Rapid detection of viable Listeria monocytogenes in chilled pork by real-time reverse-transcriptase PCR
Rapid detection of viable Listeria monocytogenes in chilled pork by real-time reverse-transcriptase PCR
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实时逆转录PCR快速检测冷却猪肉中活的单增李斯特氏菌
DOI：
10.1016/j.foodcont.2011.10.025
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发表时间：
2012-05-01
期刊：
影响因子：
6
通讯作者：
Zhou, Guanghong
中科院分区：
文献类型：
--
作者：
Ye, Keping;Zhang, Qiuqin;Zhou, Guanghong
本研究旨在建立一种基于RNA的实时荧光定量PCR（real-time RT-PCR）检测冷却猪肉中单核细胞增生李斯特菌的方法，该方法不需要预富集步骤，并采用基于DNA的实时荧光定量PCR和传统培养方法对该方法进行了验证和评价。对于特异性测试，在所使用的条件下，从与肉类物种相关的41种其他细菌菌株中的任何一种中获得了缺乏扩增信号并且在类似于78.37 ° C处没有Tm峰。用10倍系列稀释法建立的标准曲线的R-2和效率分别为0.995和90.1%，低于DNA法。在纯培养物和人工污染的冷却猪肉样品中，检出限均达10（0）cfu/mL。定量检测结果表明，当样品经过处理（如高压）时，基于RNA的检测方法获得了相对准确的结果，但未经处理，结果与平板计数相比略有偏差。本研究建立的RNA依赖的实时荧光定量RT-PCR方法快速、灵敏，可用于可靠地检测活乳杆菌。冷冻猪肉中的非细胞基因，特别是对于预处理样品。然而，这种方法不能被推荐用于准确地定量单核细胞增生李斯特菌，而只能显示活细胞的存在并近似预测其污染水平。(c)2011爱思唯尔有限公司保留所有权利。
The objective of this study was to develop an RNA-dependent real-time reverse-transcriptase PCR (real-time RT-PCR) method for the detection of Listeria monocytogenes in chilled pork without the need for pre-enrichment steps, and the soundness of the method was simultaneously validated and evaluated by DNA-based real-time PCR and traditional culture methods. For specificity testing, a lack of amplification signals and no Tm peak at similar to 78.37 degrees C were obtained from any of 41 other bacterial strains associated with meat species under the conditions used. The R-2 and efficiency of standard curves constructed by ten-fold serial dilutions of pure L monocytogenes were respectively 0.995 and 90.1%; lower than that of the DNA-based assay. The detection limit was up to 10(0) cfu/mL in both pure culture and in artificially contaminated chilled pork samples. Quantitative detection showed that the RNA-based assay obtained relatively accurate results when samples had undergone treatments (such as high pressure), but without treatment, the results showed a slight deviation compared with plate counts. The RNA-dependent real-time RT-PCR method developed in this study was found to be rapid and sensitive and should be useful for reliable detection of viable L. rnonocytogenes in chilled pork, especially for pre-treated samples. However, this method cannot be recommended to accurately quantify L monocytogenes, but only to show the presence of live cells and approximately predict its level of contamination. (c) 2011 Elsevier Ltd. All rights reserved.