發(fā)布時(shí)間：2018-02-27 11:53

  本文關(guān)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(diǎn)雜交　出處：《大連醫(yī)科大學(xué)》2007年碩士論文　論文類(lèi)型：學(xué)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(dǎo)致人類(lèi)死亡的最顯著原因之一.。近年來(lái),隨著人口流動(dòng)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(dá)國(guó)家和發(fā)展中國(guó)家再度肆虐的態(tài)勢(shì),每年在世界范圍內(nèi)導(dǎo)致大約200萬(wàn)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(shì)。利福平(Rifarmpicin,RFP)作為最有效的抗結(jié)核藥物之一,對(duì)其耐藥,尤其對(duì)RFP耐藥與其它藥物耐藥相關(guān)時(shí),通常會(huì)導(dǎo)致臨床上結(jié)核病的復(fù)燃。由于異煙肼和利福平是臨床最常用的藥物,有報(bào)道約90%的結(jié)核分枝桿菌耐利福平株同時(shí)也耐異煙肼,所以單獨(dú)進(jìn)行利福平敏感性分析也可能作為耐多藥的篩選指標(biāo)[2-4]。因此,對(duì)主要抗結(jié)核藥物如利福平的耐藥性快速檢測(cè)對(duì)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?lè)聪螯c(diǎn)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(duì)其采用生化反應(yīng)方法進(jìn)行菌種鑒定并用比例法進(jìn)行藥物敏感性試驗(yàn)。其后用聚合酶鏈反應(yīng)(Polymerase Chain Reaction,PCR)擴(kuò)增rpoB基因“熱點(diǎn)突變區(qū)”附近區(qū)域進(jìn)行DNA測(cè)序分析,了解我國(guó)結(jié)核分枝桿菌rpoB基因突變情況,明確其突變位點(diǎn)的分布,主要突變位點(diǎn)及其發(fā)生率。選取特征性突變位點(diǎn),設(shè)計(jì)檢測(cè)探針,建立快速檢測(cè)結(jié)核分枝桿菌rpoB基因突變的以PCR為基礎(chǔ)的寡核苷酸探針?lè)聪螯c(diǎn)雜交方法。采用SPSS 11.0統(tǒng)計(jì)軟件對(duì)實(shí)驗(yàn)數(shù)據(jù)進(jìn)行分析,探討該方法能否作為篩查方法用于臨床檢測(cè)結(jié)核分枝桿菌對(duì)利福平的敏感性,并評(píng)價(jià)其臨床應(yīng)用價(jià)值。 首先,采用生化培養(yǎng)基對(duì)從結(jié)核病人分離的536株分枝桿菌臨床分離株進(jìn)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(duì)篩選出來(lái)的482株結(jié)核分枝桿菌臨床分離株進(jìn)行藥物敏感性試驗(yàn),結(jié)果顯示對(duì)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(dá)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(duì)結(jié)核分枝桿菌利福平耐藥的相關(guān)基因rpoB基因設(shè)計(jì)引物,應(yīng)用DNA序列分析對(duì)結(jié)核分枝桿菌標(biāo)準(zhǔn)株H37Rv及300株已經(jīng)明確耐藥背景的臨床分離株進(jìn)行耐藥基因突變檢測(cè)。經(jīng)DNA序列分析,181株RFP耐藥株中,531位點(diǎn)突變占所有突變的53.61%,526位點(diǎn)突變占27.71%,516位點(diǎn)突變占9.64%,511位點(diǎn)突變占6.02%,這四種突變位點(diǎn)的突變頻率之和占到所有突變類(lèi)型的96.99%;多位點(diǎn)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(gè)氨基酸密碼子插入和5株單堿基或整個(gè)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(duì)敏感株中,有2株發(fā)生511、526位點(diǎn)突變。 在此基礎(chǔ)上,利用反向點(diǎn)雜交技術(shù)對(duì)300株結(jié)核分枝桿菌臨床分離株進(jìn)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(wú)突變,52株RFP相對(duì)敏感株中有2株檢測(cè)到突變;181利福平耐藥株中,165株檢測(cè)到突變,16株未見(jiàn)突變。與DNA測(cè)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(cè)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(yáng)性預(yù)測(cè)值為98.20%(164/167),陰性預(yù)測(cè)值為96.99%(129/133)。有293株反向點(diǎn)雜交的檢測(cè)結(jié)果與其DNA測(cè)序結(jié)果相符,二者的符合度可達(dá)97.67%(293/300),經(jīng)×2檢驗(yàn),差異無(wú)統(tǒng)計(jì)學(xué)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(diǎn),且92.82%的耐藥株發(fā)生突變,進(jìn)一步證實(shí)了結(jié)核分枝桿菌耐利福平與rpoB基因突變有關(guān),同時(shí)也提示了還有其他的機(jī)制參與了利福平耐藥。本研究所采用的以PCR為基礎(chǔ)的反向點(diǎn)雜交技術(shù),一次可檢測(cè)多個(gè)位點(diǎn)的突變,與DNA序列分析的符合率可達(dá)97.67%,經(jīng)х2檢驗(yàn),兩者結(jié)果的差異在統(tǒng)計(jì)上無(wú)意義。該方法具有快速、靈敏、特異度高、操作簡(jiǎn)便、價(jià)格低廉等特點(diǎn),可以適用于結(jié)核分枝桿菌臨床分離株對(duì)利福平敏感性的大批量初篩實(shí)驗(yàn),其作為基因芯片的技術(shù)基礎(chǔ),具有廣闊的臨床應(yīng)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(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R378

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

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

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本文編號(hào)：1542563