嗜水气单胞菌菌落多重PCR方法的建立及应用

Establishment and application of colony multiplex PCR for Aeromonas hydrophila

摘要:

【背景】嗜水气单胞菌(Aeromonas hydrophila)对水产动物、畜禽和人类均有致病性。基因表达的溶血素、气溶素和肠毒素是重要毒力因子，在致病性嗜水气单胞菌早期检测及防治中尤为重要。目前采用菌落直接提取DNA用于多重PCR研究的相关报道较少。【目的】基于菌落PCR方法建立针对嗜水气单胞菌溶血性基因、肠毒素基因和16s rRNA基因特异性片段(5个基因片段)的多重PCR快速检测方法。【方法】采用选择性RS (Rimler-Shotts)培养基对样品中嗜水气单胞菌有效富集分离和辨认，建立并优化嗜水气单胞菌16s rRNA、ast、alt、aerA、act这5个基因的多重PCR方法，比较菌落PCR中DNA模板不同提取方法对多重PCR扩增结果的影响，并检测该方法对维氏气单胞菌、温和气单胞菌、杀鲑气单胞菌的特异性。【结果】通过对RS培养基上单菌落的16s rRNA基因鉴定，初步判定嗜水气单胞菌和其他可培养菌的菌落形态，对其富集程度进行可视化辨别。多重PCR反应体系优化结果显示，引物浓度最优配比为16s rRNA:ast:alt:aerA:act=1:2:2:3:4。菌落PCR结果显示，新鲜菌液采用煮沸冷冻离心法和煮沸离心法均能扩增出清晰条带，采用单菌落处理则需煮沸冷冻离心法。该多重PCR方法具有特异性。【结论】利用菌落多重PCR可以不通过试剂盒抽提获得DNA模板，简便、直观地检测嗜水气单胞菌及其毒力基因，具有特异性。

Abstract:

[Background] Aeromonas hydrophila is pathogenic to aquatic animals, livestock, poultry, and human. Hemolysin, aerolysin, and enterotoxin are major virulence factors particularly important in the early detection and prevention of A. hydrophila. There are few reports using colony PCR to extract templates for multiplex PCR. [Objective] Based on colony PCR, a multiplex PCR assay was established for rapid detection of five genes including hemolysin gene, enterotoxin gene, and 16s rRNA gene of A. hydrophila. [Methods] We used the selective Rimler-Shotts (RS) medium to enrich, isolate, and identify A. hydrophila in the sample. Subsequently, we established and optimized the multiplex PCR conditions for the 16S rRNA, ast, alt, aerA, and act of A. hydrophila and compared the results of multiplex PCR with the DNA templates extracted by different methods in colony PCR. Finally, we evaluated the specificity of the established method by using A. veronii, A. sobria and A. salmonicida. [Results] After 16S rRNA identification of single colonies on RS plates, the colony morphology of A. hydrophila and other cultivable bacteria was preliminarily characterized, and the enrichment degree of A. hydrophila was visually identified. The optimization results of the multiplex PCR system showed that the optimal primer concentration ratio was 16s rRNA:ast:alt:aerA:act=1:2:2:3:4. PCR for fresh bacterial liquids treated with both boiling-refrigerated centrifugation method and boiling-centrifugation method could produce clear bands, while the PCR of single colony required the former method. The established multiplex PCR method was specific. [Conclusion] Colony multiplex PCR can be used to easily and intuitively detect A. hydrophila and its virulence genes without the need of kit extraction of DNA templates, and it was specific.