聚合酶链反应和地高辛标记的核酸杂交技术用于钩端螺旋体的检测

目的为钩端螺旋体快速诊断和流行病学调查建立一种比较理想的方法.方法根据钩端螺旋体赖株DNA合成一对flaB引物,用PCR技术对钩端螺旋体菌株、疫区现场动物标本等进行flaB基因扩增,用地高辛（DIG）标记flaB基因探针,用琼脂糖凝胶电泳和斑点杂交技术进行检测.结果纯化钩端螺旋体DNA 5pg经flaB-PCR扩增后,琼脂糖凝胶电泳可以目测.用DIG标记的flaB探针可以检测到5fg及以下的DNA扩增产物.疫区70份蛙肾标本,分离细菌8株,阳性率11.43%.flaB扩增阳性14份,阳性率20%;DIG标记探针斑点杂交检测,阳性19份,阳性率为27.14%.结论PCR-斑点杂交是一种灵敏、特异、快速的钩端螺旋体检测方法,既可用于快速检测和早期诊断,也可用于疫情监测和流行病学调查.

The detection of Leptospira interrogans by polymerase chain reaction and hybridization of nucleic acid with digoxiugenin-labled flaB probe

Objective To establish a promising and powerful technique for early diagnosis and epidemiological investigation on leptospirosis. Methods flaB of Leptospira was amplified by polymerase chain reaction (PCR) with the pair of oligonucleotide primers of flaB and flaB probe was labeled with digoxiugenin.70 kidney specimens collected from epidemic area of leptospirosis were detected by isolation, PCR or Dot blotting respectively. Then amplification products were analyzed by agarose gel electrophoresis and Dot hybridization. Results Results showed that 5 pg of purified DNA of leptospira could lead to a positive PCR by agarose gel electrophoresis and 5 fg by digoxiugenin-labled (DIG) specific probe hybridization. Among 70 samples of frog kidneys, 8 by culture, 14 by PCR and 19 by Dot blotting were positive,with positive rates was 11.43% ,20% and 27.14% respectively. Conclusion flaB-PCR combined with Dot blotting of flaB probe labeled with DIG seemed a high sensitive,specific and rapid technique both in early diagnosis of leptospirosis and in epidemiological investigation of Leptospira.