Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh
Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh
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DOI：
10.1016/s0167-7012(99)00037-8
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发表时间：
1999-06-01
影响因子：
2.2
通讯作者：
Kaysner, CA
中科院分区：
文献类型：
--
作者：
Bej, AK;Patterson, DP;Kaysner, CA
副溶血性弧菌是一种重要的人类病原体，当进食生的或半熟的海鲜时，可导致肠胃炎。针对副溶血性弧菌tl编码的不耐热溶血素、tdh编码的耐热直接溶血素和耐热直接溶血素相关的trh基因，建立了一种基于多重PCR扩增的副溶血性弧菌总株和强毒株检测方法。在使用靶向tl、tdh和trh基因的寡核苷酸引物进行优化后，将多重PCR应用于来自27个临床、43个海产品、15个环境、7个从不同实验室获得的菌株和19个从牡蛎植物获得的副溶血性弧菌。所有111株副溶血性弧菌分离株均显示tl基因的PCR扩增;然而，仅60株分离株显示tdh扩增，43株分离株显示trh基因扩增。另外，18株菌株显示tdh基因扩增，但这些菌株未显示trh基因扩增。然而，一个菌株表现出扩增的trh，但不是tdh基因，这表明这两个基因需要在PCR扩增反应中的目标，以检测所有溶血素生产菌株的这种病原体。该方法成功地应用于牡蛎组织匀浆中副溶血性弧菌的检测。所有三个靶基因片段的检测灵敏度至少在10（1）-10（2）cfu/10 g碱性蛋白胨水富集的种牡蛎组织匀浆之间。在预富集的8小时内检测该病原体的这种高水平的灵敏度完全在国家海产品卫生计划指南建议的行动水平（10（4）cfu/1 g贝类）内。与传统的微生物培养方法相比，该方法快速、可靠，可用于贝类副溶血性弧菌的全面检测。(C)1999 Elsevier Science B. V.保留所有权利。
Vibrio parahaemolyticus is an important human pathogen which can cause gastroenteritis when consumed in raw or partially-cooked seafood. A multiplex PCR amplification-based detection of total and virulent strains of V. parahaemolyticus was developed by targeting thermolabile hemolysin encoded by tl, thermostable direct hemolysin encoded by tdh, and thermostable direct hemolysin-related trh genes. Following optimization using oligonucleotide primers targeting tl, tdh and trh genes, the multiplex PCR was applied to V. parahaemolyticus from 27 clinical, 43 seafood, 15 environmental, 7 strains obtained from various laboratories and 19 from oyster plants. All 111 V. parahaemolyticus isolates showed PCR amplification of the tl gene; however, only 60 isolates showed amplification of tdh, and 43 isolates showed amplification of the trh gene. Also, 18 strains showed amplification of the tdh gene, but these strains did not show amplification of the trh gene. However, one strain exhibited amplification for the trh but not the tdh gene, suggesting both genes need to be targeted in a PCR amplification reaction to detect all hemolysin-producing strains of this pathogen. The multiplex PCR approach was successfully used to detect various strains of V. parahaemolyticus in seeded oyster tissue homogenate. Sensitivity of detection for all three target gene segments was at least between 10(1)-10(2) cfu per 10 g of alkaline peptone water enriched seeded oyster tissue homogenate. This high level of sensitivity of detection of this pathogen within 8 h of pre-enrichment is well within the action level (10(4) cfu per 1 g of shell stock) suggested by the National Seafood Sanitation Program guideline. Compared to conventional microbiological culture methods, this multiplex PCR approach is rapid and reliable for accomplishing a comprehensive detection of V. parahaemolyticus in shellfish. (C) 1999 Elsevier Science B.V. All rights reserved.