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Short communication: Quantitative PCR coupled with sodium dodecyl sulfate and propidium monoazide for detection of culturable Escherichia coli in milk

文献类型： 外文期刊

作者： Dong, Lei 1 ; Liu, Huimin 1 ; Meng, Lu 1 ; Xing, Mengru 1 ; Lan, Tu 1 ; Gu, Mei 1 ; Zheng, Nan 1 ; Wang, Cheng 4 ; Chen, He; 1 ;

作者机构： 1.Chinese Acad Agr Sci, Inst Anim Sci, Minist Agr & Rural Affairs, Lab Qual & Safety Risk Assessment Dairy Prod, Beijing 100193, Peoples R China

2.Minist Agr & Rural Affairs, Milk & Dairy Prod Inspect Ctr, Beijing 100193, Peoples R China

3.Qingdao Agr Univ, Coll Food Sci & Engineer, Qingdao 266109, Shandong, Peoples R China

4.Xinjiang Acad Agr Sci, Inst Qual Stand & Testing Technol, Urumqi 830091, Peoples R China

关键词： propidium monoazide; sodium dodecyl sulfate; Escherichia coli; quantitative real-time PCR; internal amplification control

期刊名称：JOURNAL OF DAIRY SCIENCE （ 2020影响因子：4.034； 五年影响因子：4.354 ）

ISSN： 0022-0302

年卷期： 2019 年 102 卷 8 期

页码：

收录情况： SCI

摘要： Escherichia coli has been frequently reported as a major foodborne bacterium contaminating raw milk or pasteurized milk. Therefore, the aim of this study was to explore a quantitative real-time PCR (qPCR) technique combined with sodium dodecyl sulfate (SDS) and propidium monoazide (PMA) to detect culturable E. coli in milk. An internal amplification control was also added into this reaction system as an indicator of false-negative results. The inclusivity and exclusivity of the primers were tested using DNA from 7 E. coli and 14 other bacterial strains. The concentrations of SDS and PMA were determined according to plate counts and quantitative cycle values of qPCR, respectively. A standard curve was established using series diluted E. coli DNA. The reliability and specificity of this method were further determined by the detection of E. coli in spiked milk. The results showed that the optimal concentrations of SDS and PMA were 100 mu g/mL and 40 mu M, respectively. A standard curve with a good linear relationship (coefficient of determination = 0.997; amplification efficiency = 100.5%) was obtained. Compared with conventional PCR and PMA-qPCR, the SDS-PMA-qPCR assay was more specific and sensitive in culturable E. coli detection. Therefore, we evaluated and improved the SDS-PMA-qPCR method for detecting culturable E. coli in milk.

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