DAO 117:133-143 (2015)  -  DOI: https://doi.org/10.3354/dao02942

Shewanella putrefaciens in cultured tilapia detected by a new calcein-loop-mediated isothermal amplification (Ca-LAMP) method

Rungkarn Suebsing1, Jantana Kampeera1, Sarawut Sirithammajak1, Padmaja Jayaprasad Pradeep3, Sarocha Jitrakorn2,3, Narong Arunrut1, Pakkakul Sangsuriya2, Vanvimon Saksmerprome2,3, Saengchan Senapin2,3, Boonsirm Withyachumnarnkul2,4,5, Wansika Kiatpathomchai1,*

1Bioengineering and Sensing Technology Laboratory, 2BIOTEC, National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Phahonyothin Rd., Klong Neung, Klong Luang, Pathum Thani 12120, Thailand
3CENTEX Shrimp, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand
4Department of Anatomy, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand
5Aquatic Animal Biotechnology Research Center, Faculty of Science and Industrial Technology, Prince of Songkla University, Suratthani Campus, Surat Thani 84100, Thailand
*Corresponding author:

ABSTRACT: Shewanella putrefaciens is being increasingly isolated from a wide variety of sources and is pathogenic to many marine and freshwater fish. For better control of this pathogen, there is a need for the development of simple and inexpensive but highly specific, sensitive, and rapid detection methods suitable for application in field laboratories. Our colorogenic loop-mediated isothermal amplification (LAMP) assay combined with calcein (Ca-LAMP) for unaided visual confirmation of LAMP amplicons is a simple method for fish pathogen detection in cultured tilapia. Here, we describe the detection of S. putrefaciens using the same platform. As before, the method gave positive results (orange to green color change) in 45 min at 63°C with sensitivity 100 times higher than that of a conventional PCR assay, with no cross-amplification of other known fish bacterial pathogens tested. Using the assay with 389 samples of gonads, fertilized eggs, and fry of farmed Nile and red tilapia Oreochromis spp., 35% of samples were positive for S. putrefaciens. The highest prevalence was found in samples of gonads (55%) and fertilized eggs (55%) from adult breeding stocks, indicating that S. putrefaciens could be passed on easily to fry used for stocking production ponds. Tissue tropism assays revealed that the spleen showed the highest colonization by S. putrefaciens in naturally infected tilapia and that it would be the most suitable organ for screening and monitoring fish stocks for presence of the bacteria.


KEY WORDS: Colorimetric · LAMP · Calcein · Oreochromis spp. · Tilapia · Tissue tropism


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Cite this article as: Suebsing R, Kampeera J, Sirithammajak S, Pradeep PJ and others (2015) Shewanella putrefaciens in cultured tilapia detected by a new calcein-loop-mediated isothermal amplification (Ca-LAMP) method. Dis Aquat Org 117:133-143. https://doi.org/10.3354/dao02942

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