军团菌双重PCR检测方法的建立及应用

Establishment and application of duplex PCR assay for detecting Legionella

目的建立双重PCR以检出环境水体中的军团菌。方法设计2对引物,分别扩增军团菌的5SrRNA和Mip基因,扩增片段长各为10 4bp和996bp。结果该方法检测军团菌的灵敏度为5 8×102 cfu/ml, 6株标准嗜肺军团菌均扩增出104bp和996bp两条带, 4株非嗜肺军团菌均扩增出104bp条带, 4株非军团菌无条带;检测71份环境水样, 5份出现2条条带, 2份可见104bp条带,阳性率为7 0%。结论该方法快速、灵敏、特异,为水体中的嗜肺军团菌检测提供了有效方法。

Objective:To establish a duplex PCR assay to detect Legionella in environmental water.Methods:Two different sets of oligonucleotide primers were simultaneously used to amplify 104 bp from 16SrRNA gene and 996 bp from Mip gene.Results:The sensitivity of this assy was 5.8×~102 cfu/ml ,104 bp and 996 bp fragments could be amplified from 6 reference Legionella pneumophila strains and 104 bp from non-Legionella pneumophila strains,no DNA product was observed in 4 non-Legionella strains .Of 71 water samples,104 bp and 996 bp fragments were observed in 5 samples,only 104 bp fragment in 2 samples,the positive rate was 7.0%.Conclusion:This assay has high sensitivity and specificity for detecting Legionella pneumophila in waters . [