聚合酶链式反应技术快速检测原料乳中荧光假单胞菌

Rapid detection of Pseudomonas fluorescens in raw milk based on polymerase chain reaction assay

中文关键词:  嗜冷菌  荧光假单胞菌  原料乳  聚合酶链式反应技术

英文关键词:psychrophilic bacteria  Pseudomonas fluorescens  raw milk  polymerase chain reaction assay

中文摘要:

      目的 利用聚合酶链式反应技术(polymerase chain reaction, PCR)技术建立一种快速、准确检测原料乳中最常见有害嗜冷微生物荧光假单胞菌的方法。方法 以荧光假单胞菌蛋白酶基因aprX为检测靶标, 设计特异性PCR简并引物, 建立原料乳中荧光假单胞菌PCR检测体系, 对该体系的特异性及检出限进行评价。结果 筛选到特异性引物F3: 5'-WSNGGNGGNGAYTTYCAYATGAC-3'; R3: 5'-RTCRTTNCCNCCNCCRTCCC-3', 建立了最佳PCR检测体系: 引物浓度0.5 μmol/L、Taq DNA聚合酶添加量0.4 μL、dNTPs浓度0.16 mmol/L、Mg2+浓度1.6 mmol/L, 退火温度56.4℃, 扩增35个循环。此检测方法对荧光假单胞菌具有特异性, 检出限为2.57×103 CFU/mL。结论 本方法操作简便, 特异性强, 适用于快速检测原料乳中的荧光假单胞菌。

英文摘要:

      Objective To establish a rapid and accurate method for determination of Pseudomonas fluorescens, the most common harmful psychrophilic microorganisms in raw milk, by polymerase chain reaction (PCR) technology. Methods The Pseudomonas fluorescens protease gene aprX was used as the detection target gene to design specific PCR degenerate primers, and a PCR assay system of Pseudomonas fluorescens in raw milk was established and the specificity and the limit of detection of the system were evaluated. Results Specific primers F3: 5'-WSNGGNGGNGAYTTYCAYATGAC-3', R3: 5'-RTCRTTNCCNCCNCCRTCCC-3' were screened. The best PCR detection system was established: Primer concentration of 0.5 μmol/L, Taq DNA polymerase content of 0.4 μL, dNTPs concentration of 0.16 mmol/L, Mg2+ concentration of 1.6 mmol/L, annealing temperature of 56.4℃, amplification of 35 cycles. The detection method was specific for Pseudomonas fluorescens, and the limit of detection was 2.57×103 CFU/mL. Conclusion This method is simple, specific and suitable for rapid detection of Pseudomonas fluorescens in raw milk.