DAO 120:39-47 (2016)  -  DOI: https://doi.org/10.3354/dao03021

Duplex PCR assay and in situ hybridization for detection of Francisella spp. and Francisella noatunensis subsp. orientalis in red tilapia

Ha T. Dong1,3, Warachin Gangnonngiw2,3, Kornsunee Phiwsaiya2,3, Walaiporn Charoensapsri2,3, Vuong V. Nguyen1,4, Pål Nilsen5, Padmaja J. Pradeep6, Boonsirm Withyachumnarnkul2,7,8, Saengchan Senapin2,3,*, Channarong Rodkhum1,*

1Department of Veterinary Microbiology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand
2National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani 12120, Thailand
3Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, 272 Rama VI Road, Bangkok 10400, Thailand
4National Broodstock Center for Mariculture Species, Research Institute for Aquaculture No. 1, Tu Son, Bac Ninh, Vietnam
5PHARMAQ AS, PO Box 26, 0213 Oslo, Norway
6Aquatic Animal Biotechnology Research Center, Faculty of Science and Industrial Technology, Prince of Songkla University, Surat Thani Campus, 84100, Thailand
7Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
8Shrimp Genetic Improvement Center, Surat Thani 84100, Thailand

ABSTRACT: Conventional isolation and identification based on phenotypic characteristics is challenging with the highly fastidious, intracellular bacterium Francisella noatunensis subsp. orientalis (Fno). Here, we developed a duplex PCR method for simultaneous detection of the Francisella genus and Fno in one PCR reaction and an in situ hybridization method for paraffin section based diagnosis of Fno. The PCR results showed genus- and species-specific bands (1140 and 203 bp) from Fno but only one genus-specific band (1140 bp) from F. noatunensis subsp. noatunensis. Sensitivity of the duplex PCR assay revealed a detection limit of 20 to 200 fg genomic DNA (~10 to 100 genome equivalents) depending on DNA template extraction methods. The newly developed duplex PCR assay could be used to detect Fno from clinically sick fish exhibiting signs of visceral granulomas and would also be able to detect Fno infection in naturally diseased fish without symptoms of francisellosis, indicating potential application for diagnosis of field samples. The in situ hybridization assay using Fno species-specific probe revealed positive signals in multiple organs including the spleen, liver, kidney, gills and intestine of infected fish.


KEY WORDS: Molecular diagnostic assay · Francisellosis · ISH · Histology · Oreochromis · Aquaculture


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Cite this article as: Dong HT, Gangnonngiw W, Phiwsaiya K, Charoensapsri W and others (2016) Duplex PCR assay and in situ hybridization for detection of Francisella spp. and Francisella noatunensis subsp. orientalis in red tilapia. Dis Aquat Org 120:39-47. https://doi.org/10.3354/dao03021

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