罗非鱼无乳链球菌的DIG-cfb原位杂交检测方法的建立

Construction of Digoxigenin-labeled cfb in Situ Hybridization Method of Streptococcus agalactiae from Tilapia

为建立一种批量检测罗非鱼无乳链球菌(Streptococcus agalactiae)的快速敏感的原位杂交方法,本研究依据罗非鱼无乳链球菌cfb基因序列,选取其132-599 bp之间的序列为靶序列,采用PCR法制备了特异性地高辛标记的DNA探针DIG-cfb(Digoxigenin-labelled cfb probe,430 bp),同时对所制备的探针进行了测序鉴定及特异性和敏感性检测。结果表明,探针测序序列与实际Gen Bank序列相符,所制备的DIG-cfb探针与无乳链球菌基因组杂交呈阳性,与患病罗非鱼的其他细菌病原(嗜水气单胞菌、金黄色葡萄球菌、类志贺邻单胞菌以及海豚链球菌)基因组,以及与无乳链球菌亲缘关系较近的链球菌其他种(牛源链球菌、变异链球菌),斑点反应则均为阴性,d NTP对照组也为阴性,表明该方法特异性强;该方法可实现对批量样品的快速检测,具有较高的灵敏度,可检测无乳链球菌基因组的下限为1.5 ng/μL DNA。对采集于广东、海南及广西等不同地点的无乳链球菌样品,均可获得较明显的斑点反应圈,对患链球菌病的罗非鱼肝、脑组织基因组也可特异的检测出无乳链球菌。表明本研究建立的DIG-cfb原位杂交法对无乳链球菌基因组的检测具有快速、特异、敏感等特点,适合罗非鱼无乳链球菌的批量检测。

In order to establish a specific in situ hybridization method to rapidly batch detect Streptococcus agalactiae from diseased tilapia, a digoxigenin labeled DNA probe was produced by PCR method. Thesequence between 132 bp and 599 bp of cfb gene in Streptococcus agalactiae was used as the target sequence.The digoxigenin-labelled cfb probe was verified through sequencing and its length was 430 bp. Meanwhile, thespecificity and sensitivity of the probe were also analyzed. Results showed that the probe was able to hybridizewith S. agalactiae. And no hybridization signals were observed with other tilapia pathogenic strains（Aeromonas hydrophila, Streptococcus iniae, Plesimonas shigelloides and Staphylococcus aureus）, and other homologous Streptococcus strains（Streptococcus bovis and Streptococcus mutans）, indicating that the method possessed goodspecificity. The method could rapidly batch detect Streptococcus agalactiae sensitively, and could detect atemplate concentration as low as 1.5 ng/μL. Hybridization signals were also observed with other Streptococcus agalactiae from tilapia in Guangdong, Hainan and Guangxi Provinces, and from the diseased tilapia liver andbrain. It demonstrated that the DIG-cfb in situ hybridization method possessed the characteristics of rapidity,specificity, sensitivity and could be applied to the batch detection of S. agalactiae from tilapia.