Premium
Colorimetric Method of Loop‐Mediated Isothermal Amplification with the Pre‐Addition of Calcein for Detecting Flavobacterium columnare and its Assessment in Tilapia Farms
Author(s) -
Suebsing Rungkarn,
Kampeera Jantana,
Sirithammajak Sarawut,
Withyachumnarnkul Boonsirm,
Turner Warren,
Kiatpathomchai Wansika
Publication year - 2015
Publication title -
journal of aquatic animal health
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.507
H-Index - 52
eISSN - 1548-8667
pISSN - 0899-7659
DOI - 10.1080/08997659.2014.966212
Subject(s) - tilapia , nile tilapia , loop mediated isothermal amplification , biology , oreochromis , calcein , bass (fish) , flavobacterium , microbiology and biotechnology , veterinary medicine , fishery , bacteria , fish <actinopterygii> , dna , genetics , medicine , membrane , pseudomonas
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 . Received May 21, 2014; accepted August 10, 2014