Establishment of LAMP detection method for Streptococcus iniae based on cpsA gene
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S941.4

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    Abstract:

    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.

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孙国荣,马少鸿,林桂香,万小菊,黄郁葱,简纪常,蔡双虎.基于cpsA基因的海豚链球菌LAMP检测方法的建立[J].上海海洋大学学报,2023,32(1):31-39.
SUN Guorong, MA Shaohong, LIN Guixiang, WAN Xiaoju, HUANG Yucong, JIAN Jichang, CAI Shuanghu. Establishment of LAMP detection method for Streptococcus iniae based on cpsA gene[J]. Journal of Shanghai Ocean University,2023,32(1):31-39.

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History
  • Received:August 27,2021
  • Revised:January 09,2022
  • Adopted:January 10,2022
  • Online: January 12,2023
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