四种单增李斯特菌常用核酸检测方法的评价与应用

Evaluation and application of four nucleic acid detection methods for Listeria monocytogenes

目的应用聚合酶链式反应(PCR)、实时荧光PCR(Real-time PCR)、环介导等温扩增(LAMP)和多交叉置换扩增(MCDA)4种核酸检测方法,对单增李斯特菌进行检测,探索特异、灵敏、高效,且适用于肉类样本中单增李斯特菌检测的核酸检测方法。方法比较文献报道的以单增李斯特菌特异性基因lmo0733作为靶标的4种核酸检测方法,评价这4种方法在特异性、灵敏度、检出速率方面的差异,并应用于单增李斯特菌模拟样本和猪肉样本中单增李斯特菌的检测。结果传统PCR、Real-time PCR、LAMP和MCDA方法都能准确检测单增李斯特菌,其中MCDA方法检测下限为10 fg/反应,且能在30 min内完成对样本核酸的检测,灵敏度和检出速率优于其它3种核酸诊断方法;MCDA方法对于模拟样本和实际样本的检测效率优于其它3种核酸检测方法和传统分离培养方法,能够提高样本检测的阳性率。结论MCDA方法作为一种特异、灵敏和高效的核酸检测方法,可用于肉制品中单增李斯特菌检测时的快速筛查,并能提高传统分离培养方法对标本中单增李斯特菌的阳性检出率。

Polymerase chain reaction(PCR),real-time fluorescence polymerase chain reaction(Real-time PCR),loop-mediated isothermal amplification(LAMP)and multiple cross displacement amplification(MCDA)were used to detect Listeria monocytogenes in pork samples,then a rapid,sensitive and specific nucleic acid detection method was selected for purpose of reducing detection time and increasing detection rate.According to the references,four nucleic acid methods based on the specific gene lmo0733 of L.monocytogenes were used for detection of the pathogen in the artificially contaminated samples and the raw meat samples.The reaction rate,detection sensitivity and detection specificity of four methods were compared.Our data indicated that the four nucleic acid methods could accurately detect L.monocytogenes in pure nucleic acid,artificially contaminated samples and raw pork meat samples.The reaction rate(30 minutes per whole process)and sensitivity with limit of detection(LOD)of 10 fg of DNA template per reaction for MCDA method are better than that of the other three methods.In conclusion,MCDA assay could be used as a rapid,sensitive and efficient method for detection of L.monocytogenes in meat product.