Sensitive detection of viable Listeria monocytogenes by reverse transcription-PCR
Sensitive detection of viable Listeria monocytogenes by reverse transcription-PCR
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DOI：
10.1128/aem.63.11.4441-4448.1997
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发表时间：
1997-11-01
影响因子：
4.4
通讯作者：
Juneja, VK
中科院分区：
文献类型：
--
作者：
Klein, PG;Juneja, VK
针对PCR检测污染食品中致病菌时，扩增非活细胞DNA而导致假阳性的问题，建立了一种基于逆转录-PCR（RT-PCR）扩增mRNA的单增李斯特菌活细胞特异性检测方法。检测单核细胞增多症基因lap、hly和prfA，以确定用于RT-PCR扩增的合适靶标。分离单核细胞增生症活菌，经DNase酶处理后，分别用RT-PCR和PCR方法扩增其RNA，用地高辛标记的基因探针进行Southern杂交检测，结果表明，这3个基因的表达水平存在明显差异，lap基因为建立活菌特异性检测方法提供了良好的靶基因。富集1小时后，从纯培养物中检测到371-bp lap特异性产物，灵敏度为约10至15 CFU/ml。1小时后，713-bp lly特异性扩增子的检测灵敏度降低约4，000倍，而508-bp prfA产物的检测显示出最低水平的灵敏度，直到5小时富集期后才观察到检测，lap mRNA的扩增对L.单核细胞增多症。总的来说，该试验可在约54 h内完成。结果表明，RT-PCR扩增的mRNA可作为一种灵敏的检测活李斯特菌的方法。单核细胞增多症，并表明这种方法可能证明是有用的，在即食，冷藏肉制品中检测这种病原体。
Detection of pathogens in contaminated food products by PCR can result in false-positive data due to the amplification of DNA from nonviable cells, A new method based on reverse transcription-PCR (RT-PCR) amplification of mRNA for the specific detection of viable Listeria monocytogenes was developed, The expression of three L. monocytogenes genes, lap, hly, and prfA, was examined to determine a suitable target for amplification of RT-PCR, Total RNA from L. monocytogenes was isolated, and following DNase treatment, the RNA was amplified by both RT-PCR and PCR with primers specific for the three genes, Amplicon detection was accomplished by Southern hybridization to digoxigenin-labeled gene probes, The levels of expression of these three genes differed markedly, and the results indicated that the lap gene would provide a good target for development of a specific method for detection of viable L. monocytogenes based on RT-PCR amplification, After a l-h enrichment, the 371-bp lap-specific product was detected with a sensitivity of ca, 10 to 15 CFU/ml from pure culture, Detection of the 713-bp hly-specific amplicon was ca, 4,000 times less sensitive after 1 h, whereas detection of the 508-bp prfA product showed the lowest level of sensitivity, with detection not observed until after a 5-h enrichment period, The amplification of the lap mRNA was specific for L. monocytogenes. Overall, the assay could be completed in ca, 54 h, The use of RT-PCR amplification for the detection of viable L. monocytogenes was validated in artificially contaminated cooked ground beef, Following a 2-h enrichment incubation, the lap-specific amplification product could be detected in a cooked meat sample that was originally inoculated with ca, 3 CFU/g, These results support the usefulness of RT-PCR amplification of mRNA as a sensitive method for the specific detection of viable L. monocytogenes and indicate that this method may prove useful in the detection of this pathogen in ready-to-eat, refrigerated meat products.