Multiplex PCR detection of clinical and environmental strains of Vibrio vulnificus in shellfish
Multiplex PCR detection of clinical and environmental strains of Vibrio vulnificus in shellfish
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DOI：
10.1139/w04-085
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发表时间：
2004-11-01
影响因子：
2.8
通讯作者：
Bej, AK
中科院分区：
文献类型：
--
作者：
Panicker, G;Vickery, MCL;Bej, AK
在本研究中，我们开发了一种基于PCR的快速检测临床上重要的创伤弧菌致病菌株的方法。在所有 22 个测试的临床分离株中均观察到 504 bp viuB 片段的阳性扩增，但仅在 33 个环境分离株中的 8 个中观察到。物种特异性 205 bp vvh 片段与 viuB 在多重 PCR 中的组合使我们能够确认创伤弧菌潜在致病菌株的存在。其他弧菌属物种没有扩增。或非弧菌的证据表明，表明该方法的检测具有高特异性。两个目标基因的检测灵敏度为 10 pg 纯化 DNA，与 1 mL 纯培养物或 1 g 未富集的种子牡蛎组织匀浆中的 10(3) 创伤弧菌 CFU 相关。在过夜富集的样品中，这种灵敏度提高至每克牡蛎组织匀浆 1 CFU。还开发了基于 SYBR Green I 的实时 PCR 方法，该方法可产生与传统 PCR 方法一致的结果。将多重实时 PCR 应用到天然牡蛎组织匀浆中，主要在夏季收集的样本中，51% 的 vvh 呈阳性检测；然而，只有 15% 的 vvh 阳性样本表现出 viuB 扩增子。对贝类中临床上重要的致病性创伤弧菌进行快速、灵敏和特异的检测将有利于减少该病原体引起的疾病和死亡。
In this study, we developed a PCR-based rapid detection method for clinically important pathogenic strains of Vibrio vulnificus. Positive amplification of the 504-bp viuB fragment was seen in all 22 clinical isolates tested but only in 8 out of 33 environmental isolates. The combination of the species-specific 205-bp vvh fragment along with viuB in a multiplexed PCR enabled us to confirm the presence of potentially pathogenic strains of V. vulnificus. No amplification of other Vibrio spp. or non-Vibrio bacteria was evidenced, suggesting a high specificity of detection by this method. The sensitivity of detection for both targeted genes was 10 pg of purified DNA, which correlated with 10(3) V. vulnificus CFU in 1 mL of pure culture or 1 g un-enriched seeded oyster tissue homogenate. This sensitivity was improved to 1 CFU per gram of oyster tissue homogenate in overnight-enriched samples. A SYBR Green I based real-time PCR method was also developed that was shown to produce results consistent with the conventional PCR method. Application of the multiplexed real-time PCR to natural oyster tissue homogenates exhibited positive detection of vvh in 51% of the samples collected primarily during the summer months; however, only 15% of vvh positive samples exhibited viuB amplicons. The rapid, sensitive, and specific detection of clinically important pathogenic V. vulnificus in shellfish would be beneficial in reducing illnesses and deaths caused by this pathogen.