title: Colorimetric Method of Loop-Mediated Isothermal Amplification with the Pre-Addition of Calcein for Detecting Flavobacterium columnare and its Assessment in Tilapia Farms.
abstract: Flavobacterium columnare, the causative agent of columnaris disease in fish, affects many economically important freshwater fish species. A colorimetric method of loop-mediated isothermal amplification with the pre-addition of calcein (LAMP-calcein) was developed and used to detect the presence of F. columnare in farmed tilapia (Nile Tilapia Oreochromis niloticus and red tilapia [Nile Tilapia × Mozambique Tilapia O. mossambicus]) and rearing water. The detection method, based on a change in color from orange to green, could be performed within 45 min at 63°C. The method was highly specific, as it had no cross-detections with 14 other bacterial species, including other fish pathogens and two Flavobacterium species. The method has a minimum detection limit of 2.2 × 10(2) F. columnare CFU; thus, it is about 10 times more sensitive than conventional PCR. With this method, F. columnare was detected in gonad, gill, and blood samples from apparently healthy tilapia broodstock as well as in samples of fertilized eggs, newly hatched fry, and rearing water. The bacteria isolated from the blood were further characterized biochemically and found to be phenotypically identical to F. columnare. The amplified products from the LAMP-calcein method had 97% homology with the DNA sequence of F. columnare.
authors.affiliation: a Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University , Rama VI Road, Bangkok 10400 , 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: Boonsirm
authors.initials: B
authors.lastname: Withyachumnarnkul
authors.forename: Warren
authors.initials: W
authors.lastname: Turner
authors.forename: Wansika
authors.initials: W
authors.lastname: Kiatpathomchai
mesh_terms: D000818:Animals; D017756:Aquaculture; D003124:Colorimetry / Q000379:methods / Q000662:veterinary*; D005393:Fish Diseases / Q000175:diagnosis / Q000382:microbiology*; D045826:Flavobacteriaceae Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D005417:Flavobacterium / Q000145:classification / Q000302:isolation & purification*; D005452:Fluoresceins / Q000737:chemistry*; D021141:Nucleic Acid Amplification Techniques / Q000662:veterinary*; D017210:Tilapia
chemical_list: D005452:Fluoresceins; C007740:fluorexon