Department of Fish Processing Technology, Fisheries College and Research Institute, Tamil Nadu Veterinary and Animal Sciences University, Tuticorin 628 008, India.
Appl Microbiol Biotechnol. 2011 May;90(3):1111-8. doi: 10.1007/s00253-011-3175-9. Epub 2011 Mar 1.
A multiplex polymerase chain reaction (MPCR)-based assay was developed for the simultaneous detection of Vibrios using the genus-specific RNA polymerase subunit A (rpoA) gene and specific detection of toxin-producing Vibrio cholerae strains using two sets of primer based on cholera toxin subunit A (ctxA) and repeat in toxin subunit A (RtxA)-producing genes. The MPCR method developed is applicable to both the simultaneous and the two-step detection of genus Vibrio total and toxigenic V. cholerae species. This assay was specific as no amplification occurred with the other bacterial pathogens tested. The sensitivity of the assay was tested by artificially spiking the shrimp homogenate with the toxigenic strain of V. cholerae (NICED 16582) in different dilutions. The developed MPCR assay could detect three cells of V. cholerae in 12 h pre-enrichment in APW. The proposed method is rapid, sensitive, and specific for the detection of Vibrio genus as well as toxin-producing V. cholerae strains in environmental samples.
建立了一种基于多重聚合酶链反应（MPCR）的方法，用于使用属特异性 RNA 聚合酶亚基 A（rpoA）基因同时检测弧菌，并使用基于霍乱毒素亚基 A（ctxA）和重复毒素亚基 A（RtxA）基因的两组引物特异性检测产毒霍乱弧菌菌株。所开发的 MPCR 方法适用于同时和两步检测属弧菌总群和产毒霍乱弧菌。该检测方法具有特异性，因为用其他测试的细菌病原体进行扩增时没有发生扩增。通过用不同稀释度的产毒霍乱弧菌（NICED 16582）人工添加到虾匀浆中，测试了该检测方法的灵敏度。开发的 MPCR 检测方法可在 12 小时预富集 APW 中检测到 3 个霍乱弧菌细胞。该方法快速、灵敏、特异性强，可用于检测环境样本中的弧菌属以及产毒霍乱弧菌菌株。
