应用PCR技术检测致病性钩端螺旋体

唐雨德; 周东明; 陈永红; 李越希; 陶开华

应用PCR技术检测致病性钩端螺旋体

Detection of all serogroups of pathogenic leptospires epidemic by PCR

摘要

摘要:
目的建立一种能特异检测我国致病性钩端螺旋体所有血清型的PCR方法。
方法从Genbank中选取钩体23SrDNA序列, 设计一对种特异性引物。通过Blast验证引物的特异性和广谱性后, 用PCR检测我国流行的所有18个血清群钩体代表株, 观察其敏感性和特异性。探讨简便的样品处理方法, 并对模拟标本进行检测。
结果 18个血清群的25株钩体均出现单一482bp的特异性扩增产物, 而双曲钩体及其他螺旋体、微生物、空白对照均无任何DNA扩增条带。PCR单一反应最小检出钩体数为8条。用煮沸法、试剂盒、SiO高盐吸附法及氯仿苯酚混合法处理样品对PCR结果无明显影响。对模拟标本的检测符合临床实际要求。
结论 PCR方法是一种灵敏度高、特异性强、检测谱广的快速检测钩体的有效方法。

Abstract:
Objective To develop a polymerase chain reaction(PCR)for specifically detecting all serogroups of leptospira interrogans epidemic in China.
Methods A pair of species-specific primers were designed from an 23s rDNA sequence of leptospira interrogans from Genbank.After the primers were identified they were specific and universal by Blast, 18 serogroups of Leptospira interrogans and other pathohens or microbes were detected by PCR.Its sensitivity and specifity were tested.The different sample preparating method was developed, and sera and water samples were tested.
Results A specific amplification product with 482 bp length which could be found from eighteen serogroups of leptospira interrogans by PCR, and agarose gel electrophoresis was demonstrated to have a sensitivity of detecting DNAs of eight L.Interrogans.None of DNA amplification products was found from other microbes.
Conclusion PCR was a rapid, specific and sensitive and universal method for detecting leptospira in the epidemic regions.

HTML全文

参考文献(6)

施引文献

资源附件(0)