一步法逆转录荧光定量PCR检测食源性沙门氏菌

doi:10.7506/spkx1002-6630-201312038

食品科学

一步法逆转录荧光定量PCR检测食源性沙门氏菌

周晓清，李苗云，刘杰，赵改名*，柳艳霞，田玮，黄现青，高晓平

河南省肉制品加工与质量安全控制重点实验室，河南农业大学食品科学技术学院，河南郑州 450002

出版日期:2013-06-25 发布日期:2013-06-17

One-Step Real-Time Reverse Transcription PCR for Detection of Food-Borne Salmonella

ZHOU Xiao-qing，LI Miao-yun，LIU Jie，ZHAO Gai-ming*，LIU Yan-xia，TIAN Wei，HUANG Xian-qing，GAO Xiao-ping

Henan Key Laboratory of Meat Processing and Quality Safety Control, College of Food Science and Technology, Henan Agricultural University, Zhengzhou 450002, China

Online:2013-06-25 Published:2013-06-17

摘要/Abstract

摘要：

建立采用一步法逆转录荧光定量聚合酶链式反应(PCR)检测食源性沙门氏菌的检测方法并初步应用。以沙门氏菌的invA基因为目的基因设计引物，优化PCR反应体系和反应条件，同时进行特异性、灵敏度和重复性实验；并用所建立的方法检测人工污染的猪肉样品，通过与传统计数方法的结果对比验证所建立方法的适用性。优化后的逆转录荧光定量PCR检测条件为：42℃、5min，95℃、10s，95℃、5s，67℃、34s；此步骤进行40个循环；上、下游引物的最佳终浓度均为0.16μmol/L。结果表明：该方法的特异性为100%；重复性良好；对沙门氏菌的检测下限为150CFU/mL；检测人工污染食品样品的结果100%符合传统计数结果。建立的检测方法具有快速、灵敏、简单等特点，非常适用于食源性沙门氏菌的检测。

关键词: 沙门氏菌, RNA, 逆转录荧光定量聚合酶链式反应

Abstract:

The objective of this study was to develop and apply a one-step real-time reverse transcription PCR (real-time RTPCR)
assay for the specific detection of Salmonella. A pair of primers was designed using a Salmonella spp. specific 284-bp
fragment of the invA gene (GeneBank ID: AE008832) as the target sequence. The reaction system and conditions of real-time
RT-PCR were optimized. The specificity, sensitivity and repeatability of the RT-PCR method were analyzed. Pork artificially
contaminated with Salmonella were detected using the new method and traditional method, and the results obtained were
compared. The optimal reaction conditions were 42 ℃ for 5 min, 95 ℃ for 10 s, 95 ℃ for 5 s, and then 67 ℃ for 34 s for 40
cycles; both the optimal concentrations of forward primer and reverse primer were 0.16 μmol/L. Results showed that the new
method was 100% specific. The sensitivity of the method was sufficient to detect 1.5 × 102 CFU/mL of Salmonella in pure
culture with good stability and repeatability. The results of detection of artificially contaminated foods were in accordance
with those obtained with the traditional method. This method could prevent RNAnase effectively and it was proved to be a
simple method for the detection of food-borne Salmonella.

Key words: Salmonella, RNA, real-time reverse transcription polymerase chain reaction (real-time RT-PCR)

周晓清，李苗云，刘杰，赵改名*，柳艳霞，田玮，黄现青，高晓平. 一步法逆转录荧光定量PCR检测食源性沙门氏菌[J]. 食品科学, doi: 10.7506/spkx1002-6630-201312038.

ZHOU Xiao-qing，LI Miao-yun，LIU Jie，ZHAO Gai-ming*，LIU Yan-xia，TIAN Wei，HUANG Xian-qing，GAO Xiao-ping. One-Step Real-Time Reverse Transcription PCR for Detection of Food-Borne Salmonella[J]. FOOD SCIENCE, doi: 10.7506/spkx1002-6630-201312038.

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一步法逆转录荧光定量PCR检测食源性沙门氏菌

One-Step Real-Time Reverse Transcription PCR for Detection of Food-Borne Salmonella

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