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目的建立一种能够快速准确地检测致病性嗜水气单胞菌的四重PCR方法。方法根据致病性嗜水气单胞菌的丝氨酸蛋白酶(ahpA)基因、气溶素(aerA)基因和溶血素(hlyA)基因的保守序列以及嗜水气单胞菌的16SrRNA基因种属特异性序列分别设计4对特异性引物,通过优化各引物浓度、退火温度和Mg2+浓度,确定了四重PCR的最佳反应条件;然后进行特异性和敏感性试验,并比较其与常规微生物学方法对不同菌株和临床样本的检出率。结果该方法特异性好,能特异性检测并鉴别出嗜水气单胞菌致病性菌株;检测灵敏度高,最低可检测到约100fg的嗜水气单胞菌基因组DNA;对9株嗜水气单胞菌的检测结果与常规微生物学方法一致,对56份送检的水产动物样品的检出率为21.4%,高于常规微生物学方法的16.1%,两者检测结果符合率为94.6%。结论成功建立了能同时检测嗜水气单胞菌16SrRNA、aerA、ahpA和hlyA基因的四重PCR方法,能快速、准确地对嗜水气单胞菌进行检测并区分致病性与非致病性菌株。
Objective To establish a quadruple PCR method for rapid and accurate detection of pathogenic Aeromonas hydrophila. Methods Based on the serine protease (ahpA) genes of the pathogenic Aeromonas hydrophila, the conserved sequences of the aerA and hlyA genes and the 16S rRNA gene of Aeromonas hydrophila Four pairs of specific primers were designed and sequenced respectively. The optimal reaction conditions for quadruple PCR were determined by optimizing the primer concentration, annealing temperature and Mg2 + concentration. Specific and sensitivity tests were performed and compared with conventional microbiological methods Detection rate of different strains and clinical samples. Results The method was specific and specific, and could detect and identify the pathogenic strains of Aeromonas hydrophila. The genomic DNA of Aeromonas hydrophila with the highest detection sensitivity and the lowest detectable concentration was about 100 fg. Aeromonas test results consistent with conventional microbiological methods, the detection rate of 56 samples submitted for aquatic animal samples was 21.4%, 16.1% higher than the conventional microbiological methods, the two test results coincide rate of 94.6% . Conclusion A quadruple PCR method for simultaneous detection of Aeromonas hydrophila 16SrRNA, aerA, ahpA and hlyA genes was successfully established, which can rapidly and accurately detect Aeromonas hydrophila and distinguish pathogenic and non-pathogenic Sexual strains.