PCR detection of a newly emerged pandemic Vibrio parahaemolyticus O3:K6 pathogen in pure cultures and seeded waters from the Gulf of Mexico
PCR detection of a newly emerged pandemic Vibrio parahaemolyticus O3:K6 pathogen in pure cultures and seeded waters from the Gulf of Mexico
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DOI：
10.1128/aem.69.4.2194-2200.2003
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发表时间：
2003-04-01
影响因子：
4.4
通讯作者：
Bej, AK
中科院分区：
文献类型：
--
作者：
Myers, ML;Panicker, G;Bej, AK
本研究对PCR参数进行了优化，并对大量微生物进行了检测，以建立一种基于PCR的方法，在墨西哥湾（海湾水）的纯培养物和种子水中检测新出现的大流行副溶血性弧菌03:K6菌株。所选择的开放阅读框8 (ORF8) dna特异性寡核苷酸引物对35株致病性副溶血性弧菌03:K6大流行分离株均有特异扩增作用，而对1996年爆发前分离的2株副溶血性弧菌03:K6、122株非o3:K6、198株非副溶血性弧菌、198株非副溶血性弧菌、122株副溶血性弧菌、122株副溶血性弧菌、122株副溶血性弧菌、122株副溶血性弧菌、122株副溶血性弧菌、122株副溶血性弧菌、122株副溶血性弧菌、122株副溶血性弧菌、122株副溶血性弧菌、122株副溶血性弧菌、122株副溶血性弧菌、198株副溶血性弧菌的dna特异性引物均无特异扩增作用。PCR方法的最低检出量为1 pg纯化的基因组DNA或10(2)个orf8阳性V副溶血性03:K6细胞在100 ml水中。本方法对环境样品中orf8阳性分离株的检测效果进行了试验，并在10倍连续稀释的海湾水中进行了试验。达到了每100毫升海湾水中10(3)个细胞的检测水平。并通过在不同温度下培养28 d的海湾水中病原菌的检测，验证了该方法的适用性。该方法可在要求的敏感性范围内以高特异性监测新出现的致病性副溶血性弧菌03:K6菌株在沿海、海洋和船舶压载水中的发生和分布。早期发现副溶血性弧菌03:K6将有助于提高海产品的安全性，降低由该病原体引起的传染病暴发的风险。
This study describes the optimization of PCR parameters and testing of a wide number of microbial species to establish a highly specific and sensitive PCR-based method of detection of a newly emerged pandemic Vibrio parahaemolyticus 03:K6 strain in pure cultures and seeded waters from the Gulf of Mexico (gulf water). The selected open reading frame 8 (ORF8) DNA-specific oligonucleotide primers tested were found to specifically amplify all 35 pathogenic V. parahaemolyticus 03:K6 pandemic isolates, whereas these primers were not found to detectably amplify two strains of V. parahaemolyticus 03:K6 that were isolated prior to the 1996 outbreaks, 122 non-O3:K6 strains of V. parahaemolyticus, 198 non-V parahaemolyticus spp., or 16 non-Vibrio bacterial spp. The minimum level of detection by the PCR method was I pg of purified genomic DNA or 10(2) ORF8-positive V parahaemolyticus 03:K6 cells in 100 ml of water. The effectiveness of this method for the detection of ORF8-positive isolates in environmental samples was tested in gulf water seeded with 10-fold serial dilutions of this pathogen. A detection level of 10(3) cells per 100 ml of gulf water was achieved. Also, the applicability of this methodology was tested by the detection of this pathogen in gulf water incubated at various temperatures for 28 days. This PCR approach can potentially be used to monitor with high specificity and well within the required range of sensitivity the occurrence and distribution of this newly emerged pathogenic V. parahaemolyticus 03:K6 strain in coastal, marine, and ship ballast waters. Early detection of V. parahaemolyticus 03:K6 will help increase seafood safety and decrease the risk of infectious outbreaks caused by this pathogen.