), respectively; no significant difference was observed between two groups (χ2=0.352, P=0.569). Using the clinical diagnosis as the standard, the sensitivity and specificity of VNTR were 86.9% (113/130) and 100% (200/200), and those of FQ-PCR were 87.7% (114/130) and 99.0% (198/200), respectively; the difference was not statistically significant (χ2=0.030, P=0.862). In 113 VNTR positive samples, the molecular codes differed from each other in 98.2% samples (111/113); only 2 samples had identical code (5-4-6-8-5-5-3-8). Conclusion: The study suggests that VNTR provides a promising method for diagnosis of clinical tuberculosis.
Key words： Mycobacterium tuberculosis/isolation &    purification    Tuberculosis, pulmonary/diagnosis    Sputum/microbiology    Mycobacterium tuberculosis/genetics    Genotype    Tandem repeat sequences
收稿日期: 2015-05-04      出版日期: 2015-12-12
CLC:  R378.91  
基金资助:

国家自然科学基金(81272544,81371775)
通讯作者: 陈婷梅(1969-),女,博士,副教授,主要研究方向为肿瘤微环境与肿瘤细胞侵袭转移、肿瘤免疫抑制;E-mail:chentingmei@sohu.com;http//orcid.org/0000-0003-0111-0837     E-mail: chentingmei@sohu.com
作者简介: 苏敏(1990-),男,硕士研究生,主要研究方向为临床结核快速诊断;E-mail:sumin0506@126.com;http://orcid.org/0000-0001-8651-8908
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　

引用本文:

苏敏 等. 基于可变数目串联重复序列的痰液结核分枝杆菌检测方法建立及初步应用[J]. 浙江大学学报（医学版）, 2016, 45(1): 61-67.
SU Min, CHEN Jin, BAI Bing, HUANG Yunxiu, WEI Lan, LIU Minyan, CHEN Tingmei. Establishment and preliminary application of detection of Mycobacterium tuberculosis in sputum based on variable number tandem repeat. Journal of ZheJiang University(Medical Science), 2016, 45(1): 61-67.

链接本文:

http://www.zjujournals.com/xueshu/med/CN/10.3785/j.issn.1008-9292.2016.01.10      或      http://www.zjujournals.com/xueshu/med/CN/Y2016/V45/I1/61

[1] HANRAHAN C F, SHAH M. Economic challenges associated with tuberculosis diagnostic development[J]. Expert REV Pharmacoecon Outcomes Res, 2014, 14(4):499-510.
[2] CHIANG C Y, VAN WEEZENBEEK C, MORI T, et al. Challenges to the global control of tuberculosis[J]. Respirology, 2013, 18(4):596-604.
[3] WEYER K, CARAI S, NUNN P. Viewpoint TB diagnostics:what does the world really need?[J]. J Infect Dis, 2011, 204(Suppl 4):1196-1202.
[4] SHINGADIA D. The diagnosis of tuberculosis[J]. Pediatr Infect Dis J, 2012, 31(3):302-305.
[5] KIK S V, DENKINGER C M, CHEDORE P, et al. Replacing smear microscopy for the diagnosis of tuberculosis:what is the market potential?[J]. Eur Respir J, 2014, 43(6):1793-1796.
[6] SCHERER L C, SPERHACKE R D, JARCZEWSKI C, et al. Comparison of two laboratory-developed PCR methods for the diagnosis of pulmonary tuberculosis in Brazilian patients with and without HIV infection[J]. BMC Pulm Med, 2011, 11:15.
[7] MEHTA P K, RAJ A, SINGH N, et al. Diagnosis of extrapulmonary tuberculosis by PCR[J]. FEMS Immunol Med Microbiol, 2012, 66(1):20-36.
[8] ZHOU K, AERTSEN A, MICHIELS C W. The role of variable DNA tandem repeats in bacterial adaptation[J]. FEMS Microbiol Rev, 2014, 38(1):119-141.
[9] GEMAYEL R, VINCES M D, LEGENDRE M, et al. Variable tandem repeats accelerate evolution of coding and regulatory sequences[J]. Annu Rev Genet, 2010, 44:445-477.
[10] SCHURCH A C, VAN SOOLINGEN D. DNA fingerprinting of Mycobacterium tuberculosis:from phage typing to whole-genome sequencing[J]. Infect Genet Evol, 2012, 12(4):602-609.
[11] LUO T, YANG C, PANG Y, et al. Development of a hierarchical variable-number tandem repeat typing scheme for Mycobacterium tuberculosis in China[J]. PLoS One, 2014,9(2):e89726.
[12] ALONSO M, HERRANZ M, MARTINEZ LIROLA M, et al. Real-time molecular epidemiology of tuberculosis by direct genotyping of smear-positive clinical specimens[J]. J Clin Microbiol, 2012, 50(5):1755-1757.
[13] 中华医学会结核病分会. 中国结核病分类法[J]. 中华结核和呼吸杂志, 1998, 21(12):716-717. TB branch of Chinese Medical Association. China's TB taxonomy[J]. Chinese Journal of Tuberculosis and Respiratory Diseases, 1998, 21(12):716-717. (in Chinese)
[14] 中国防痨协会基础专业委员会. 结核病诊断实验室检验规程[M]. 北京:中国教育文化出版社,2006:36. Professional committee of Chinese antituberculosis association. TB diagnosis laboratory test procedures[M]. Beijing:Chinese Education Culture Press, 2006:36. (in Chinese)
[15] LIU R X, LI Q Z, XING L L, et al. Genotyping of clinical Mycobacterium tuberculosis isolates based on eight loci of MIRU-VNTR[J]. Int J Tuberc Lung Dis, 2013, 17(2):243-245.
[16] LUO T, YANG C, GAGNEUX S, et al. Combination of single nucleotide polymorphism and variable-number tandem repeats for genotyping a homogenous population of Mycobacterium tuberculosis Beijing strains in China[J]. J Clin Microbiol, 2012, 50(3):633-639.
[17] SCHERER L C, SPERHACKE R D, ROSSETTI M L, et al. Usefulness of the polymerase chain reaction dot-blot assay, used with Ziehl-Neelsen staining, for the rapid and convenient diagnosis of pulmonary tuberculosis in human immunodeficiency virus-seropositive and-seronegative individuals[J]. Infect Dis Rep, 2011, 3(1):e3.
[1] 毛小荣, 张立婷, 蒋妮, 肖萍, 彭雪彬, 张有成. 丙型肝炎病毒基因型在中国大陆汉族慢性丙型肝炎患者中的分布特征[J]. 浙江大学学报（医学版）, 2015, 44(4): 417-422.
[2] 张弘，谷卫，贾敏月，等. 原发性醛固酮增多症遗传学研究进展[J]. 浙江大学学报（医学版）, 2014, 43(5): 612-.
[3] 蔡坚，等. Wnk1基因多态性与汉族人缺血性卒中的关联分析[J]. 浙江大学学报（医学版）, 2014, 43(1): 43-50.
[4] 毛小荣, 彭雪彬, 张有成, 李林平. 甘肃地区不同基因型丙肝感染与糖脂铁代谢异常的相关性研究[J]. 浙江大学学报（医学版）, 2013, 42(3): 345-349.
[5] 吴亦斐, 孙爱华, 赵金方, 葛玉梅, 严杰. 细菌药物钝化酶基因分布及其表达诱导与抑制机制的研究[J]. 浙江大学学报（医学版）, 2013, 42(2): 131-140.
[6] . CASP3基因多态及单体型分布与乳腺癌危险性的关联研究[J]. 浙江大学学报（医学版）, 2011, 40(3): 259-264.
[7] . XPC基因Lys939Gln和Ala499Val多态性与乳腺癌易感性的关联研究[J]. 浙江大学学报（医学版）, 2011, 40(3): 252-258.
[8] . miR-605和miR-149基因多态性以及相关因素与肺癌易感性的关联研究[J]. 浙江大学学报（医学版）, 2011, 40(3): 265-271.
[9] 何颖蕾;朱敏;金笑平;周元林. ALOX5AP基因SG13S114A/T多态性与动脉粥样硬化性脑梗死的关系[J]. 浙江大学学报（医学版）, 2009, 38(6): 630-633.
[10] 赵四海;刘恩岐;成大欣;薛欣;楚雍烈. HIV患者合并感染基因1和4型丙型肝炎病毒治疗效果的Meta分析[J]. 浙江大学学报（医学版）, 2009, 38(3): 315-319.
[11] 蒋汉梁;张甦;周林福;陈峰;朱海红;陈智. 湖州地区妇女宫颈病变人乳头瘤病毒亚型感染情况分析[J]. 浙江大学学报（医学版）, 2007, 36(6): 549-552.
[12] 胡农;蔡挺;张顺;周林福;许小敏;陈琳. 耐药鲍曼不动杆菌扩增片段长度多态性分析指纹图谱数据库的建立[J]. 浙江大学学报（医学版）, 2007, 36(6): 537-542.
[13] 顾珊智；仲靖芳；沐志强；李生斌. 中国阿昌族5个X-STR位点多态性研究[J]. 浙江大学学报（医学版）, 2007, 36(2): 185-190.
[14] 施育平%孟伟华%单江%傅国胜%徐耕. 血管紧张素转换酶基因多态性与冠心病及其冠状动脉粥样硬化病变程度的关系[J]. 浙江大学学报（医学版）, 2006, 35(3): 287-291.
[15] 茅海燕；方平楚；叶少菁；尤建飞；朱永良；俞建国. 慢性胃炎和消化性溃疡中幽门螺杆菌babA2和cagA及vacA基因型分析[J]. 浙江大学学报（医学版）, 2003, 32(1): 29-32.