Development of 16S rRNA targeted PCR methods for the detection and differentiation of Vibrio vulnificus in marine environments
Development of 16S rRNA targeted PCR methods for the detection and differentiation of Vibrio vulnificus in marine environments
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DOI：
10.1016/s0044-8486(00)00495-6
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发表时间：
2001-02-15
期刊：
影响因子：
4.5
通讯作者：
Jeong, HD
中科院分区：
文献类型：
--
作者：
Kim, MS;Jeong, HD
利用针对16 SrRNA可变区的组合引物，建立了一种创伤弧菌的PCR检测和鉴别方法。根据弧菌科16 S rRNA的两个可变区（对应于大肠杆菌16 S rRNA中的核苷酸编号1006至1023和1278至1258）设计的引物通过PCR被发现对创伤弧菌具有种特异性。此外，16 S rRNA的三引物PCR用于区分创伤弧菌菌株进行了评价。虽然第三引物来源于可变区，位置454至473，不能区分创伤弧菌与其他细菌，但它用于避免在PCR中检测到该生物体的B型16 S rRNA。在存在创伤弧菌特异性的273 bp片段的情况下，在三引物PCR中得到的825 bp片段清楚地区分了A型16 S rRNA菌株和B型菌株。用最大概然数法（MPN）对5支添加多粘菌素B的碱性蛋白胨水预增菌管进行计数，阳性管用16 S rRNA基因扩增或在mCPC琼脂上划线确认。与MPN方法中PCR方法确认的数量相比，选择性培养基确认的推定创伤弧菌数量更高，这表明环境样本中可能存在一些无法在mCPC琼脂上与创伤弧菌区分开来的细菌。在牡蛎样品中的创伤弧菌分离株的生物分型中，大多数菌株（92.5%）属于生物型1。生物型2占7.5%。海洋环境中创伤弧菌16 SrRNA的三引物PCR结果显示，A型占35%，B型占65%。这些结果表明，海洋环境可以作为创伤弧菌生物型1和生物型2的储存库，并且在该环境中16 S rRNA B型菌株比A型菌株更常见。(C)2001 Elsevier Science B. V.保留所有权利。
A PCR method for the detection and differentiation of Vibrio vulnificus strains was developed as an alternative to culture methods by using combined primers directed against the variable regions of 16S rRNA. Primers designed from two variable regions of the Vibrionaceae 16S rRNA (corresponding to nucleotide numbers 1006 to 1023 and 1278 to 1258 in Escherichia coli 16S rRNA) was found to be species-specific for V. vulnificus by PCR. Additionally, tri-primer PCR of 16S rRNA was evaluated for the differentiation of V. vulnificus strains. Although the third primer, which was derived from the variable region, positions 454 to 473, cannot discriminate V. vulnificus from other bacteria, it was used to avoid the detection of type B 16S rRNA of this organism in PCR. The resulting 825 bp fragment in the presence of the 273 bp fragment, which is specific to V. vulnificus, in tri-primer PCR clearly differentiated type A 16S rRNA strains from type B. Enumeration of V. vulnificus in the samples of oyster and environmental samples was done by most probable numbers (MPN) method of five preenrichment tubes of alkaline peptone water supplemented with polymyxin B following up the confirmation of positive tubes by streaking the samples onto mCPC agar or by 16S rRNA gene amplification. Higher numbers of presumptive V. I vulnificus confirmed by selective media compared with those confirmed by PCR method in MPN method suggested that there would be some bacteria that cannot be discriminated from V. vulnificus on mCPC agar in environmental samples. In the biotyping of the V. vulnificus isolates in oyster samples, the majority of the strains (92.5%) belonged to biotype 1. and 7.5% of the strains belonged to biotype 2. However, strains of 16S rRNA of V. vulnificus isolates in the marine environment determined by tri-primer PCR appeared to be 35% type A and 65% type B. These results implied that the marine environment can serve as reservoir of both V. vulnificus biotypes 1 and 2, and strains of 16S rRNA type B were more frequent than strains of type A in that environment. (C) 2001 Elsevier Science B.V. All rights reserved.