抗酸染色阳性痰涂片刮削物扩增分枝杆菌rpoB基因的初步探索

发布时间：2018-12-11 05:45

【摘要】：目的:探讨直接从抗酸染色阳性痰涂片刮削物中扩增分枝杆菌rpoB基因的方法。方法:(1)使用引物Primer1:5′-CGTACGGTCGGCGAGCTGATC-3′,与Primer2:5′-AGGGGTTTCGATC GGGCACATC-3′;Primer3:5′-TGTTCTTCAAGGAGAAGCG-3′,与Primer4:5′-TCGTCGGCGGTCAGGTA-3′分别扩增18株临床分离结核分枝杆菌的rpoB基因,以检测引物设计的合理性。(2)分别采用普通PCR、Touchdown PCR、巢式PCR、二次PCR对直接从3+~4+抗酸染色痰涂片刮削物中提取的80例分枝杆菌基因组DNA进行rpoB基因扩增。(3)对80例抗酸染色痰涂片刮削物基因组DNA行实时荧光定量PCR以检测其拷贝数。(4)对18例临床分离菌株基因组DNA 100、101、102、103、104、105、106、107倍稀释物行实时荧光定量PCR与普通PCR(Primer1与Primer2,Primer3与Primer4),以比较二者检出限。结果:(1)两对引物对18株临床分离结核杆菌rpoB基因扩增均有明亮目的条带,并经测序证实。(2)80例痰涂片刮削物基因组DNA扩增均未见目的条带。(3)80例痰涂片刮削物基因组中,其中20例DNA的拷贝数为102~103copies/μL,60例DNA的拷贝数为104~105copies/μL。(4)两组引物能够扩增出rpoB基因的最高稀释倍数分别为104、103倍,其对应的荧光定量PCR检测拷贝数分别为106copies/μL、107copies/μL。结论:从抗酸染色阳性痰涂片刮削物中扩增分枝杆菌rpoB基因理论上可行,但在实际操作中尚存在一些问题有待进一步探索。抗酸染色涂片制作过程对基因提取及扩增的影响,痰涂片刮屑物基因组提取过程中多次离心造成的DNA量部分丢失均可能是本实验失败需要考虑的因素之一。
[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.
【作者单位】：广州医科大学;广州市胸科医院结核科国家呼吸重点实验室;
【基金】：广东省科技计划项目(编号:2011B061300085) 广州市医药卫生科技重点项目(编号:201102A212009)
【分类号】：R521

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