Soltani M, Pirali E, Shayan P, Eckert B, Rouholahi S, Sadr Shirazi N
Department of aquatic animal health, faculty of veterinary medicine, university of Tehran, Tehran, Iran.
Iran J Microbiol. 2012 Jun;4(2):70-4.
Streptococcosis/lactococcosis is the cause of high morbidity and mortality in aquaculture sector and to date a number of species of Streptococcus and Lactococcus genera including S. iniae, S. agalactiae, S. dysagalactiae, S. parauberis, S. feacalis, L. garvieae and L. lactis have been discriminated as the cause of disease in aquatic animals. Despite the use of diagnostic molecular methods for each of these bacterial species, no data is available on a suitable, rapid and simple simultaneous detection tool for these pathogens. This paper describes a simultaneous detection method which is PCR based on a reverse line blot (RLB) for rapid detection and differentiation of four species of genera of Streptococcus and Lactococcus genera consisting of S. iniae, S. agalactiae, S. parauberis and L. garvieae the most important agents of the disease in fish.
A reverse line blot (RLB) assay was developed for the simultaneously identification of four species of Streptococcus/lactococcusconsisting of S. iniae, S. parauberis, S. agalactiaeand Lactococcusgarvieae. The assay employs one set of primer pair for specific amplification of the 16S rRNA gene. These were designed based on the nucleotide sequences of 16S rRNA gene sharing a homology region with Streptococcus spp. and Lactococcus spp. DNA was extracted from the pure bacterial colonies and amplified. A membrane was prepared with specific oligonucleotide for each bacterial species. PCR products were then hybridized to a membrane.
The amplification resulted in PCR product of 241 bp in length. No cross-reactions were observed between any of the tested bacterial species, and mixed DNAs from these four bacterial species were correctly identified. This RLB method is a suitable technique for a simultaneous detection of these species of bacterial fish pathogens that are some of the main causes of streptococcal/lactococcal infections in both freshwater and marine aquatic animals, and so we recommend its use for integrated epidemiological monitoring of streptococcosis/lactococcis in aquaculture industry.
链球菌病/乳球菌病是水产养殖业中高发病率和高死亡率的病因，迄今为止，包括海豚链球菌、无乳链球菌、停乳链球菌、副乳房链球菌、粪链球菌、格氏乳球菌和乳酸乳球菌在内的多种链球菌属和乳球菌属物种已被鉴别为水生动物疾病的病因。尽管针对这些细菌物种中的每一种都使用了诊断分子方法，但尚无关于适合、快速且简单的同时检测这些病原体的工具的数据。本文描述了一种基于反向线杂交（RLB）的PCR同时检测方法，用于快速检测和区分链球菌属和乳球菌属的四个物种，即海豚链球菌、无乳链球菌、副乳房链球菌和格氏乳球菌，它们是鱼类疾病的最重要病原体。
开发了一种反向线杂交（RLB）检测方法，用于同时鉴定海豚链球菌、副乳房链球菌、无乳链球菌和格氏乳球菌这四种链球菌/乳球菌。该检测方法使用一组引物对特异性扩增16S rRNA基因。这些引物是根据与链球菌属和乳球菌属共享同源区域的16S rRNA基因的核苷酸序列设计的。从纯细菌菌落中提取DNA并进行扩增。用针对每种细菌物种的特异性寡核苷酸制备膜。然后将PCR产物与膜杂交。
扩增产生了长度为241 bp的PCR产物。在任何测试的细菌物种之间均未观察到交叉反应，并且来自这四种细菌物种的混合DNA被正确鉴定。这种RLB方法是一种适用于同时检测这些细菌鱼类病原体物种的技术，这些病原体是淡水和海洋水生动物中链球菌/乳球菌感染的一些主要原因，因此我们建议将其用于水产养殖业链球菌病/乳球菌病的综合流行病学监测。
