基于cpsA基因的海豚链球菌LAMP检测方法的建立

Establishment of LAMP detection method for Streptococcus iniae based on cpsA gene

基于荚膜多糖cpsA基因设计引物,建立海豚链球菌可视化环介导等温扩增(Loop-mediated isothermal amplification,LAMP)技术,以快速检测鱼类养殖中的海豚链球菌。结果表明,LAMP最佳反应条件为65℃反应20 min,镁离子浓度为1.2 mmol/L、dNTPs浓度为0.64 mmol/L、内外引物比例为16∶1。特异性检测结果表明:该方法能特异性检出海豚链球菌,对无乳链球菌和其他14种菌检测结果均呈阴性;灵敏度检测结果表明,该LAMP方法灵敏度为2.12×10^(-5)ng/μL,比PCR检测方法灵敏度高100倍;适用性分析结果表明,该LAMP方法在模板中存在鱼类基因组干扰下也能正确完成检测。研究中建立的LAMP检测方法为海豚链球菌的检测提供一种可视化、灵敏、成本低的快速检测技术。

Streptococcus iniae is bacterial pathogen that cause streptococcosis in many fish species.Rapid,low-cost,and user-friendly strategies are urgently needed for early disease diagnosis and timely treatment,particularly for on-site screening of pathogens in aquaculture.Loop-mediated isothermal amplification(LAMP)technique is an established diagnostic tool that can be conveniently used to screen pathogens in routine or field station laboratories.In this study,primers were designed based on the capsular polysaccharide cpsA gene to establish a visual LAMP technique for rapidly detecting S.iniae in fish culture.The results showed that the best reaction condition for LAMP is 65℃for 20 min,the concentration of Mg2+is 1.2 mmol/L,the concentration of dNTPs is 0.64 mmol/L,and the ratio of internal to external primers is 16∶1.The specific detection results showed that this method could specifically detect S.iniae and could not detect S.agalactiae and other 14 kinds of bacteria.The sensitivity test results showed that the sensitivity of the LAMP method is 2.12×10^(-5)ng/μL,which is 100 times higher than the PCR method.The result of applicability analysis showed that the LAMP method could also correctly complete the detection in the presence of fish genome interference in the template.The LAMP detection method established in this study provides a visual,sensitive,and low cost rapid detection technique to detect S.iniae.It could be such preliminary data provided for the screening broodstock before breeding and/or the specific-pathogen-free production.