四重PCR检测致病性嗜水气单胞菌

Detection of pathogenic Aeromonas hydrophila by quadruple PCR

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。

Aeromonas hydrophila can infect an array of animals, including fish, amphibians, reptiles, birds and mam mals, and lead to hemorrhagic septicaemia. To develop a rapid and specific quadruple PCR method for the detection of patho genic Aeromonas hydrophila, four pairs of primers were designed based on the conservative sequences of 16S rRNA gene, ser ineprotease （ahpA） gene, aero[ysin（aerA） gene and hemolysin（hlyA） gene of Aeromonas hydrophila. After optimalizing the reaction conditions, specificity, sensitivity and detection rate of the quadruple PCR method were studied. Results indicated that this method has a high specificity in detecting pathogenic strains of Aeromonas hydrophila but not other irrelative bacteria, and can detect as less as 100 fg DNA of Aeromonas hydrophila. Nine Aerornonas hydrophila strains and fifty six clinical samples were tested by this quadruple PCR and conventional microbiology methods, and both of the methods could identify these nine Aeromonas hydrophila strains the detection rate of the fiftysix clinical samples by quadruple PCR was 21.4%, which was higher than that by conventional microbiology methods （16.1%）, and their coherence was 94.6%. It could be concluded that the quadruple PCR method which can simultaneously detect 16S rRNA gene and three virulent genes of pathogenic Aeromonas hydrophila was well established, and this method is a reliable and convenient method to detect Aeromonas hydrophila and i dentify specific type of pathogenic or nonpathogenic strains.