title: Shewanella putrefaciens in cultured tilapia detected by a new calcein-loop-mediated isothermal amplification (Ca-LAMP) method.
abstract: Shewanella putrefaciens is being increasingly isolated from a wide variety of sources and is pathogenic to many marine and freshwater fish. For better control of this pathogen, there is a need for the development of simple and inexpensive but highly specific, sensitive, and rapid detection methods suitable for application in field laboratories. Our colorogenic loop-mediated isothermal amplification (LAMP) assay combined with calcein (Ca-LAMP) for unaided visual confirmation of LAMP amplicons is a simple method for fish pathogen detection in cultured tilapia. Here, we describe the detection of S. putrefaciens using the same platform. As before, the method gave positive results (orange to green color change) in 45 min at 63°C with sensitivity 100 times higher than that of a conventional PCR assay, with no cross-amplification of other known fish bacterial pathogens tested. Using the assay with 389 samples of gonads, fertilized eggs, and fry of farmed Nile and red tilapia Oreochromis spp., 35% of samples were positive for S. putrefaciens. The highest prevalence was found in samples of gonads (55%) and fertilized eggs (55%) from adult breeding stocks, indicating that S. putrefaciens could be passed on easily to fry used for stocking production ponds. Tissue tropism assays revealed that the spleen showed the highest colonization by S. putrefaciens in naturally infected tilapia and that it would be the most suitable organ for screening and monitoring fish stocks for presence of the bacteria.
authors.affiliation: Bioengineering and Sensing Technology Laboratory, BIOTEC, National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Phahonyothin Rd., Klong Neung, Klong Luang, Pathum Thani 12120, Thailand.
authors.forename: Rungkarn
authors.initials: R
authors.lastname: Suebsing
authors.forename: Jantana
authors.initials: J
authors.lastname: Kampeera
authors.forename: Sarawut
authors.initials: S
authors.lastname: Sirithammajak
authors.forename: Padmaja Jayaprasad
authors.initials: PJ
authors.lastname: Pradeep
authors.forename: Sarocha
authors.initials: S
authors.lastname: Jitrakorn
authors.forename: Narong
authors.initials: N
authors.lastname: Arunrut
authors.forename: Pakkakul
authors.initials: P
authors.lastname: Sangsuriya
authors.forename: Vanvimon
authors.initials: V
authors.lastname: Saksmerprome
authors.forename: Saengchan
authors.initials: S
authors.lastname: Senapin
authors.forename: Boonsirm
authors.initials: B
authors.lastname: Withyachumnarnkul
authors.forename: Wansika
authors.initials: W
authors.lastname: Kiatpathomchai
mesh_terms: D000818:Animals; D017756:Aquaculture; D005393:Fish Diseases / Q000175:diagnosis / Q000382:microbiology*; D016905:Gram-Negative Bacterial Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D020614:Shewanella putrefaciens / Q000302:isolation & purification*; D017210:Tilapia; D014871:Water Microbiology