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目的：针对目前结核性疾病实验室诊断的局限性，探索一种更为敏感和特异的结核分枝杆菌DNA检测新方法。方法：选取 10 株江苏地区流行的结核分支杆菌（MTB）菌株，选取临床其他常见菌株及分枝杆菌菌株作为对照组，分别提取DNA作为随机引物的模板。参考国内、外文献设计12 条随机引物，并分别对MTB 及对照菌株进行单个引物随机扩增，2%的琼脂糖凝胶电泳对扩增产物进行分离并切胶纯化，通过TA克隆将纯化片段连接到质粒pEASYTM-T5 Zero 并进行测序，通过BLAST-nr 比对验证是否为MTB DNA片段。按照所确定的MTB 片段序列，在其内部设计、合成一对特异性引物。用此特异性引物扩增对应的随机引物扩增产物，获得MTB 特异性条带图谱。并将该方法检测的敏感性和特异性与临床上常用的real-time PCR进行比较。结果：经 BLAST-nr比对，随机引物IS986F，S535 及IS986R 扩增的条带与MTB DNA 有高度同源性（均为99%）。随机引物IS986F、S535 和IS986R分别联合其特异性引物可以检测稀释105倍、105倍和103倍的MTB DNA，其特异性分别为100%、90%和80%。常规 real-time PCR可检测出稀释104倍的MTB DNA。结论：随机引物IS986F联合其特异性引物检测结核分枝杆菌的灵敏度和特异性优于S535、IS986R两组，特异性为100%，且灵敏度优于常规real-time PCR 法。

英文摘要:

Objective:To explore a more sensitive and specific molecular biology method for the diagnosis of tuberculosis disease, on account of the limitation of currently laboratory diagnosis of tuberculosis.Methods:Ten strains of Mycobacterium tubeculosis (2× 105-2× 106/ml) being popular in Jiangsu area were extracted. DNA was diluted according to 1:10 series dilution as templates to evaluate sensitivity. Specificity were evaluated using the related bacteria and Mycobacterium as control. Referring to the relevant domestic and foreign literature, twelve randomprimers were selected for RAPD (Random Amplified Polymorphic DNA). 12 tubes of RAPD products were run by electrophoresis in 2%agarose gel stained with DuRed and visualized in UV light. The high brightness and clear bands were selected and excised fromthe 2%agarose gel by means of Gel-out kit. The products were extracted and cloned into the TA cloning vector (pEASYTM-T5 Zero) and sequenced. The sequences obtained were blasted against the NCBI database to verify whether a MTB DNA fragment or not. A pair of MTB specific primers were designed and synthesized according to the determined sequence. The specific primers based PCR were performed using RAPD products as temples. The specific MTB bands were showed by PAGE. The sensitivity and specificity were compared between this novel method and routine real-time PCR test in our hospital.Results:Three DNA sequences from bands amplified with the random primer IS986F, S535 and IS986R and MTB sequence from BLAST-nr were highly homologous. MTB DNA diluted in 1:105 can be detected by random primer IS986F combined its internal specific primers with 100% of specificity. MTB DNA diluted in 1:105 can be detected by random primer S535 combined its internal specific primers with 90.0% of specificity. While MTB DNA diluted in 1:103 can be detected by random primer IS986R combined its internal specific primers with 80% of specificity. MTB DNA diluted in 1:104 can be detected by routine real-time PCR.Conclusion:A new method for the detection of MTB based randomly combined specific PCRhas been developed and has more sensitivity than routine real-time PCRwith 100%of specificity.

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