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  本文關鍵詞： 結(jié)核分枝桿菌 利福平耐藥 rpoB基因 DNA序列分析 反向點雜交　出處：《大連醫(yī)科大學》2007年碩士論文　論文類型：學位論文

【摘要】： 結(jié)核分枝桿菌(Mycobacterium tuberculosis,MTB)的感染是因感染性因素導致人類死亡的最顯著原因之一.。近年來,隨著人口流動增加,人口密度增高,結(jié)核分枝桿菌多重耐藥菌株及同人體免疫缺陷病毒雙重感染的出現(xiàn),更加重了結(jié)核病在發(fā)達國家和發(fā)展中國家再度肆虐的態(tài)勢,每年在世界范圍內(nèi)導致大約200萬人死亡[1]。隨著結(jié)核病發(fā)病人數(shù)的上升,耐多藥結(jié)核病(Multi-Drug Resistance Tuberculosis,MDR-TB)的發(fā)病率也呈上升趨勢。利福平(Rifarmpicin,RFP)作為最有效的抗結(jié)核藥物之一,對其耐藥,尤其對RFP耐藥與其它藥物耐藥相關時,通常會導致臨床上結(jié)核病的復燃。由于異煙肼和利福平是臨床最常用的藥物,有報道約90%的結(jié)核分枝桿菌耐利福平株同時也耐異煙肼,所以單獨進行利福平敏感性分析也可能作為耐多藥的篩選指標[2-4]。因此,對主要抗結(jié)核藥物如利福平的耐藥性快速檢測對于有效治療結(jié)核病和控制耐多藥株的流行是必不可少的。 為較好建立寡核苷酸探針反向點雜交方法,本研究從西藏、湖南、河南、四川、福建、安徽和陜西七省收集結(jié)核分枝桿菌臨床分離菌株,首先對其采用生化反應方法進行菌種鑒定并用比例法進行藥物敏感性試驗。其后用聚合酶鏈反應(Polymerase Chain Reaction,PCR)擴增rpoB基因“熱點突變區(qū)”附近區(qū)域進行DNA測序分析,了解我國結(jié)核分枝桿菌rpoB基因突變情況,明確其突變位點的分布,主要突變位點及其發(fā)生率。選取特征性突變位點,設計檢測探針,建立快速檢測結(jié)核分枝桿菌rpoB基因突變的以PCR為基礎的寡核苷酸探針反向點雜交方法。采用SPSS 11.0統(tǒng)計軟件對實驗數(shù)據(jù)進行分析,探討該方法能否作為篩查方法用于臨床檢測結(jié)核分枝桿菌對利福平的敏感性,并評價其臨床應用價值。 首先,采用生化培養(yǎng)基對從結(jié)核病人分離的536株分枝桿菌臨床分離株進行了菌種鑒定,482株被鑒定為結(jié)核分枝桿菌(MTB),占89.93%;19株為牛結(jié)核分枝桿菌(M. bovis),3.54%;33株為非結(jié)核分枝桿菌(NTM),6.16%;2株為結(jié)核分枝桿菌加非結(jié)核分枝桿菌的混合感染,0.37%。 采用比例法對篩選出來的482株結(jié)核分枝桿菌臨床分離株進行藥物敏感性試驗,結(jié)果顯示對5種抗結(jié)核藥物全部敏感的菌株為126株,占26.14%;耐藥菌株356株,耐藥率達75.52%,其中,單耐藥菌株88株,18.26%;多耐藥菌株268株,55.60%。 其次,針對結(jié)核分枝桿菌利福平耐藥的相關基因rpoB基因設計引物,應用DNA序列分析對結(jié)核分枝桿菌標準株H37Rv及300株已經(jīng)明確耐藥背景的臨床分離株進行耐藥基因突變檢測。經(jīng)DNA序列分析,181株RFP耐藥株中,531位點突變占所有突變的53.61%,526位點突變占27.71%,516位點突變占9.64%,511位點突變占6.02%,這四種突變位點的突變頻率之和占到所有突變類型的96.99%;多位點聯(lián)合突變15株,占所有突變的9.04%,且發(fā)現(xiàn)1株發(fā)生整個氨基酸密碼子插入和5株單堿基或整個氨基酸密碼子缺失菌株。67株全敏感株中未發(fā)現(xiàn)突變,52株RFP相對敏感株中,有2株發(fā)生511、526位點突變。 在此基礎上,利用反向點雜交技術對300株結(jié)核分枝桿菌臨床分離株進行rpoB基因突變分析顯示:119株利福平敏感株中,67株全敏感株均無突變,52株RFP相對敏感株中有2株檢測到突變;181利福平耐藥株中,165株檢測到突變,16株未見突變。與DNA測序結(jié)果比較,168株中有164株被判定為耐藥株,4株判定為敏感株;132株測序未發(fā)現(xiàn)突變的菌株中129株被判定為敏感株,3株被判定為耐藥株。其敏感度為97.62%(164/168),特異度為97.73%(129/132),陽性預測值為98.20%(164/167),陰性預測值為96.99%(129/133)。有293株反向點雜交的檢測結(jié)果與其DNA測序結(jié)果相符,二者的符合度可達97.67%(293/300),經(jīng)×2檢驗,差異無統(tǒng)計學意義�YP0.05�Z。 綜上所述,RFP耐藥基因rpoB的突變主要發(fā)生在531、526、516、511位點,且92.82%的耐藥株發(fā)生突變,進一步證實了結(jié)核分枝桿菌耐利福平與rpoB基因突變有關,同時也提示了還有其他的機制參與了利福平耐藥。本研究所采用的以PCR為基礎的反向點雜交技術,一次可檢測多個位點的突變,與DNA序列分析的符合率可達97.67%,經(jīng)х2檢驗,兩者結(jié)果的差異在統(tǒng)計上無意義。該方法具有快速、靈敏、特異度高、操作簡便、價格低廉等特點,可以適用于結(jié)核分枝桿菌臨床分離株對利福平敏感性的大批量初篩實驗,其作為基因芯片的技術基礎,具有廣闊的臨床應用前景。
[Abstract]:Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) infection is caused by infectious factors leading to the death of a human being one of the most significant reasons. In recent years, with the increase of population, population density increased, the emergence of multiple drug-resistant strains of Mycobacterium tuberculosis and human immunodeficiency virus with double infection, more TB in developed countries and again the developing trend, leading to about 2 million deaths in [1]. with the rise in the number of TB each year in the world, multi drug resistant tuberculosis (Multi-Drug Resistance, Tuberculosis, MDR-TB) the incidence rate is rising. Li Fuping (Rifarmpicin, RFP) as one of the most effective anti tuberculosis drugs, the drug resistance, especially related to RFP drug resistance and other drug resistance, usually leads to clinical TB resurgence. Because isoniazid and Li Fuping are the most commonly used clinical medicine Things have reported about 90% of Mycobacterium tuberculosis resistant strains of Li Fuping also resistant to isoniazid alone, so Li Fuping sensitivity analysis may also be used as a screening index for [2-4]. multi drug resistance so the main anti tuberculosis drugs such as Li Fuping's resistance to rapid detection for effective treatment of tuberculosis and control of multidrug-resistant strains is essential.
To better establish oligonucleotide probe reverse dot blot hybridization method, this study from Tibet, Hunan, Henan, Sichuan, Fujian, Anhui and Shaanxi Province, collected seven clinical isolates of Mycobacterium tuberculosis from the first, using biochemical methods of species identification and drug sensitivity test was carried out using the proportion method. By using polymerase chain reaction (Polymerase Chain Reaction PCR), rpoB gene amplification "hotspot region" near the area DNA sequencing analysis, my understanding of the rpoB gene mutation of Mycobacterium tuberculosis, the clear distribution of mutations, mutation sites and the incidence of major mutations. Feature selection, design of detection probe, oligonucleotide probe reverse point to establish the rapid detection of Mycobacterium tuberculosis rpoB gene mutation of PCR based hybrid method. The experimental data were analyzed by SPSS 11 statistical software, explore the method can make The screening method is used to detect the sensitivity of Mycobacterium tuberculosis to rifampin, and to evaluate its clinical value.
First of all, using biochemical medium isolated from 536 strains of Mycobacterium tuberculosis clinical isolates were identified, 482 strains were identified as Mycobacterium tuberculosis (MTB), accounting for 89.93%; 19 strains of Mycobacterium tuberculosis (M. bovis), 3.54%; 33 strains were non tuberculous mycobacteria (NTM), 6.16%; 2 strains of Mycobacterium tuberculosis bacillus mixed non Mycobacterium tuberculosis infection, 0.37%.
For screening out of 482 clinical isolates of Mycobacterium tuberculosis drug sensitivity test was carried out using proportion method, the results showed that for all 5 kinds of anti tuberculosis drug sensitive strains accounted for 26.14% and 126 strains, 356 strains; drug resistance, drug resistance rate was 75.52%, among them, the single drug resistant strains of 88, 18.26%; 268 strains of multi drug resistant strains 55.60%..
Secondly, according to the related gene of Mycobacterium tuberculosis rifampin resistant rpoB gene primers of Mycobacterium tuberculosis standard strain H37Rv and 300 strains have clear background resistance of clinical isolates resistant gene mutation detection using DNA sequence analysis. The result of DNA sequence analysis, 181 strains of RFP resistant strains, 531 site mutations accounted for 53.61% of all mutations 526 mutation, 516 mutation accounted for 27.71%, accounted for 9.64%, accounted for 6.02% of the 511 mutation, four mutation frequency and mutation accounted for 96.99% of all types of mutations; multilocus mutation combined 15 strains accounted for 9.04% of all mutations, and found that 1 strains had the amino acid codon insertion and 5 strains of single the base or the entire amino acid codon deletion mutant.67 strains sensitive strains of mutations were found in 52 strains, RFP relatively sensitive strains, 2 strains had 511526 mutation.
On this basis, the use of reverse dot blot in 300 clinical isolates of Mycobacterium tuberculosis rpoB gene mutation analysis showed that 119 rifampin sensitive strains, 67 strains of sensitive strains had no mutation, 52 strains of RFP relatively sensitive strains in 2 strains detected mutations; 181 rifampin resistant strains, 165 strains were detected the mutation, no mutation was found in 16 strains. Compared with DNA sequencing, 168 strains in 164 strains were determined as resistant strains, 4 strains were determined as sensitive strains; 132 strains sequenced 129 strains were judged to be not found in sensitive strains mutation strains, 3 strains were determined as resistant strains. The sensitivity was 97.62% (164/168), the specificity was 97.73% (129/132), the positive predictive value was 98.20% (164/167), the negative predictive value was 96.99% (129/133). The detection results of 293 strains of reverse dot blot and DNA sequencing results are consistent, up to 97.67% with the two X 2 (293/300), after inspection, the difference was not statistically significant P0.0? 5.?
In summary, the mutation of RFP resistance gene of rpoB mainly occurred in 531526516511 sites, and 92.82% strains of resistance mutations, further confirmed that the rpoB gene mutation and rifampin resistance of Mycobacterium tuberculosis, but also suggests that there are other mechanisms involved in resistance to rifampin. Hybridization technique used in this study is based on PCR and reverse. A mutation detection of multiple loci, and DNA sequence analysis of the coincidence rate was 97.67%, the 2 were test results no difference in statistics. The method is rapid, sensitive, high specificity, simple operation, low price and other characteristics, can be applied to clinical isolates of Mycobacterium tuberculosis in the bulk of rifampicin sensitivity screening test, the gene chip technology, it has broad prospects of clinical application.

【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R378

【引證文獻】

相關碩士學位論文 前1條

1 沈興華;蘇州市結(jié)核病耐藥流行病學調(diào)查及INH和RFP耐藥結(jié)核分枝桿菌相關基因突變特點的研究[D];蘇州大學;2011年

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本文編號：1542563