Location: Aquatic Animal Health ResearchTitle: Optimized reverse primer for 16S-RFLP analysis and genomovar assignment of Flavobacterium columnare Author
|Dong, Ha - Chulalongkorn University|
|Waldbieser, Geoffrey - Geoff|
|Rodkhum, Channarong - Chulalongkorn University|
|Wong, Fong - Merck Animal Health|
|Chang, Siow - Merck Animal Health|
Submitted to: Genbank
Publication Type: Other
Publication Acceptance Date: 9/9/2016
Publication Date: 12/23/2016
Citation: Lafrentz, B.R., Garcia, J.C., Dong, H.T., Waldbieser, G.C., Rodkhum, C., Wong, F.S., Chang, S.F. 2016. Optimized reverse primer for 16S-RFLP analysis and genomovar assignment of Flavobacterium columnare. Genebank. Accession Nos. KX711893-KX711900.
Technical Abstract: Flavobacterium columnare is the causative agent of columnaris disease which severely impacts channel catfish production in the USA and has emerged as an important pathogen in the US rainbow trout industry. Our laboratory previously standardized a genetic typing system for F. columnare in which a portion of the 16S rRNA gene is amplified by PCR using specific primers and then digested with an enzyme that will cut the DNA at specific sites. Based on the number and size of DNA fragments generated, isolates of the bacterium are assigned to a genomovar (i.e., genetic type). During routine use of the typing system, it was observed that the 16S rRNA gene of some isolates did not amplify by PCR and could not be assigned to a genomovar. It was hypothesized that there was nucleotide differences in these isolates at the primer binding sites that accounted for the lack of amplification. To test this hypothesis, the 16S rRNA gene from these isolates were sequenced. Each sequence (n = 8) was deposited in GenBank, and the results demonstrated that these isolates exhibited nucleotide differences at the reverse primer binding site. A new reverse primer was designed, tested, and validated for the ability to amplify the 16S rRNA gene from these isolates as well as others. The new primer can be used in place of the original and should allow for genomovar assignment of all F. columnare isolates.