致病性维罗纳气单胞菌检测方法的建立

Development of a PCR method for the detection of pathogenic Aeromonas veronii

摘要:

发掘维罗纳气单胞菌特异性更强的检测靶点和毒力相关基因靶点, 建立能够检测致病性维罗纳气单胞菌的PCR检测方法。通过序列比对分析气单胞菌的16S rRNA基因序列, 筛选对维罗纳气单胞菌特异的引物, 用于检测种特异性, 利用气单胞菌气溶素基因保守引物, 检测菌株的致病性, 并进行反应条件和反应体系的优化, 灵敏度试验和特异性试验。发掘并设计的维罗纳气单胞菌16S rRNA特异性引物结合气单胞菌气溶素基因保守引物建立的检测方法, 对12株气单胞菌和10株非气单胞菌的检测结果显示, 所有致病性维罗纳气单胞菌都能扩增到大小分别为343 bp和232 bp的特异性条带, 而非维罗纳气单胞菌的致病性气单胞菌只能扩增到232 bp的气溶素基因特异性条带, 其它菌株都不能扩增到目的条带。灵敏度试验表明, 该反应体系的检测灵敏度为1.35×10?3 mg/L。我们建立的致病性维罗纳气单胞菌检测方法能特异地检测致病性维罗纳气单胞菌, 并具有高度灵敏性。

Abstract:

The aim of the study was explore a set of new detective targets in pathogenic Aeromonas veronii which was more specific than others at present, to develop a new duplex PCR method which could effectively detection the pathogenic A. veronii. Comparison analysis of the 16S rRNA gene sequences of Aeromonas spp. was used to explore A. veronii specific targets, which were then evaluated and used to design specific primers, and select the conservative primers of aerolysin genes in Aeromonas spp. to explore pathogenic targets. PCR parameters were optimized, its reaction system was developed, and its sensitivity and specificity were checked. 12 Aeromonas stains and 10 non-Aeromonas stains were tested for pathogenic A. veronii by duplex PCR method. The duplex PCR can clearly identify A. veronii from Aeromonas species, and can identify aerolysin-producing stains of A. veronii. The detection limit of genomic DNA by the duplex PCR was 1.35×10?3 mg/L. A novel duplex PCR method was successfully developed in this study, which could effectively detect pathogenic A. veronii with high accuracy and sensitivity.