基于毒力基因的罗非鱼无乳链球菌三重PCR检测方法的建立及应用

The Application and Detection of Streptococcus agalactiae Isolated from Tilapia with a Triple PCR Based on Virulence Genes

关键词:  罗非鱼  无乳链球菌  毒力基因  三重PCR

Key words:  Tilapia  Streptococcus agalactiae  Virulence gene  Triple PCR

摘要:

无乳链球菌(Streptococcus agalactiae)作为罗非鱼主要病原，传染力强、致死率高，部分鱼从发病到死亡无明显病症，难以确定鱼体是否携带该病原菌，建立适用于生产一线的无乳链球菌检测技术势在必行。根据GenBank中无乳链球菌透明质酸酶基因(hylB)、青霉素结合蛋白基因(ponA)和CAMP因子基因(cfb)的保守序列，设计3对特异性引物，对多重PCR的反应条件和体系进行优化，建立基于毒力基因的罗非鱼无乳链球菌三重PCR检测方法，并运用该方法检测来自广东省不同地区的罗非鱼组织样品。构建的三重PCR检测方法仅在无乳链球菌中扩增出3条特异性条带，而在罗非鱼和常见的水产病原菌菌株中均未扩增出任何条带，表现出良好的特异性；以无乳链球菌基因组DNA浓度7.24×10–5~5.65 ng/μl为模板进行扩增，该三重PCR能检测到的最低模板浓度为1.81×10–3 ng/μl，表现出较高的灵敏度；运用该方法检测188个罗非鱼组织样品，其阳性检出率与常规细菌分离鉴定阳性率一致，以常规细菌分离鉴定为标准，对该三重PCR检测方法进行评价，其诊断敏感性(Dse)和诊断特异性(Dsp)均为100%。结果表明，构建的三重PCR检测方法不仅显著提高了检测的准确度和灵敏度，还可在同一反应中同时检测3种无乳链球菌毒力基因，为无公害水产品中无乳链球菌的快速检疫、水产养殖病害早期预警提供了一种快速、精准和高效的检测技术。

Abstract:

Streptococcus agalactiae has become one of the most important emerging pathogens with strong infectivity and virulence, causing high mortalities and large economic losses in tilapia farming industries in China. There are no obvious symptoms in some tilapia infected by S. agalactiae, so it can be difficult to determine if a fish carried the pathogen. Thus, it was necessary to establish an effective method for detection of S. agalactiae in the breeding process of tilapia. Three pairs of specific primers were designed based on the conserved sequence of hylB, ponA, and cfb genes encoding hyaluronidase, penicillin binding protein, and CAMP factor of S. agalactiae published in GenBank, respectively. After the optimization of the reaction conditions and reaction system of multiplex PCR, the triple PCR method based on the three virulence genes was developed for detection of S. agalactiae isolated from tilapia. Furthermore, the established method was applied to detect tissue samples of tilapia collected from different farming areas in Guangdong Province. The detection result of S. agalactiae only amplified three specific bands, however there were no bands in the host and the other common bacterial pathogen strains in aquaculture, which indicated that the method had good specificity. The dynamic range of template concentration of S. agalactiae was 7.24×10-5~5.65 ng/μl. Sensitivity tests showed that the detection limit of the S. agalactiae in genomic DNA was 1.81×10-3 ng/μl, with a higher sensitivity. The positive rate for detection of 188 tissue samples isolated from tilapia using the triple PCR method was consistent with the positive rate by the conventional bacterial identification method. The triple PCR method was evaluated with the conventional method as the standard; diagnostic sensitivity (Dse) and diagnostic specificity (Dsp) were 100%. In summary, the results showed that the triple PCR method not only improved the accuracy and sensitivity of the detection, but also detected three virulence genes of S. agalactiae in the same reaction system. Therefore, this method provided a rapid, accurate, and efficient detection technology for monitoring S. agalactiae infection in harmless aquatic products and early warning of aquaculture diseases.