聚合酶链反应检测致病性钩端螺旋体DNA的研究

以黄疸出血型ＲＧＡ菌株对所有致病性钩端螺旋体特异的ＤＮＡ克隆片段序列为基础合成聚合酶链反应引物Ｇ１Ｇ２。用该对引物扩增各群型致病性钩端螺旋体微量ＤＮＡ，均获阳性结果，对非致病性钩端螺旋体和其他细菌不扩增。纯化的钩端螺旋体ＤＮＡ５ｐｇ经聚合酶链反应后，琼脂糖凝胶电泳可以目测。用地高辛配基标记的特异性探针可检测到５ｆｇ即相当于１条钩端螺旋体ＤＮＡ量的扩增产物。将该对引物扩增早期钩体病患者血清标本，阳性率为８２％，显著高于ＭＡＴ和ｄｏｔ－ＥＬＩＳＡ阳性率。由此表明，ＰＣＲ技术是灵敏、特异、快速的钩端螺旋体病早期诊断方法，还可用于流行病学调查。

Detection of DNA of Leptospira Interrogans by Polymerase Chain Reaction

Leptospirosis is a severe zoonosis in the world.The methods for detecting leptospira are not sensitive and specific so far,The problems in early diagnosis for leptospirosis remain unsolved.According to the sequence of the DNA specific fragment from RGA strain,the pair of polymerase chain reaction(PCR) oligonucleolide primers were synthesized,named primers G1,G2.PCRs were carried out with these primers for detecting the microquantity of various serovars,serogroups of leptospira. All the DNA of Leptospira interrogans could be amplified by primers G1,G2 and the DNA of Leptospira biflexa and other bacterum had no amplification at all.The samples obtaining 5 pg DNA could give a positive result in the PCR assay by agarose gel electrophresis and obtaining 5 fg DNA by DIG probe hybridization. Detecting sera from early leptospirosis patients by PCR,positive rate was 82% and much more sensitive than by MAT and dotELISA;The results demonstrated that PCR was a very sensitive and specific technique of DNA Amplification,which could be used as a powerful tool in the early diagnosis and epidemiological investiga-tion of leptospirosis.