食品中荧光假单胞菌聚合酶链式反应检测体系的建立和评价

摘要

建立用聚合酶链式反应（polymerase chain reaction,PCR）检测食品中荧光假单胞菌的方法。分别针对16～23S rRNA基因间隔区序列、gyrB基因以及通过生物信息学方法发掘到的4个种特异性基因设计6对检测引物,通过初步特异性实验,筛选出一对种特异性最佳的引物。最终建立以gyrB基因为检测靶点的PCR扩增体系,并对体系进行系统评价。结果表明：该方法可特异检测荧光假单胞菌的存在,纯DNA检测灵敏度为14.9fg/μL（2～3拷贝/μL）,纯培养物检测灵敏度为2.8×102CFU/mL。豆奶样品经15h充分增菌可提高检测灵敏度至0.28CFU/25g。%A polymerase chain reaction（PCR） assay for the detection of Pseudomonas fluorescens in foods was developed in this study.Six pairs of detection primers were designed for the 16-23S rDNA internal transcribed spacer sequence,the gyrB gene and 4 specific genes obtained by bioinformatics,respectively.Primary specificity experiments were used to screen the best primer pair out of them.A PCR amplification system targeting the gyrB gene was constructed and evaluated.The results indicated that the developed assay could specifically detect Pseudomonas fluorescens.The specificity was 14.9 fg/μL for pure DNA,2.8 × 102 CFU/mL for pure culture and 0.28 CFU/ 25 g for soybean milk with 15 h enrichment.