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首页> 外文期刊>Applied and Environmental Microbiology >Multiplex PCR Method for Detection of Three Aeromonas Enterotoxin Genes
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Multiplex PCR Method for Detection of Three Aeromonas Enterotoxin Genes

机译：多重PCR检测三种气单胞菌肠毒素基因

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A novel multiplex PCR method using three sets of specific primers was developed for the detection of the cytotoxic (act), heat-labile (alt), and heat-stable (ast) enterotoxin genes in Aeromonas spp. This assay was used to characterize 35 reference strains as well as 537 food-borne isolates. A total of seven gene pattern combinations were encountered, including act, alt, act/alt, act/alt/ast, act/alt/148-bp amplicon, alt/ast, and alt/148-bp amplicon. The alt gene was detected with 34 reference strains (97%) and occurred singly in 14% of these strains. The frequency of occurrence of the act/alt, act/alt/ast, and alt/ast gene patterns in reference strains was 14 (40%), 2 (6%), and 2 (6%), respectively. An unpredicted amplicon was detected in 11 reference strains (31%). Characterization of this amplicon showed that its size was 148 bp, as generated by the AHLF and AHLR primers, and that it uniquely aligned with the Aeromonas salmonicida A449 genome sequence (GenBank accession number CP000644). This amplicon was named Aeromonas salmonicida subsp. salmonicida hypothetical protein amplicon (AssHPA). In the 537 food-borne isolates, the act and alt genes were most dominant and were detected in 349 (65%) and 452 (84%) isolates, respectively, either alone or in combinations. The act and alt genes occurred singly in 30 (6%) and 128 (24%) of these strains, respectively. The act/alt gene pattern occurred in 315 isolates (59%), whereas the ast gene was always linked to strains exhibiting the act/alt/ast and alt/ast gene combinations in 4 (0.7%) and 5 (0.9%) isolates, respectively. The uniplex amplification of three enterotoxin genes separately confirms the specificity of the unique selected primers. This multiplex PCR is rapid and simple and can detect the presence of three Aeromonas enterotoxin genes in a single assay.
机译：开发了一种使用三组特异性引物的新型多重PCR方法，用于检测Aeromonas spp中的细胞毒素（act），热不稳定（alt）和热稳定（ast）肠毒素基因。该测定法用于表征35种参考菌株以及537种食源性分离株。总共遇到了七个基因模式组合，包括act，alt，act / alt，act / alt / ast，act / alt / 148-bp扩增子，alt / ast和alt / 148-bp扩增子。在34个参考菌株（97％）中检测到了alt基因，并在其中14％的菌株中单独出现了alt基因。参考菌株中act / alt，act / alt / ast和alt / ast基因模式的发生频率分别为14（40％），2（6％）和2（6％）。在11个参考菌株（31％）中检测到了无法预测的扩增子。该扩增子的表征表明，其大小为148 bp（由AHLF和AHLR引物生成），并且与鲑鱼气单胞菌A449基因组序列（GenBank登录号CP000644）唯一对齐。该扩增子被命名为鲑鱼气单胞菌亚种。鲑鱼假说蛋白扩增子（AssHPA）。在537种食源性分离株中，act和alt基因是最主要的，分别在349（65％）和452（84％）分离株中单独或组合检测到。 act和alt基因分别在这些菌株中的30（6％）和128（24％）中发生。 act / alt基因模式出现在315个分离株中（59％），而ast基因始终与表现出act / alt / ast和alt / ast基因组合的菌株相关，分别为4（0.7％）和5（0.9％）。 ％）分离物。三个肠毒素基因的单重扩增分别证实了唯一选择的引物的特异性。这种多重PCR快速简便，可以在一次测定中检测到三种气单胞菌肠毒素基因的存在。

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