分子分型方法在结核分枝杆菌复合群分离株中的研究和应用

发布时间：2018-05-26 22:30

本文选题：结核分枝杆菌复合群 + 聚合酶链反应　；参考：《第三军医大学》2007年硕士论文

【摘要】： 目的: 1.建立快速鉴定结核分枝杆菌复合群(MTBC)菌种的方法。 2.建立Spoligotyping和VNTR两种MTBC分离株基因分型方法,评价其应用价值及在我国应用的可行性。方法: 1.将7对MTBC各菌种特异性引物(Mtbcl-7)进行聚合酶链反应(PCR),通过对扩增产物的不同带型进行分析,对己知标准株和96株临床分离株进行菌种鉴定。 2.将被鉴定的结核分枝杆菌菌株进行Spoligotyping和VNTR两种分型,其结果进行比较。结果: 1.用3株MTBC标准株,9株非分枝杆菌标准株以及27株非结核分枝杆菌(NTM)标准株对7对MTBC各菌种特异性引物进行特异性鉴定,结果证明引物Mtbc1能鉴定出分枝杆菌和非分枝杆菌;引物Mtbc2能鉴定出NTM和MTBC;引物Mtbc3能鉴定出微小分枝杆菌;引物Mtbc4能从MTBC中识别出牛分枝杆菌和卡介苗;引物Mtbc5能鉴定出非洲分枝杆菌Ⅱ型和结核分枝杆菌;引物Mtbc6和引物Mtbc4结合能鉴定出卡介苗;引物Mtbc7和引物Mtbc5联合能鉴定出M.canettii分枝杆菌。96株临床分离株经7对引物扩增,扩增结果显示NTM 20株,牛分枝杆菌1株,卡介苗1株,M.canettii分枝杆菌3株,微小分枝杆菌3株,非洲分枝杆菌Ⅰ型3株,M.caprae分枝杆菌1株,结核分枝杆菌或非洲分枝杆菌Ⅱ型64株。 2.用Spoligotyping方法对59株结核分枝杆菌菌株进行基因分型,得到11种基因型,其中49株属北京基因型菌株,而用VNTR分型方法可将这些菌株分为53种基因型。49株北京基因型菌株可以分为43种不同的VNTR基因型。10个VNTR位点的多态性分析表明各位点有较大的区别,其中8个位点显示了较高的多态性,2个位点显示了中等程度的多态性。结论: 1.应用PCR技术鉴定MTBC菌种结果准确可靠,且具有简便和快速等优点,有较大的分子流行病学应用价值。 2.Spoligotyping分型法只需要少量的DNA可直接用于标本的检测,但分辨率不高,在北京家族株流行的地区,该方法不适用。VNTR分型方法简单快速,其数字化结果清晰可靠,是一种有效的结核分枝杆菌菌株基因分型方法。 3.Spoligotyping及VNTR两种非常有效的分型方法进行结核病流行病学调查和分子流行病学研究具有重要意义,尤其是VNTR技术,便于普及,可以在结核病分子流行病学调查研究上发挥重要作用。
[Abstract]:Purpose :

1 . A method for rapidly identifying Mycobacterium tuberculosis complex group ( MTBC ) strain is established .

2 . To establish the genotyping method of two MTBC isolates of Spoligotyping and VNTR , to evaluate its application value and the feasibility of its application in China .

Method :

1 . 7 pairs of MTBC strain specific primers ( Mtbcl - 7 ) were subjected to polymerase chain reaction ( PCR ) .

2 . Spoligotyping and VNTR typing were performed on the identified Mycobacterium tuberculosis strains , and the results were compared .

Results :

1 . The specific primers of MTBC standard strains , 9 strains of non - mycobacterium and 27 strains of non - Mycobacterium tuberculosis were identified . The results showed that the primers Mtbc2 could identify Mycobacterium tuberculosis and Mycobacterium tuberculosis ; the primers Mtbc2 could identify Mycobacterium tuberculosis and M.canettii . The primers Mtbc4 were able to identify M . canettii . The primers Mtbc4 and Mtbc4 were able to identify M . canettii . The primers Mtbc4 and Mtbc4 were able to identify M . canettii . The primers Mtbc4 and Mtbc4 were able to identify Mycobacterium tuberculosis .

2 . By using Spoligotyping method , 59 strains of Mycobacterium tuberculosis were genotyped to obtain 11 genotypes , 49 of them belong to Beijing genotype strains , and the strains can be divided into 53 genotypes by VNTR typing method . The 49 Beijing genotype strains can be divided into 43 different VNTR genotypes .

Conclusion :

1 . Using PCR technique to identify MTBC strain results is accurate and reliable , and has the advantages of simplicity and rapidity , and has great molecular epidemiology application value .

2 . Spoligotyping typing method requires only a small amount of DNA to be directly used for specimen detection , but the resolution is not high . The method is not applicable in the area where the Beijing family is popular . The VNTR typing method is simple and rapid , the digitization result is clear and reliable , and is an effective genotyping method for Mycobacterium tuberculosis strains .

3 . Both Spoligotyping and VNTR are important for epidemiological investigation of tuberculosis and molecular epidemiology , especially VNTR technology , which can play an important role in epidemiological investigation of tuberculosis .
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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