本文選題：結(jié)核分枝桿菌 + 基因分型　； 參考：《天津醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 目的：初步探討15個可變數(shù)目串聯(lián)重復(fù)(Variable Number Tandem Repeat，VNTR)位點(diǎn)在天津地區(qū)結(jié)核分枝桿菌(Mycobacterium tuberculosis，MTB)中的多態(tài)性、天津地區(qū)的主要流行菌株以及VNTR分型方法在結(jié)核分子流行病學(xué)中的主要應(yīng)用。 方法：選取天津市結(jié)核病控制中心參比實(shí)驗(yàn)室菌種庫中2005年6月至2006年4月的100株MTB，收集這100例確診肺結(jié)核患者的一般資料和實(shí)驗(yàn)室資料。將菌株復(fù)蘇傳代，提取其基因組DNA。采用PCR方法擴(kuò)增結(jié)核菌基因組中的15個VNTR位點(diǎn)，包括由12個結(jié)核分枝桿菌散在重復(fù)單元(Mycobacterial Interspersed Repetitive Unit，MIRU)和5個精確串聯(lián)重復(fù)序列(Exact Tandem Repeats，ETR)(其中的兩個與MIRU位點(diǎn)一致)。擴(kuò)增的產(chǎn)物經(jīng)瓊脂糖凝膠電泳后，應(yīng)用Bio-Rad凝膠成像系統(tǒng)觀察PCR產(chǎn)物并通過Quantity One4.4分析軟件確定PCR產(chǎn)物的片段長度并與標(biāo)準(zhǔn)菌株H_(37)Rv比較計算得到每個菌株不同串聯(lián)重復(fù)位點(diǎn)的拷貝數(shù)，將部分菌株的VNTR位點(diǎn)PCR擴(kuò)增產(chǎn)物經(jīng)純化后直接測序；評價每個VNTR位點(diǎn)的多態(tài)性以及MIRU位點(diǎn)和ETR位點(diǎn)對天津地區(qū)結(jié)核菌株的綜合分辨能力。應(yīng)用PAUP4.0軟件繪制系統(tǒng)樹圖，揭示菌株之間的相互關(guān)系。根據(jù)VNTR基因分型結(jié)果，設(shè)計調(diào)查表，收集成簇菌株的患者資料，用以追蹤同一簇患者間的相互傳播途徑。應(yīng)用SPSS11.5進(jìn)行卡方檢驗(yàn)和單因素Logistic回歸分析。 結(jié)果： 1、基本資料描述：本次研究中的100株MTB分別來自100名肺結(jié)核患者，患者的男女比例為1.9：1，，年齡的中位數(shù)為36歲(15-82歲)，以20-29歲患者比例最高。市內(nèi)六區(qū)患者占患者總數(shù)的81％，初治患者占81％，有卡介苗接種史的患者占68％。 2、本次研究中58株MTB的5個ETR-VNTR位點(diǎn)拷貝數(shù)編碼均為42435，另有兩株編碼分別為42436和42433，均屬于具有北京家族VNTR分型特點(diǎn)的菌株，共占實(shí)驗(yàn)菌株總數(shù)的60％。 3、5個ETR-VNTR位點(diǎn)僅將菌株分為23個基因型，獨(dú)立株17株，6個簇，最大簇包含58株菌；12個MIRU-VNTR位點(diǎn)將菌株分為45個基因型，獨(dú)立株36株，9個簇，最大簇包含34株菌；MIRU和ETR-VNTR相結(jié)合后將菌株可被分為52個基因型，獨(dú)立株41株，11個簇，最大簇包含29株菌。成簇組與非成簇組患者在性別，年齡，住址，治療史，卡介苗接種史等方面的差異均無統(tǒng)計學(xué)意義(P＞0.05)。 4、VNTR位點(diǎn)等位基因多樣性(h)分析結(jié)果顯示：MIRU-26和MIRU-31位點(diǎn)多態(tài)性較好h分別為0.54和0.45；其次為MIRU-10，40和ETR-A，0.3＜h＜0.4；MIRU-39，27，0.2＜h＜0.3；MIRU-4，16，23和ETR-C，0.1＜h＜0.2；MIRU-2，20，24和ETR-B的多樣性最低h＜0.1。不同VNTR位點(diǎn)組合對結(jié)核分枝桿菌的分辨能力比較結(jié)果顯示：單獨(dú)使用5個ETR位點(diǎn)、12個MIRU位點(diǎn)和兩者結(jié)合后分辨能力HGDI分別為0.6503，0.8622和0.9093。 5、PAUP分析表明，100株MTB可被分為三組(Ⅰ，Ⅱ，Ⅲ)，其中89株菌屬于第Ⅲ組，屬于Ⅰ，Ⅱ兩組的菌株分別為6株和5株。 6、對成簇組的結(jié)核病患者資料進(jìn)一步調(diào)查發(fā)現(xiàn)，其中2名患者為相繼發(fā)病的某中學(xué)同班同學(xué)，可以確定這兩名患者處于同一條傳播鏈上。 結(jié)論：15個VNTR位點(diǎn)中MIRU-10，26，31，40和ETA五個位點(diǎn)在天津地區(qū)流行的MTB中多態(tài)性較好，其余位點(diǎn)對菌株的分辨能力欠佳。MIRU和ETR-VNTR位點(diǎn)還不足以達(dá)到理想的分型效果，還應(yīng)再引入其他多態(tài)性位點(diǎn)。天津地區(qū)可能是北京家族菌株的高流行區(qū)，應(yīng)加強(qiáng)對該類菌株的預(yù)防和控制。VNTR分型方法可廣泛用于結(jié)核病的分子流行病學(xué)研究。
[Abstract]:Objective: To investigate the polymorphism of the 15 Variable Number Tandem Repeat (VNTR) loci in the Mycobacterium tuberculosis (MTB) in Tianjin area, and the main epidemic strains in Tianjin and the main application of the VNTR typing method in the epidemiology of tuberculosis.
Methods: 100 strains of MTB from June 2005 to April 2006 in the reference laboratory of the Tianjin tuberculosis control center were selected to collect the general data and laboratory data of the 100 cases of pulmonary tuberculosis. The strains were resuscitation, and the genomic DNA. was extracted by PCR method to amplify 15 VNTR loci in the genome of tuberculosis bacteria, including 12 Mycobacterium tuberculosis scattered in the repeating unit (Mycobacterial Interspersed Repetitive Unit, MIRU) and 5 precise series repeats (Exact Tandem Repeats, ETR). (two of them are in agreement with the MIRU loci). The amplified products were analyzed by the agarose gel electrophoresis, and the PCR products were observed and analyzed by the Bio-Rad gel imaging system. The software determined the length of the fragment of the PCR product and compared with the standard strain H_ (37) Rv to calculate the copy number of the different tandem reset points of each strain. The PCR amplification products of the VNTR loci of some strains were directly sequenced, and the polymorphism of each VNTR site and the synthesis of the MIRU and ETR loci in the synthesis of the tuberculosis strains in Tianjin region were evaluated. PAUP4.0 software was used to map the system tree map to reveal the relationship between the strains. According to the results of the VNTR genotyping, the questionnaire was designed and the patient data of the cluster strains were collected to track the communication pathways of the same cluster of patients. SPSS11.5 was used for chi square test and single factor Logistic regression analysis.
Result:
1, basic data description: 100 MTB in this study were from 100 tuberculosis patients, the proportion of men and women was 1.9:1, the median age was 36 years (15-82 years old), the highest proportion was 20-29 years old. The six patients in the city accounted for 81% of the total, the initial treatment was 81%, and the patients with the history of BCG vaccination accounted for 68%..
2, in this study, the number of 5 ETR-VNTR loci copies of 58 MTB was 42435, and the other two strains were 42436 and 42433, all of which belonged to the VNTR typing of the Beijing family, which accounted for 60%. of the total number of experimental strains.
3,5 ETR-VNTR loci only divide the strain into 23 genotypes, 17 independent strains, 6 clusters and 58 strains. 12 MIRU-VNTR sites divide the strains into 45 genotypes, 36 isolated strains, 9 clusters, and 34 strains, and the strain can be divided into 52 genotypes, 41 strains of independent strains, 11 clusters, and the largest clusters after MIRU and ETR-VNTR. There were 29 strains. There was no significant difference in the sex, age, address, treatment history and BCG vaccination history between the cluster group and the non clustered group (P > 0.05).
4, VNTR allele diversity (H) analysis showed that the better h of MIRU-26 and MIRU-31 loci were 0.54 and 0.45, followed by MIRU-10,40 and ETR-A, 0.3 < h < 0.4, MIRU-39,27,0.2 < 0.3, MIRU-4,16,23 and ETR-C, 0.1 < 0.2. The results of the resolution of Mycobacterium tuberculosis showed that 5 ETR loci alone, 12 MIRU loci and the binding post resolution HGDI were 0.6503,0.8622 and 0.9093., respectively.
5, PAUP analysis showed that 100 strains of MTB could be divided into three groups (I, II, III), of which 89 strains belonged to group III, belonging to group I and II two strains were 6 strains and 5 strains respectively.
6, a further investigation of the data of tuberculosis patients in the cluster group found that 2 of the patients were classmate of a secondary school who had been attacked successively, and the two patients could be determined to be in the same transmission chain.
Conclusion: the five loci in the 15 VNTR loci are better polymorphic in the popular MTB in Tianjin area, and the other loci are not good enough to distinguish the strains. The.MIRU and ETR-VNTR loci are not enough to achieve the ideal typing effect, and the other polymorphic loci should be reintroduced. The Tianjin region may be the high prevalence of the Beijing family strain. The prevention and control of this strain should be strengthened. The.VNTR typing method can be widely used in molecular epidemiological studies of tuberculosis.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R181.3

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