荧光假单胞菌、沙门氏菌和单增李斯特菌多重PCR检测方法的建立

针对肉制品中易污染的荧光假单胞菌、沙门氏菌和单增李斯特菌等有害微生物，通过3 种标准菌株及肉制品建立多重聚合酶链式反应方法，实现肉制品中这3 种菌的同时、快速检测。利用荧光假单胞菌的gyrB基因、沙门氏菌的invA基因和单增李斯特菌的hlyA基因设计3 对特异性引物，在确定引物特异性的基础上，对3 种标准菌株及在冷却肉上过夜富集后进行灵敏度检测。结果表明，该多重聚合酶链式反应方法对于同时检测这3 种有害微生物具有高度的特异性；同时检测这3 种菌时，纯菌DNA检测限可达1 pg/μL；将3 种菌一起接种到冷却肉中35 ℃过夜培养后，荧光假单胞菌、沙门氏菌和单增李斯特菌的检测限分别可达到9、5、70 CFU/mL。

A Multiplex PCR Method for Simultaneous Detection of Pseudomonas fluorescens,Salmonella and Listeria monocytogenes

Pseudomonas fluorescens (P. fluorescens), Salmonella and Listeria monocytogenes (L. monocytogenes) are 3
species of bacteria that contaminate meat products. The aim of this work was to establish a feasible multiplex polymerase
chain reaction (m-PCR) protocol for simultaneous and rapid detection of these 3 species of bacteria. The specific primers
for the gyrB gene of P. fluorescens, the invA gene of Salmonella and the hlyA gene of L. monocytogenes were designed and
their specificities were determined. In addition, the sensitivities for detecting 3 standard strains and these strains enriched
on meat overnight were tested as well. The results indicated that the m-PCR method had high specificity and sensitivity. For
simultaneous detection of these target microorganisms, the limit of detection (LOD) for pure DNA was 1 pg/μL, and the
LOD for P. fluorescens, Salmonella and L. monocytogenes were 9, 5, and 70 CFU/mL, respectively, by inoculating these 3
strains onto duck meat and overnight culturing at 35 ℃.