Real-time PCR快速检测钩端螺旋体方法的建立

Rapid detection of pathogenic leptospira spp by quantitative real-time PCR

目的:采用新型TaqMan探针建立检测钩端螺旋体的实时荧光定量PCR。方法:根据钩端螺旋体16s rDNA设计的一对引物及探针,以钩体标准菌株的核酸为模板,在荧光定量PCR检测仪(ABI 7500)建立实时荧光定量PCR。结果:建立的定量标准曲线的循环CT值与模板拷贝数呈现良好的线性关系(r=0.998),与普通PCR相比,荧光PCR的灵敏度是其1000倍,检测其他细菌DNA,未检测到信号。结论:本研究中检测钩端螺旋体的实时荧光定量PCR具有高度的特异性和敏感性,特别适合检测样本中微量的钩端螺旋体。

Objective： To establish quantitative real-time PCR method for the detection of leptospira spp by using new TaqMan probe.Methods： According to the 16s rDNA to design a pair of primers and probes,and then take the nuclear acid of standard strain of leptospira spp as template to set up quantitative real-time PCR on detector（ABI 7500）.Results： The relationship between the values of threshold cycle（CT） and the DNA copy number was found to be linear（r=0.998）.The sensitivity of real-time PCR was about 1000 times of the convention PCR when used to detect Leptospira DNA,accompanied by high species-specificity.Conclusion： This results suggest that the quantitative real-time PCR is hightly specific and sensitive for detection of Leptospira,especially useful for detection of tiny leptospira spp of samples.