flaB-PCR在钩端螺旋体病检测中应用  

Application of flaB-PCR to leptospirosis detection

作　　者：王兆芬[1] 蒋秀高[2] 李秀文[2] 聂一新[2] 顾黎莉[3] 耿排力[1] 

机构地区：[1]青海医学院公共卫生系流行病教研室,西宁810001 [2]中国疾病预防控制中心传染病预防控制所 [3]安徽省疾病预防控制中心

出　　处：《中国公共卫生》2006年第3期291-293,共3页Chinese Journal of Public Health

基　　金：国家863计划项目(2003AA223030)

摘　　要：目的探讨flaB-PCR对组织标本中钩端螺旋体检测的可行性,为钩端螺旋体流行病学调查提供一种快速、有效的技术手段。方法根据黄疸出血型钩体赖株高度保守序列flaB设计一对引物,用PCR方法对实验动物标本及疫区动物标本中的钩端螺旋体DNA进行flaB基因片断扩增,扩增产物用琼脂糖凝胶电泳进行检测。结果该引物可特异性扩增钩端螺旋体DNA,而对本实验所用其它细菌均不扩增。肾组织中含10条钩端螺旋体经flaB-PCR扩增后,琼脂糖凝胶电泳可以目测到扩增产物。26份人工感染钩端螺旋体的动物脏器标本,flaB-PCR扩增阳性10份,阳性率38.46%;细菌分离阳性2份,阳性率7.69%,2种方法比较差异有统计学意义(χ2=6.93,P<0.01)。疫区70份蛙肾标本,分离细菌8株,阳性检出率11.43%;flaB-PCR扩增产物琼脂糖凝胶电泳阳性14份,阳性检出率20%。细菌分离阳性的标本flaB-PCR均为阳性。结论flaB-PCR灵敏、特异、快速,是钩端螺旋体检测的有效方法,可用于钩体病疫情监测和流行病学调查。Objective To study the feasibility of flaB-PCR in the detection of leptospira existed in animal tissues and to establish a rapid and useful technique for the use of epidemiological investigation of leptospirorosis, which better than those methods traditionally used. Methods A pair of oligonucleotide primers of flaB were synthesized according to the sequence of flaB, which high conserved in Lai strain of leptospira icterohaemorrhagiae, the DNA of leptospira in experimental animal samples and in frog kidney specimens were detected by PCR with this pair of flaB primers. Results The DNA of leptospira interrogans could be amplified specifically by the primers. Whereas, the DNA of other bacteria used in this study had no amplification at all. 10 leptospira in samples could give a positive PCR result on 1.5% agarose gel electrophoresis. 10 of 26 tissues of experimental animals infected with leptospira were positive by flaB-PCR, the positive rate was 38.46 %. However only 2 strains were isolated from them and with the positive rate of 7.69 %, the difference between flaB-PCR and isolation was sig- nificant（X^2 = 6.63, P〈 0.01 ）. 70 specimens of frog kidney from Anhui province were tested by flaB-PCR and isolation synchronously. 8 of them were positive by culure and 14 by PCR, the positive rate was 11.43 % and 20 % respectively. Conclusion flaB-PCR is a high sensitive, specific and rapid detecting mean, which can be used as a powerful tool in surveillance of epidemic situation and epidemiological investigation of leptospirosis.

关 键 词：flaB基因 聚合酶链反应 钩端螺旋体 

分 类 号：R514.4[医药卫生—内科学]