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Jitrakorn S.,National Science and Technology Development Agency | Jitrakorn S.,Mahidol University | Arunrut N.,Bioengineering and Sensing Technology Laboratory | Sanguanrut P.,Mahidol University | And 5 more authors.
Aquaculture | Year: 2016

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is also known as Penaeus stylirostris densovirus (PstDNV). Since it is listed as a reportable crustacean disease by the World Organization for Animal Health (OIE), many countries require testing for IHHNV in shrimp stocks imported for aquaculture, necessitating rapid and highly sensitive detection methods to guarantee and maintain absence of IHHNV in traded stocks. Here we describe a new, highly sensitive method for detection of IHHNV in shrimp tissues. It involves in situ loop-mediated DNA amplification (IS-LAMP) employing digoxigenin-11-dUTP (DIG-dUTP) as part of the deoxynucleotide triphosphate (dNTP) mix. This resulted in DIG-labeling of the DNA amplicons (i.e., in situ, DIG-labeling LAMP or ISDL) allowing direct in situ, immuno-histochemical detection of IHHNV using an anti-DIG antibody conjugated with alkaline phosphatase, and eliminating the lengthy hybridization step of conventional ISH. The target region for the method is at the 3'end of the IHHNV genome that has been reported least likely to give rise to endogenous viral elements (EVE) in the shrimp genome. When compared to the results for IHHN detection by conventional in situ hybridization (ISH) using a DIG-labeled oligonucleotide probe, signals from the ISDL method were stronger and also gave positive results with some tissues unreactive by the conventional ISH method. Additional comparison of the two methods revealed that the proteinase-K digestion step needed for normal ISH was unnecessary for ISDL. This further shortened the assay time and improved the tissue integrity necessary to clearly indicate the location of positive hybridization signals. The ISDL method gave no cross- reactions with tissues of shrimp infected with other common pathogens, and the total time needed to carry out ISDL was one-third of that needed for conventional ISH. Statement of relevance: This work describes an in situ, DIG-labeling LAMP (ISDL) method, as a new, highly sensitive method for detection of IHHNV in shrimp tissues. The developed method can be used as a confirmatory test carried out with tissue sections to confirm the results from molecular assays, especially when immune-histochemical methods are not available to do so. ISDL is especially important for molecular detection from a new or unexpected location and for molecular detection in newly suspected carriers to determine whether they are actually infected or just mechanical carriers. © 2016 Elsevier B.V.

Suebsing R.,Bioengineering and Sensing Technology Laboratory | Kampeera J.,Bioengineering and Sensing Technology Laboratory | Sirithammajak S.,Bioengineering and Sensing Technology Laboratory | Pradeep P.J.,Mahidol University | And 12 more authors.
Diseases of Aquatic Organisms | Year: 2015

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. © Inter-Research 2015.

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