食品中荧光假单胞菌双重PCR法检测体系的建立和评价

Development and evaluation of duplex pcr assays for detection of Pseudomonas fluorescens in foods

摘要: 根据荧光假单胞菌(Pseudomonas fluorescens)促旋酶(gyrase)的B亚单位基因以及金属蛋白酶(apr)基因设计出两对引物,经过反应条件的优化,建立了用于荧光假单胞菌快速检测的双重聚合酶链式反应(PCR)方法,并对该体系进行评价。结果显示,Pseudomonas fluorescens菌株经普通PCR扩增均可见2条特异性条带(384 bp和194 bp),而其它阴性对照菌株PCR扩增均为阴性。基于基因组DNA的双重PCR检测灵敏度为16.9 fg/μL(3~4拷贝/μL),1.8CFU/m L的UHT牛奶样品(250 m L)经增菌24 h后用该双重PCR方法均可检出。本研究建立的双重PCR检测方法具有良好的特异性和灵敏度,能克服乳制品样品基质的干扰,可应用于乳制品中荧光假单胞菌的快速检测。

Abstract: Duplex PCR assays were developed for the detection of Pseudomonas fluorescens,based on its gyr B gene and apr gene. Among all the bacterial strains used in this research,duplex PCR amplified two specific products( 384 bp and 194 bp) from the strains of Pseudomonas fluorescens,but not from the negatives. The specifity of our assays was 16.9 fg / μL for pure DNA and 1.8 CFU / m L for UHT milk( 250 m L) with 24 h enrichment.So,the methods described in our study can be adopted to detect Pseudomonas fluorescens in food samples with higher sensitivity and specificity.