应用实时荧光PCR检测各种环境标本中的嗜肺军团菌

Detection of Legionella pneumophilain various environmental samples by real-time PCR

目的评价实时荧光PCR在检测各种环境标本中嗜肺军团菌的应用效果。方法选择针对mip基因的引物和探针,优化实时荧光PCR的反应条件,并对嗜肺军团菌和其他非嗜肺军团菌进行检测,验证方法的敏感性、特异性和重复性。将实时荧光PCR检测各种环境标本中嗜肺军团菌的效果与传统培养法进行比较。结果实时荧光PCR法最低检测限达6CFU/mL。该方法特异性好,嗜肺军团菌呈现阳性结果,而非嗜肺军团菌均为阴性结果。重复性好,Ct值变异系数较小。从菌株核酸的提取至检测完成仅需2h左右。实时荧光PCR法和传统培养法的检测嗜肺军团菌的阳性率差异有统计学意义(P<0.05),实时荧光PCR检测敏感性优于传统培养法。结论实时荧光PCR具有较好的敏感性、特异性和快速的特点,适于各种环境标本中嗜肺军团菌污染状况调查及应急事件的快速检测。

Objective To evaluate the effect of the detection of Legionella pneumophila in various environmental samples by re-al-time PCR.Methods A pair of primer and probe were selected to identify the mip gene of Legionellapneumophila,and the PCR reaction conditions were optimized.By using this method,Legionella pneumophila and non- Legionella pneumophila were detec-ted.The sensitivity,specificity and reproducibility of the assay were tested and verified.The detection of Legionella pneumophila in various environmental samples by real-time PCR was compared with the detection by traditional culture.Results The lowest de-tectable limit of real-time PCR was 6 CFU/mL.This assay had high specificity for detecting Legionella pneumophila but not to non-Legionella pneumophila.The reproducibility of this assay was high,the coefficient of variation of Ct values was low.The whole process from extraction of genomic DNA of strains to assessment of the results could be done in about 2 h.There was statis-tically significant between the positive rate which was detected by real-time PCR and detected by traditional culture（P〈0.05）,real-time PCR was more sensitive than traditional culture for the detection of Legionella pneumophila.Conclusion The real-time PCR provides a sensitive,specific and rapid method for detection of Legionellapneumophila.It is helpful for the rapid detection of envi-ronment source of Legionella pneumophila pollution and emergency.