title: Evaluation of colorimetric loop-mediated isothermal amplification assay for visual detection of Streptococcus agalactiae and Streptococcus iniae in tilapia.
abstract: Streptococcus agalactiae and Strep. iniae are bacterial pathogens that cause streptococcosis in many fish species. An accelerated colorimetric loop-mediated isothermal amplification (LAMP) assay with pre-addition of calcein was established, and the transmission and detection of Strep. agalactiae and Strep. iniae in tilapia under natural aquatic environment were investigated. A positive reaction was observed by a colour change from orange to green through the naked eyes after completion at 63°C for 30 min with 10 times higher sensitivity than that of nested PCR assays and without cross-amplification with other fish bacterial pathogens. All sample types of Nile and red tilapia (broodstock, fertilized egg, fry) were Strep. agalactiae- and Strep. iniae positive by this new method, implying that they could be vertically transmitted. With its application for screening broodstock and fry before stocking and for monitoring fish health in grow-out ponds, the method would become very useful in fish farming industry.
  
  
  CONCLUSIONS
  The application of colorimetric LAMP with pre-addition of calcein offers simple, rapid and sensitive technique with applicability for small field laboratories. This technique explored the possible vertical transmission mode of Strep. agalactiae and Strep. iniae under natural aquatic environment. It could be such preliminary data provided for the screening broodstock before breeding and/or the specific-pathogen-free production.
authors.affiliation: Center of Excellence for Shrimp Molecular Biology and Biotechnology (CENTEX Shrimp), Faculty of Science, Mahidol University, Bangkok, Thailand; National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathum Thani, Thailand.
authors.forename: R
authors.initials: R
authors.lastname: Suebsing
authors.forename: J
authors.initials: J
authors.lastname: Kampeera
authors.forename: B
authors.initials: B
authors.lastname: Tookdee
authors.forename: B
authors.initials: B
authors.lastname: Withyachumnarnkul
authors.forename: W
authors.initials: W
authors.lastname: Turner
authors.forename: W
authors.initials: W
authors.lastname: Kiatpathomchai
mesh_terms: D000818:Animals; D003124:Colorimetry / Q000379:methods*; D005393:Fish Diseases / Q000175:diagnosis / Q000382:microbiology*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D013290:Streptococcal Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D013291:Streptococcus / Q000235:genetics / Q000302:isolation & purification*; D013292:Streptococcus agalactiae / Q000235:genetics / Q000302:isolation & purification; D017210:Tilapia / Q000382:microbiology
keywords: Streptococcus agalactiae; Streptococcus iniae; calcein; loop-mediated isothermal amplification; tilapia; transmission