發(fā)布時(shí)間：2018-10-05 18:18

【摘要】：目的運(yùn)用實(shí)時(shí)定量PCR(quantitative real-time, qRT-PCR)檢測(cè)結(jié)核分枝桿菌蛋白Ag85B、38kDa及MPT64編碼基因(fbpB, pstsl, mpt64)的表達(dá)水平,探討結(jié)核分枝桿菌耐藥菌株和敏感株,北京基因型和非北京基因型的上述蛋白抗原在轉(zhuǎn)錄水平的差異性及其意義。 方法根據(jù)GenBank提供的序列,設(shè)計(jì)目的基因fbpB, pstsl, mpt64及內(nèi)參基因Siga的特異性引物,將fbpB, pstsl, mpt64及Siga擴(kuò)增片段克隆入載體pMD18-T simple,重組質(zhì)粒經(jīng)純化及梯度稀釋,應(yīng)用SYBR GreenⅠ熒光染料建立檢測(cè)fbpB, pstsl, mpt64基因表達(dá)水平的實(shí)時(shí)定量PCR方法。選用46株結(jié)核分枝桿菌臨床分離株,包括藥物敏感菌株24株,耐藥菌株22株,北京基因型菌株26株和非北京基因型菌株20株,以及2株非結(jié)核分枝桿菌。應(yīng)用qRT-PCR檢測(cè)上述菌株、H37Rv標(biāo)準(zhǔn)株中fbpB, pstsl, mpt64基因的表達(dá)水平。 結(jié)果耐藥菌株中的mpt64基因表達(dá)水平低于敏感菌株及H37Rv標(biāo)準(zhǔn)株,有統(tǒng)計(jì)學(xué)意義(t=3.093,p0.01；t=2.545,p0.05),fbpB, pstsl基因表達(dá)水平與敏感組及H37Rv標(biāo)準(zhǔn)株無差異；北京基因型菌株中的pstsl基因表達(dá)水平低于非北京基因型菌株及H37Rv標(biāo)準(zhǔn)株,結(jié)果有統(tǒng)計(jì)學(xué)意義(t=2.567,p0.05；t=2.373,p0.05),fbpB, mpt64基因表達(dá)與非北京基因型組及H37Rv標(biāo)準(zhǔn)株表達(dá)無差異。結(jié)核分枝桿菌活菌的擴(kuò)增結(jié)果呈典型的S型曲線,而結(jié)核分枝桿菌死菌和2株非結(jié)核分枝桿菌呈水平直線,沒有檢測(cè)到熒光信號(hào)。 結(jié)論結(jié)核分枝桿菌菌株中的fbpB基因的表達(dá)量在敏感菌株和耐藥菌株及北京基因型菌株和非北京基因型菌株均沒有差異；結(jié)核分枝桿菌耐藥菌株中的mpt64基因的表達(dá)量低于敏感株及H37Rv,在北京基因型菌株和非北京基因型菌株中的表達(dá)量沒有差異；結(jié)核分枝桿菌北京基因型中pstsl基因的表達(dá)量低于非北京基因型及H37Rv,在敏感菌株和耐藥菌株中的表達(dá)量沒有差異。
[Abstract]:Objective to detect the expression of mycobacterium tuberculosis protein Ag85B,38kDa and MPT64 coding gene (fbpB, pstsl, mpt64 by real-time quantitative PCR (quantitative real-time, qRT-PCR, and to explore the drug-resistant and sensitive strains of Mycobacterium tuberculosis. The difference and significance of the above-mentioned protein antigens between the Beijing genotype and non-Beijing genotype at the transcriptional level. Methods according to the sequence provided by GenBank, the specific primers for fbpB, pstsl, mpt64 and Siga were designed. The amplified fragments of fbpB, pstsl, mpt64 and Siga were cloned into the recombinant plasmid pMD18-T simple,. SYBR Green 鈪，

本文編號(hào)：2254388