【摘要】：目的:探討直接從抗酸染色陽性痰涂片刮削物中擴增分枝桿菌rpoB基因的方法。方法:(1)使用引物Primer1:5′-CGTACGGTCGGCGAGCTGATC-3′,與Primer2:5′-AGGGGTTTCGATC GGGCACATC-3′;Primer3:5′-TGTTCTTCAAGGAGAAGCG-3′,與Primer4:5′-TCGTCGGCGGTCAGGTA-3′分別擴增18株臨床分離結(jié)核分枝桿菌的rpoB基因,以檢測引物設(shè)計的合理性。(2)分別采用普通PCR、Touchdown PCR、巢式PCR、二次PCR對直接從3+~4+抗酸染色痰涂片刮削物中提取的80例分枝桿菌基因組DNA進(jìn)行rpoB基因擴增。(3)對80例抗酸染色痰涂片刮削物基因組DNA行實時熒光定量PCR以檢測其拷貝數(shù)。(4)對18例臨床分離菌株基因組DNA 100、101、102、103、104、105、106、107倍稀釋物行實時熒光定量PCR與普通PCR(Primer1與Primer2,Primer3與Primer4),以比較二者檢出限。結(jié)果:(1)兩對引物對18株臨床分離結(jié)核桿菌rpoB基因擴增均有明亮目的條帶,并經(jīng)測序證實。(2)80例痰涂片刮削物基因組DNA擴增均未見目的條帶。(3)80例痰涂片刮削物基因組中,其中20例DNA的拷貝數(shù)為102~103copies/μL,60例DNA的拷貝數(shù)為104~105copies/μL。(4)兩組引物能夠擴增出rpoB基因的最高稀釋倍數(shù)分別為104、103倍,其對應(yīng)的熒光定量PCR檢測拷貝數(shù)分別為106copies/μL、107copies/μL。結(jié)論:從抗酸染色陽性痰涂片刮削物中擴增分枝桿菌rpoB基因理論上可行,但在實際操作中尚存在一些問題有待進(jìn)一步探索。抗酸染色涂片制作過程對基因提取及擴增的影響,痰涂片刮屑物基因組提取過程中多次離心造成的DNA量部分丟失均可能是本實驗失敗需要考慮的因素之一。
[Abstract]:Objective: to study the method of direct amplification of Mycobacterium rpoB gene from acid-fast positive sputum smear scraper. Methods: (1) primer Primer1:5'-CGTACGGTCGGCGAGCTGATC-3', and Primer2:5'-AGGGGTTTCGATC GGGCACATC-3'; were used. The rpoB gene of 18 clinical isolates of Mycobacterium tuberculosis was amplified by Primer3:5'-TGTTCTTCAAGGAGAAGCG-3', and Primer4:5'-TCGTCGGCGGTCAGGTA-3' to detect the rationality of primer design. (2) ordinary PCR,Touchdown PCR, nested PCR, was used respectively. The genomic DNA of 80 cases of mycobacterium were amplified by rpoB gene amplification by secondary PCR. (3) the genomic DNA of 80 samples of acid-fast sputum scraper was detected by real-time fluorescence quantitative PCR. (4) Real-time quantitative PCR and PCR (Primer1 and Primer2,) were performed on 18 clinical isolates genomic DNA 100101102103104105106107 times dilution. Primer3 and Primer4) to compare their detection limits. Results: (1) two pairs of primers had bright target bands for amplification of rpoB gene from 18 clinical isolates of Mycobacterium tuberculosis. It was confirmed by sequencing. (2) there were no target bands in 80 sputum smear scraping genomic DNA amplification. (3) in 80 sputum smear scraper genomes, the copy number of DNA in 20 cases was 102~103copies/ 渭 L. The copy number of 60 DNA samples was 104~105copies/ 渭 L. (4) the highest dilution times of rpoB gene amplified by two sets of primers were 104103 times, respectively, and the corresponding copy number of fluorescence quantitative PCR detection was 106copies/ 渭 L ~ (107) copies/ 渭 L. Conclusion: it is theoretically feasible to amplify the rpoB gene of Mycobacterium from acid fast stain positive sputum smear scraper, but there are still some problems to be explored in practice. The effect of acid-fast smear preparation on gene extraction and amplification. The partial loss of DNA caused by multiple centrifugation during genomic extraction of sputum scraps may be one of the factors to be considered in the failure of this experiment.
【作者單位】： 廣州醫(yī)科大學(xué);廣州市胸科醫(yī)院結(jié)核科 國家呼吸重點實驗室;
【基金】：廣東省科技計劃項目(編號:2011B061300085) 廣州市醫(yī)藥衛(wèi)生科技重點項目(編號:201102A212009)
【分類號】：R521

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