肉及肉制品中沙门氏菌活细胞的PCR检测研究

食品科学 ›› 2012, Vol. 33 ›› Issue (16): 199-203.

肉及肉制品中沙门氏菌活细胞的PCR检测研究

祝儒刚1，宋立峰2

1.辽宁大学轻型产业学院，辽宁省食品生物加工工程技术研究中心，沈阳市食品生物加工与质量控制技术重点实验室 2.辽宁经济职业技术学院

收稿日期:2011-11-11 修回日期:2012-07-08 出版日期:2012-08-25 发布日期:2012-09-07
通讯作者: 祝儒刚 E-mail:zrg_luck@163.com
基金资助:
辽宁大学青年科学基金

Detection of Viable Salmonella Cells in Meat and Meat Products by PCR

Received:2011-11-11 Revised:2012-07-08 Online:2012-08-25 Published:2012-09-07

摘要/Abstract

摘要： 将荧光染料叠氮溴化丙锭(propidium monoazide，PMA)与普通聚合酶链式反应(polymerase chain reaction，PCR)结合，通过对PMA的曝光时间、浓度进行优化，确定PMA-PCR区别死活细胞的最佳条件，并制作活细胞定量标准曲线，建立肉及肉制品中沙门氏菌活细胞的PMA-PCR检测方法。结果表明：使插入死细胞DNA中的PMA活化并且光解溶液中游离PMA的最佳曝光时间为15min；不抑制沙门氏菌活细胞DNA扩增的最大PMA质量浓度为10μg/mL；完全抑制热致死细胞DNA扩增的最小PMA质量浓度为4μg/mL。经PMA处理，含有不同比例的沙门氏菌热致死细胞和活细胞的混合液中活的沙门氏菌能够通过PCR被选择性的检测，最小检测限为20CFU/PCR。而且，经研究发现在20～2×105CFU/PCR范围内，电泳条带相对荧光强度与活细胞数的对数具有线性关系。采集30份肉及肉制品样品，利用PMA-PCR方法检测出两份生肉样品中存在沙门氏菌，经过6h的富集培养后的活菌浓度分别为2.5×103CFU/mL和3.4×103CFU/mL。

关键词: 肉及肉制品, 沙门氏菌, 活细胞, PMA-PCR

Abstract: A method for the selective detection of viable cells of Salmonella in meat and meat products was developed using propidium monoazide (PMA) staining in combination with conventional PCR (PMA-PCR). Exposure time and PMA concentration were optimized to find optimal PMA-based PCR conditions for distinguishing between dead and viable cells. The results showed that the optimal light exposure time to activate PMA in dead cells and to photolyze free PMA in solution was 15 min. PMA at a dose of 10 μg/mL or less could not inhibit PCR amplification of DNA derived from viable cells of Salmonella. The minimum concentration of PMA to completely inhibit PCR amplification of DNA derived from heat-killed cells was 4 μg/mL. After PMA treatment, viable Salmonella cells in a mixture containing different proportions of viable and dead cells could be selectively detected by PCR, and the minimum detection level was 20 CFU/PCR. An excellent linear relationship was observed between relative fluorescent intensity of DNA bands and lgCFU in the range of 20－2 × 105 CFU/PCR. Among 30 meat and meat product samples, two samples were detected by the developed PMA-PCR method as being positive after 6 h enrichment with average amounts of 2.5 × 103 CFU/mL and 3.4 × 103 CFU/mL for viable Salmonella cells, respectively.

Key words: meat and meat products, Salmonella, viable cells, propidium monoazide-based polymerase chain reaction (PMA-based PCR)

中图分类号:

TS201.6

祝儒刚1，宋立峰2. 肉及肉制品中沙门氏菌活细胞的PCR检测研究[J]. 食品科学, 2012, 33(16): 199-203.

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