Identification of new target sequences for PCR detection of Vibrio parahaemolyticus by genome comparison
Identification of new target sequences for PCR detection of Vibrio parahaemolyticus by genome comparison
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DOI：
10.1111/j.1745-4581.2009.00158.x
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发表时间：
2009-03
影响因子：
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通讯作者：
D. Zhu;Min Zhou;Yiling Fan;Xianming Shi
中科院分区：
文献类型：
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作者：
D. Zhu;Min Zhou;Yiling Fan;Xianming Shi
摘要采用基因组比较法对副溶血性弧菌的特异性靶序列进行了PCR检测，并与139种其他细菌基因组的CDS值进行了比较。根据BLAST（一种比较蛋白质和核苷酸序列的新工具）的E值，发现副溶血弧菌的20个CDS具有相对特异性，并从中选出4个CDS用于PCR引物的设计。这4对引物在9株副溶血弧菌中均扩增出阳性产物，而在其他9株弧菌科菌株和4株非弧菌科菌株中均未扩增出阳性产物。检测灵敏度的评价表明，这四对引物可用于PCR检测副溶血性弧菌。实际应用本研究开发了一种自动BLAST方法，通过该方法可以快速筛选出物种特异性序列。通过这种方法，确定了新的和特异的副溶血性弧菌的基因作为PCR检测的目标序列。通过筛选，筛选出特异性和敏感性均较好的4对引物，可应用于PCR检测等分子生物学方法。这类方法在新的未来可能成为商业化的检测产品。此外，该方法也可用于其他属种的特异性序列的挖掘，如沙门氏菌、葡萄球菌等。
ABSTRACT A genome comparison method was used to identify specific target sequences for the polymerase chain reaction (PCR) detection of Vibrio parahaemolyticus, and the CDS value of this bacterium was compared with that of 139 other bacterial genomes. It was found that 20 CDS of V. parahaemolyticus were relatively specific according to their E value in BLAST (a new tool for comparing protein and nucleotide sequences), and four of them were selected for the design of PCR primers. There were positive amplification products of these four pairs of primers from nine V. parahaemolyticus strains, whereas there were no amplification products from nine other Vibrionaceae strains and four non-Vibrionaceae strains. An evaluation of detection sensitivities revealed that these four pairs of primers can be used in a PCR assay for the detection of V. parahaemolyticus. PRACTICAL APPLICATIONS An automatic BLAST method was developed in this study, by which species-specific sequences can be screened out rapidly. In this way, new and specific genes of Vibrio parahaemolyticus were identified to be used as target sequences for PCR detection. In terms of acceptable specificity and sensitivity, the four pairs of primers were selected by screening, which can be applied in PCR assays and other molecular methods. These kinds of methods might become commercial detection products in the new future. In addition, this method for searching specific DNA sequences can also be used for the mining specific sequences in other genus and species, such as Salmonella, Staphylococcus, etc.