具有产蛋白酶能力的假单胞菌是原料乳中的主要危害菌群之一,针对假单胞菌的sucD基因和aprX基因分别设计了2对特异性引物,建立了一种能够快速检测产蛋白酶假单胞菌的双重PCR方法,并对引物的特异性、灵敏度和人工污染检测限等进行了评价。结果表明,该双重PCR方法的特异性良好,对纯细菌DNA检测限可达到0.167 pg/μL。人工污染试验结果表明,经增菌培养12、24 h后,原料乳样品中产蛋白酶假单胞菌检测限分别为3.5×103、3.5 CFU/mL。因此该研究建立的双重PCR检测方法对原料乳中产蛋白酶假单胞菌的快速检测具有一定的应用前景。
Proteinase-producing Pseudomonas is the main harmful bacteria in raw milk. The aim of this study was to establish a feasible duplex PCR method for the rapid detection of proteinase-producing Pseudomonas. Two pairs of specific primers were designed according to the sequences of sucD gene and aprX gene in Pseudomonas. The primers were evaluated by their specificity, sensitivity and detection limits. The results demonstrated that this method had good specificity and the detection limit was 0.167 pg/μL for pure DNA. Meanwhile, after 12 h and 24 h enrichment cultivation, the detection limits of this method in artificially contaminated raw milk were 3.5×103 CFU/mL and 3.5 CFU/mL, respectively. In conclusion, this PCR method could be widely used for the rapid detection of proteinase-producing Pseudomonas in raw milk.
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