Detection of Live Salmonella sp Cells in Produce by a TaqMan-Based Quantitative Reverse Transcriptase Real-Time PCR Targeting invA mRNA
Detection of Live Salmonella sp Cells in Produce by a TaqMan-Based Quantitative Reverse Transcriptase Real-Time PCR Targeting invA mRNA
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DOI：
10.1128/aem.02686-08
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发表时间：
2009-06-01
影响因子：
4.4
通讯作者：
Lampel, Keith A.
中科院分区：
文献类型：
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作者：
Gonzalez-Escalona, Narjol;Hammack, Thomas S.;Lampel, Keith A.
食品中的肠道沙门氏菌污染是公众健康的重大问题。当DNA检测方法用于食品分析时，主要关注的问题之一是检测死亡细胞的假阳性结果。为了绕过这一关键问题，建立了一种带有RNA内对照的TaqMan定量实时RT-PCR(qRT-PCR)方法。用INVA RNA标准来确定该方法的检测下限，以及确定指数中期、指数晚期和稳定期细胞中INVA的mRNA水平。该方法的检测下限为每次反应40个拷贝的inva mRNA。指数中期、指数晚期和稳定期肠链球菌的inva mRNA水平分别约为每3CFU 1拷贝、每CFU 1拷贝和每10(3)CFU 4拷贝。菠菜、西红柿、辣椒和沙拉诺辣椒被4种不同的沙门氏菌血清型人工污染，污染水平分别为10(5)和小于10cfu。用定量逆转录聚合酶链式反应和食品和药物管理局的细菌分析手册沙门氏菌培养方法(G.J.Jackson等编辑的W.A.Andrews和T.S.Hammack，在线细菌分析手册，http://www.cfsan.fda.gov/similar to EBAM/BAM-5.html，2007年)对这些食品进行分析。两种方法得到的结果具有可比性。这种qRT-PCR检测只能检测到活的沙门氏菌细胞，从而避免了来自非活细胞的假阳性结果的危险。假阴性(抑制聚合酶链式反应)也通过使用RNA内部对照被排除。这种检测方法可以快速准确地检测到活的沙门氏菌。在菠菜，西红柿，以及胡椒和色拉诺辣椒中。
Salmonella enterica contamination in foods is a significant concern for public health. When DNA detection methods are used for analysis of foods, one of the major concerns is false-positive results from the detection of dead cells. To circumvent this crucial issue, a TaqMan quantitative real-time RT-PCR (qRT-PCR) assay with an RNA internal control was developed. invA RNA standards were used to determine the detection limit of this assay as well as to determine invA mRNA levels in mid-exponential-, late-exponential-, and stationary-phase cells. This assay has a detection limit of 40 copies of invA mRNA per reaction. The levels of invA mRNA in mid-exponential-, late-exponential-, and stationary-phase S. enterica cells was approximately 1 copy per 3 CFU, 1 copy per CFU, and 4 copies per 10(3) CFU, respectively. Spinach, tomatoes, jalapeno peppers, and serrano peppers were artificially contaminated with four different Salmonella serovars at levels of 10(5) and less than 10 CFU. These foods were analyzed with qRT-PCR and with the FDA's Bacteriological Analytical Manual Salmonella culture method (W. A. Andrews and T. S. Hammack, in G. J. Jackson et al., ed., Bacteriological analytical manual online, http://www.cfsan.fda.gov/similar to ebam/bam-5.html, 2007). Comparable results were obtained by both methods. Only live Salmonella cells could be detected by this qRT-PCR assay, thus avoiding the dangers of false-positive results from nonviable cells. False negatives (inhibition of the PCR) were also ruled out through the use of an RNA internal control. This assay allows for the fast and accurate detection of viable Salmonella spp. in spinach, tomatoes, and in both jalapeno and serrano peppers.