致病性钩端螺旋体TaqMan Real-timePCR检测技术的建立及其应用

Establishment and application of TaqMan Real-time PCR for the detection of pathogenicLeptospira species

目的建立致病性钩端螺旋体（钩体）TaqManReal-timePCR检测技术。方法以钩体16SrRNA基因的部分片段rrs基因作为靶基因，设计引物、TaqMan探针，PCR产物克隆到pMDl9-T载体，制作标准曲线，建立定量分析质控标准。利用中国15群15型致病性钩体参考菌株、16群21型非致病性钩体参考菌株、50株不同血清群致病性分离株及伯氏疏螺旋体、嗜肺军团菌、肺炎链球菌、脑膜炎奈瑟菌等27株其他常见致病菌检验引物、探针的灵敏性、特异性。将Real-timePCR、普通PCR同时应用于倍比稀释致病性钩体染色体DNA及25份现场鼠肾标本的检测。结果建立、优化致病性钩体Real-time PCR技术，致病性钩体扩增荧光信号阳性，非致病性钩体及其他非钩体菌均无扩增。对于倍比稀释的质粒标准品，Real-timePCR和普通PCR的最低检测下限分别是10copy／μl和10。copy／μl。对于倍比稀释的钩体染色体DNA，两者的最低检测下限分别为：100fg／μl和1ng／μl。25份现场鼠肾标本检测显示，两种方法的检测结果一致。结论以rrs为靶基因建立的Real-timePCR技术，具有较高的灵敏度和特异度，可用于致病性钩体的病原学检测。

Objective To develop and evaluate a TaqMan Real-time PCR method for thedetection of pathogenic Leptospira species. Methods rrs gene of part fragment on 16S rRNA was used to design primers and TaqMan probe. The target gene was cloned into vector pMD19-T in order to make the standard curve and be used for quality control. To determine the specificity and specificity, DNA from Chinese Leptospira strains belonging to 15 pathogenic reference strains, 21 non-pathogenic reference strains, and 50 different serotypes of pathogenic isolates as well as 27 other micro-organisms were included in this study. Eight serial DNA dilutions from pathogenic Leptospira and DNA from 25 kidney tissues were detected by Real-time PCR and conventional PCR simultaneously. Results A Real-time PCR methodology was developed and optimised. All the pathogenic Leptospira gave a positive amplification. Non-pathogenic Leptospira and all the other micro-organisms were not amplified. The plasmid sensitivity of Real-time PCR and conventional PCR were 10 copy/μl and 104 copy/μl respectively. The DNA sensitivity of Real-time PCR and conventional PCR were 100 fg/μl and 1 ng/μl respectively. The kidney tissue detection of the two methods appeared to be exactly the same. Conclusion This research project successfully developed a Real-time PCR methodology with better sensitivity and specificity for the identification of pathogenic Leptospira, using the rrs gene.