PCR结合索氏转移杂交在结核病诊断中的应用

Specific detection of mycobacterium tuberculosisin clinical material by PCR and southern blot.

利用Ｈｅｒｍａｎｓ引物扩增插入序列ＩＳ９８６所得之２４５ｂｐ片断是一结核分支杆菌复合体特异性片断，采用随机引物法将地戈辛标记该片断。将纯化结核分支杆菌ＤＮＡ经聚合酶链反应（ＰＣＲ）扩增，索氏转移后与该探针杂交，检测灵敏度可达１ｆｇＤＮＡ。通过对７９例痰标本、１４例结核性胸腔积液及２６例结核性关节腔积液的ＰＣＲ和ＤＮＡ索氏杂交试验比较，认为采用２４５ｂｐ探针，将ＤＮＡ索氏转移杂交与ＰＣＲ体外扩增相结合，不仅提高了结核病基因诊断的敏感性，同时也提高了诊断的特异性。

n amplified fragment of 245 base pairs from IS-986 sequence hybridized specifically with DNA of M·tuberculosis complex strains. The fragment was la-belled with digoxigenin. after amplification by PCRand southern blot hybridization with the dig-labelledprobe,the limit of detection of purified genomic M. tu-berculosis DNA amounted to 1 fg. We detected spu-tum,pleural and synovial fluid with active tuberculosisby PCR and southern blot, and the sensitivities were81.0%, 64.2%and 57.7%,respectively.From theresult of the experiment,it is a very useful method fordiagnosis of tuberculosis by combining PCR withSouthern blot.