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|Title:||Colorimetric method of loop-mediated isothermal amplification with the pre-addition of calcein for detecting flavobacterium columnare and its assessment in tilapia farms|
Thailand National Center for Genetic Engineering and Biotechnology
Prince of Songkla University
Nam Sai Farms
|Keywords:||Agricultural and Biological Sciences|
|Citation:||Journal of Aquatic Animal Health. Vol.27, No.1 (2015), 38-44|
|Abstract:||© American Fisheries Society 2015 Flavobacterium column are, the causative agent of columnar is 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.column are infarmed tilapia (NileTilapia 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 × 102 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.|
|Appears in Collections:||Scopus 2011-2015|
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