荧光定量PCR技术用于模拟临床标本单增李斯特菌检测的研究

Real-time PCR-based method for the detection of Listeria monocytogenes in simulated clinical sample

目的利用荧光定量PCR(real-time PCR)技术,建立针对模拟临床标本中单增李斯特菌快速检测方法。方法以单增李斯特菌特异性基因hlyO的部分片段作为靶基因,克隆到pMD18-T载体,制作标准曲线,以确定方法的检测下限。利用各种血清型单增李斯特菌以及常见致病菌(如副溶血弧菌、霍乱弧菌、沙门菌、O157∶H7大肠杆菌等)验证引物探针的特异性,通过制备模拟粪便和血液感染标本,直接提取DNA进行检测,以达到快速检测的目的。结果采用real-time PCR技术对模拟临床标本检测结果显示,只有单增李斯特菌有荧光信号,其他常见致病菌以及李斯特菌属中的无害李斯特菌均没有荧光信号;由标准曲线可知该方法可以检测到22copy/反应管的目的基因,而粪便中6.35×103CFU/g和血液中7.0×102CFU/ml的细菌含量即可被检出;粪便标本在2h内可出报告,血液标本1.5h即可出报告。结论以hlyO为靶基因的Real-time PCR单增李斯特菌的检测技术具有特异性好、敏感性高、快速易操作等优点,可用于临床标本的快速诊断、疾病监测和疫情暴发的病原调查。

To establish a. real-time PCR method for the rapid detection of Listeria monocytogenes in simulated clinical specimens, a pair of primers and Taq-Man probe were designed based on part of the hlyO gene, and the target gene was cloned into the vector pMD18-T in order to make the standard curve. The specificity of the＇primers and probe were tested by using different serotypes of L. monocytogenes strains and other common pathogenic bacteria. DNA from fecal and blood samples contaminated artificially at different concerntration of L. monocytogenes were extracted directly and tested by real-time PCR. The results of detection were positive for the samples containing L. monocytogenes, but negative for the genome of other bacteria. Standard curve showed that 22 copy L. monocytogenes per reaction could be detected by this method. The lowest detection limit of this method were 6.35 × 10^3 CFU per g of fecal sample and 7.0 × 10^2 CFU per mL of blood sample. It just took 2 hour to get the result for fecal sample and 1.5 hour for blood sample. It is concluded that the Taq-Man real-time PCR technology for detecting L. monocytogenes in simulated clinical specimen shows high specificity, sensitivity, simplicity and celerity. Therefore, this method can be used to detect the samples of patients infected by L. monocytogenes and identify the causes of the food borne list eriosis outbreak.