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Evaluation of DNA Extraction Methods for Culture-Independent Real-Time PCR-Based Detection of Listeria monocytogenes in Cheese
Food Analytical Methods ( IF 2.667 ) Pub Date : 2019-12-12 , DOI: 10.1007/s12161-019-01686-2
Minarovičová Jana,Véghová Adriana,Kaclíková Eva

Listeria monocytogenes, the causative agent of listeriosis, is a foodborne pathogen with significant public health and economic impacts. The control of pathogen presence in food requires rapid and sensitive methods. Real-time PCR is considered to be a fast and accurate tool for the detection of foodborne pathogens. A crucial step for the success of a culture-independent PCR-based detection is the template DNA extraction. Two open-formula extraction procedures and five kits were used to extract DNA prior to detection of L. monocytogenes in cheese. The extraction procedures were evaluated in quantitative PCR analyses of the total extracted bacterial DNA from cheese samples artificially contaminated with L. monocytogenes. The results of the study suggested that all seven extraction procedures can be used to obtain intact and amplifiable bacterial DNA, although with different L. monocytogenes detection limits in four types of cheese. PowerFood Microbial DNA Isolation Kit and DNeasy Mericon Food Kit performed the best with detection limits of 2.1 × 102 to 4.7 × 102 CFU/g in all analysed cheese samples. No differences in PCR detection limits of low numbers were found when different L. monocytogenes serotypes (1/2a, 1/2b, 1/2c and 4b) were used, despite the relatively lower DNA extraction efficiency observed in L. monocytogenes serotype 1/2c strain. Six out of seven evaluated methods demonstrated quantitative response with linear calibration lines and quantification limits of 4.6 × 102 to 9.3 × 103 CFU/g. The results demonstrated that culture-independent sample preparation and total DNA extraction, followed by target-specific DNA amplification have a potential to direct determination of low L. monocytogenes numbers in cheese within hours.

中文翻译:

基于DNA提取方法的奶酪中不依赖培养物的实时PCR检测单核细胞增生李斯特菌的评估

单核细胞增生李斯特菌是李斯特菌病的病原体,是一种食源性病原体,具有重大的公共卫生和经济影响。控制食品中病原体的存在需要快速而敏感的方法。实时PCR被认为是检测食源性病原体的快速准确的工具。成功地进行基于文化的,基于PCR的检测的关键步骤是模板DNA提取。在检测奶酪中的单核细胞增生李斯特氏菌之前,使用两种开放式提取程序和五种试剂盒提取DNA 。在定量PCR分析中评估了提取程序,该定量PCR分析是从人工污染了单核细胞增生李斯特菌的干酪样品中提取的细菌DNA总量研究结果表明,尽管四种奶酪的单核细胞增生李斯特氏菌的检出限不同,但全部七个提取程序均可用于获得完整且可扩增的细菌DNA 。PowerFood微生物DNA分离试剂盒和DNeasy Mericon食品试剂盒 在所有分析的奶酪样品中的检出限为2.1×10 2至4.7×10 2 CFU / g,性能最佳。尽管使用了单核细胞增生李斯特菌血清型(1 / 2a,1 / 2b,1 / 2c和4b),但在不同的单核细胞增生李斯特菌血清型中,PCR检测下限没有差异,尽管在单核细胞增生李斯特菌中观察到相对较低的DNA提取效率血清型1 / 2c菌株。七种评估方法中有六种表现出线性校准线的定量响应,定量限为4.6×10 2至9.3×10 3  CFU / g。结果表明,不依赖培养物的样品制备和总DNA提取,然后进行靶标特异性DNA扩增,有可能在几小时内直接确定奶酪中低单核细胞增生李斯特氏菌数量。
更新日期:2019-12-13
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