【摘要】：結(jié)核病(Tuberculosis, TB)是由結(jié)核分枝桿菌復(fù)合群(Mycobacterium tuberculosis complex, MTBC)引起的一種慢性傳染病,主要感染人的呼吸道系統(tǒng)。據(jù)WHO估計,世界上近三分之一的人口已感染結(jié)核分枝桿菌。雖然TB流行水平呈下降趨勢,但每年新發(fā)病例仍突破900萬例,并且,隨著耐藥菌株的不斷出現(xiàn),對結(jié)核病的治療和防控造成很大的挑戰(zhàn)。因此,結(jié)核分枝桿菌菌種的鑒定分型以及耐藥性監(jiān)測對于該病的診斷、治療以及預(yù)后都有著重要的意義。本研究針對結(jié)核分枝桿菌揚(yáng)州臨床分離株,運(yùn)用傳統(tǒng)細(xì)菌學(xué)方法和多位點PCR相結(jié)合,初步鑒定分枝桿菌的種屬,再利用MIRU-VNTR和Spoligotyping分型方法研究其流行病學(xué)特征,最后通過比例法、MIC和耐藥基因檢測分析結(jié)核分枝桿菌耐藥性情況。1揚(yáng)州市結(jié)核分枝桿菌的分離培養(yǎng)與鑒定用抗酸染色實驗確定抗酸桿菌陽性后,用PNB/TCH試驗和多位點PCR從抗酸桿菌陽性菌株中鑒定出結(jié)核分枝桿菌(Mycobacterium tuberculosis, MTB),并比較兩個方法之間的差異。結(jié)果顯示,289株抗酸染色陽性臨床分離株,經(jīng)過PNB/TCH試驗和多位點PCR進(jìn)一步鑒定,其中270株為MTB,9株為非結(jié)核分枝桿菌(nontuberculous mycobacteria, NTM),2株為M. africanum,剩余8株需進(jìn)一步鑒定。PNB/TCH試驗結(jié)果與多位點PCR結(jié)果無統(tǒng)計學(xué)顯著性差異。鑒定結(jié)果表明,揚(yáng)州市結(jié)核病例絕大多數(shù)是由結(jié)核分枝桿菌所引起的,但也存在非結(jié)核分枝桿菌引起的結(jié)核病。2結(jié)核分枝桿菌揚(yáng)州分離株的分子分型研究CTAB法提取到270株MTB分離株基因組DNA,用MIRU-VNTR和Spoligotyping進(jìn)行基因分型。Spoligotyping方法共區(qū)分出8個家族,其中北京家族233株、T1家族15株、未知家族菌株11株、相似北京家族4株、U家族3株、H3家族2株、MANU2家族1株、T2家族1株。MIRU-VNTR方法共區(qū)分出154個基因型,其中144株組成28個簇,剩余126株均為獨立基因型,該方法的分辨力為0.960。在15個VNTR位點中,不管是對全部菌株還是只對北京家族進(jìn)行分析,Mtub21位點的HGDI值均為最高,分別為0.545和0.451。MIRU-VNTR分型方法的分辨率(0.960)明顯高于Spoligotyping方法的分辨力(0.253),且兩種分型方法相結(jié)合會使分辨力更高。通過分型研究,我們發(fā)現(xiàn)結(jié)核分枝桿菌揚(yáng)州分離株具有一定的基因多態(tài)性,北京家族基因型菌株為該市的主要流行菌株,其流行趨勢具有高度集中性,同時Spoligotyping方法也發(fā)現(xiàn)了11個新基因型,豐富了SpolDB4數(shù)據(jù)庫。3結(jié)核分枝桿菌揚(yáng)州分離株的耐藥表型及耐藥基因分析通過傳統(tǒng)比例法和MIC方法對174株臨床表現(xiàn)為耐藥的分離株進(jìn)行耐藥性檢測,并針對鑒定為耐藥的菌株進(jìn)行耐藥基因的測序(主要針對四種一線藥：異煙肼INH、利福平RFP、鏈霉素SM、乙胺丁醇EMB)。傳統(tǒng)比例法耐藥性結(jié)果顯示174株MTB中有72株耐藥(41.4%),多重耐藥菌株有32株(18.4%)。對72株耐藥菌株的耐藥性檢測方面,MIC法與傳統(tǒng)比例法的結(jié)果之間無統(tǒng)計學(xué)顯著性差異。耐藥基因比對結(jié)果顯示,INH耐藥菌株中,katG突變率為72.0%(36/50),inhA突變率為6.0%(3/50)；RFP耐藥菌株中,rpoB突變率為67.5%(27/40)；EMB耐藥菌株中,embB突變率為52.6%(10/19)；SM耐藥菌株中,rrs突變率為15.4%(6/39),rpsL突變率為71.8%(28/39)。通過比對分析,耐藥基因的突變與結(jié)核分枝桿菌耐藥表型密切相關(guān),出現(xiàn)耐藥表型的菌株不一定發(fā)生耐藥基因突變,但是耐藥基因突變的菌株全部耐藥。本研究以揚(yáng)州市2012-2015年結(jié)核分枝桿菌為對象,通過分離鑒定、分子分型、藥物敏感性試驗和耐藥基因突變位點分析等檢測,在分子水平分析揚(yáng)州地區(qū)結(jié)核分枝桿菌的分布和傳播特征,為該地區(qū)結(jié)核病的防治策略的訂制提供科學(xué)參考依據(jù),同時為結(jié)核病分子流行病學(xué)研究奠定了基礎(chǔ),并為結(jié)核病耐藥監(jiān)測提供了相關(guān)背景數(shù)據(jù)。
[Abstract]:Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis complex (MTBC). According to the WHO estimates, nearly one-third of the world's population has been infected with Mycobacterium tuberculosis. Although the prevalence of TB has a downward trend, new cases of TB continue to break through 9 million cases every year, and with the continuous emergence of drug-resistant strains, the treatment and prevention and control of tuberculosis pose a great challenge. Therefore, the identification and classification of Mycobacterium tuberculosis and the monitoring of drug resistance are of great significance to the diagnosis, treatment and prognosis of the disease. In this study, for the clinical isolates of M. tuberculosis in Yangzhou, the species of M. tuberculosis were identified by the combination of traditional bacteriological method and multi-site PCR. The epidemiological characteristics of M. tuberculosis were studied by using the methods of MIRU-VNTR and Spirigotyping. The drug resistance of Mycobacterium tuberculosis was analyzed by MIC and drug-resistant gene test. The isolated culture and identification of Mycobacterium tuberculosis in Yangzhou were determined by acid-fast staining. After the positive of acid-fast bacilli, Mycobacterium tuberculosis (M. tuberculosis) was identified from the positive strain of the acid-fast bacilli by using the PNB/ TCH test and the multi-site PCR. MTB) and comparing the differences between the two methods. The results showed that 289 strains of acid-fast stain positive clinical isolates were further identified by PNB/ TCH and multi-site PCR, of which 270 were MTB and 9 were non-tuberculous mycobacteria (NTM),2 were M. aficianum, and the remaining 8 strains were further identified. There was no statistically significant difference between the results of the PNB/ TCH test and the multi-site PCR. The results showed that the majority of the tuberculosis cases in Yangzhou were caused by Mycobacterium tuberculosis, but there were no M.tuberculosis caused by M.tuberculosis. The molecular typing of the isolated strains of M. tuberculosis was extracted by CTAB method to the genomic DNA of 270 MTB isolates. Genotyping was performed with MIRU-VNTR and Spirigotyping. A total of 8 families were identified, including 233 strains of the Beijing family,15 strains of the T1 family,11 strains of the unknown family strain,4 similar to the Beijing family,3 in the U family,2 in the H3 family,1 in the MANU2 family and 1 in the T2 family. A total of 154 genotypes were identified by the MIRU-VNTR method, of which 144 were 28 clusters, and the remaining 126 were independent. The resolution of the method was 0.960. In the 15 VNTR sites, the HGDI values of the Mtb21 site were the highest, 0.545 and 0.451.The resolution (0.960) of the Mtb21 locus was significantly higher than the resolution (0.253), and the combination of the two typing methods would make the resolution higher. By typing, we found that the isolated strain of M. tuberculosis has a certain gene polymorphism, and the Beijing family genotype strain is the main epidemic strain of the city, and the epidemic trend of the strain is highly concentrated, and 11 new genotypes are also found in the Spirigoinging method. The drug-resistant phenotype and the drug-resistance gene of the isolated strain of Mycobacterium tuberculosis were analyzed by the traditional method and the MIC method, and the drug resistance of the isolates was detected by the traditional method and the MIC method. And sequencing of the drug resistant genes for the strains identified as resistant (mainly aiming at the four first-line drugs: the heterosmotic INH, the rifampin RFP, the streptomycin SM, and the ethylamine-butanol EMB). The results showed that there were 72 drug-resistant strains (41.4%) in 174 strains of MTB and 32 (18.4%) of the multiple drug-resistant strains. There was no statistically significant difference between the results of the drug resistance of 72 strains of drug-resistant strains and the results of the method of MIC and the traditional method. The results showed that the mutation rate was 72.0% (36/50), the mutation rate of inhA was 6.0% (3/50), the mutation rate of rpoB in the resistant strain of RFP was 67.5% (27/40), and the mutation rate of rpoB in the drug-resistant strain was 52.6% (10/19). In the SM-resistant strain, the mutation rate of rrs was 15.4% (6/39), and the rpsL mutation rate was 71.8% (28/39). Compared with the analysis, the mutation of the drug-resistant gene is closely related to the drug-resistant phenotype of the mycobacterium tuberculosis, and the strain of the drug-resistant phenotype does not necessarily have the drug-resistant gene mutation, but the strain of the drug-resistant gene mutation is all resistant. In this study, the distribution and transmission characteristics of Mycobacterium tuberculosis in Yangzhou area were analyzed by means of isolation and identification, molecular typing, drug sensitivity test and resistance gene mutation site analysis in Yangzhou,2012-2015. It provides scientific reference for the customization of tuberculosis control strategy in the region, and lays a foundation for the epidemiological study of tuberculosis, and provides relevant background data for tuberculosis drug resistance monitoring.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R52;R446.5

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