Sensitive and rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimps by loop-mediated isothermal amplification

A method for nucleic acid amplification, loop-mediated isothermal amplification (LAMP) is a novel, sensitive and rapid technique, which can be applied for disease diagnosis in aquaculture. Using the LAMP method, a highly specific and sensitive diagnostic system for infectious hypodermal and hematopoietic necrosis virus (IHHNV) detection was designed. A set of four primers was designed by targeting the IHHNV genome DNA. By the detection system, target DNA was amplified and visualized on agarose gel within 60min under isothermal condition at 64 degrees C. Without gel electrophoresis, the LAMP amplicon was visualized directly in the reaction tube by addition of SYBR Green I for a naked-eye inspection. The LAMP reaction was also assessed by the white turbidity of magnesium pyrophosphate (a by-product of LAMP) in the tube. The assay had a detection limit of 5-500 copies of DNA template with gel electrophoresis, SYBR Green I and white turbidity with naked-eye inspection. The detection sensitivity of LAMP was 100-fold higher than the PCR. A diagnostic procedure which is rapid and highly sensitive was developed for IHHNV detection.     

Rapid and sensitive detection of infectious hypodermal and hematopoietic necrosis virus by loop-mediated isothermal amplification combined with a lateral flow dipstick

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is an important shrimp pathogen that causes mortality in Penaeus stylirostris and stunting (called runt deformity syndrome or RDS) in Penaeus vannamei. Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acids under isothermal conditions. It can be combined with a chromatographic lateral flow dipstick (LFD) for highly specific, rapid and simple visual detection of IHHNV-specific amplicons. Using this protocol, a 30-min amplification followed by 5 min hybridization with an FITC-labeled DNA probe and 5 min LFD resulted in visualization of DNA amplicons trapped at the LFD test line. Thus, 10 min for rapid DNA extraction followed by LAMP combined with LFD detection resulted in a total assay time of approximately 50 min. Detection sensitivity was comparable to other methods used commonly for nested PCR detection of IHHNV but had the additional advantages of reduced assay time, confirmation of amplicon identity by hybridization and elimination of electrophoresis with carcinogenic ethidium bromide.     

Isolation, purification and characterization of infectious hypodermal and hematopoietic necrosis virus (IHHNV) from penaeidshrimp

A protocol was developed for the isolation and purification of the infectious hypodermal and hematopoietic necrosis virus (IHHNV) of penaeid shrimps. Cesium chloride-banded virus obtained from three sources of infected shrimps was found to have a buoyant density of 1.33 g/cm3. Also, electron microscopical studies employing negative stain revealed isometric particles having a size of 19 +/- 1 nm. Colorimetric analysis of its nucleic acid type indicated that these particles contain only RNA.     

A new virus isolate from infectious hypodermal and hematopoietic necrosis virus (IHHNV)-infected penaeid shrimps

A new virus was isolated from infectious hypodermal and hematopoietic necrosis virus (IHHNV)-infected penaeid shrimps. The virus was isolated from two species of penaeid shrimps obtained from three different sources employing a previously developed cell-culture assay. Electron-microscopical studies of both purified virus and infected cells showed bullet-shaped particles identifying it as a rhabdovirus.     

White spot syndrome virus and infectious hypodermal and hematopoietic necrosis virus simultaneous diagnosis by miniarray system with colorimetry detection

Highly sensitive and specific diagnostic tools are essential for monitoring the health status of farmed species. After the development of genomic probe diagnostic systems in the 1990s, followed by PCR-based systems, a miniarray method has been developed allowing one-step multiple detection. The miniarray method was developed to enable the accessibility of powerful array technology. To use this system, hybridisation and washing process were modified, resulting into a significant increase in the test's rapidity and sensitivity. With miniarray technology, hybridisation time is reduced to 20 min, whereas other methods require a longer hybridisation time. Hybridisation of the PCR product on a nylon membrane and revelation of the hybrids by an antibody increase considerably the ability of pathogen's detection. A first application is developed for the diagnosis of two specific viruses which are, by their geographical range and their impact on the production, very important in shrimp pathology, namely, the White Spot Syndrome Virus (WSSV) and the Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV).     

Genomic sequence of infectious hypodermal and hematopoietic necrosis virus (IHHNV) KLV-2010-01 originating from the first Korean outbreak in cultured Litopenaeus vannamei

Due to the need to track and monitor genetic diversity, the genome of the infectious hypodermal and hematopoietic necrosis virus (IHHNV) strain KLV-2010-01 in cultured Litopenaeus vannamei shrimp that originated from the first Korean outbreak in 2010 was sequenced and analyzed. The genome, with a length of 3914 nucleotides, was sequenced from the Korean IHHNV. The genome encoded three large and overlapping open reading frames: ORF1 (NS-1) of 2001 bp, ORF2 (NS-2) of 1092 bp and ORF3 (capsid protein) of 990 bp. The overall organization, size and predicted amino acid sequence of the three ORFs in Korean IHHNV were highly similar to those of members of the infectious IHHNV group, and the most closely related strains were IHHNVs described from Ecuador and Hawaii. Additionally, phylogenetic analysis showed that the Korean IHHNV was clustered with lineage III in the infectious IHHNV group and was most similar to IHHNV isolates from Ecuador, China and Taiwan.     

Rapid and sensitive detection of infectious spleen and kidney necrosis virus by loop-mediated isothermal amplification combined with a lateral flow dipstick

Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acids under isothermal conditions. In this report, a 20-min LAMP amplification of the DPOL gene of infectious spleen and kidney necrosis virus (ISKNV) using a biotin-labeled primer was combined with lateral flow dipstick (LFD) chromatography for rapid and simple visual detection of ISKNV-specific amplicons. The LFD process involves a 5-min specific hybridization with an FITC-labeled DNA probe to confirm the presence of complement ISKNV amplicons that were biotinated in LAMP. The resulting DNA duplexes, consisting of labeled probes and amplicons, migrate along the LFD strip by chromatography for 5 min and are trapped at the test line and visualized by biotin labeling. The detection limit of ISKNV by LAMP-LFD was 10 copies. The results show that the LAMP-LFD method has the advantages of better sensitivity and speed and less dependence on equipment than the standard PCR for specifically detecting low levels of ISKNV DNA, and this can be useful in the field as a routine diagnostic tool.     

Rapid detection of orf virus by loop-mediated isothermal amplification based on the DNA polymerase gene

At present, there are no effective antiviral treatments available for contagious ecthyma, and rapid diagnosis is therefore critical for effective control of the disease. Recently, the invention of a novel LAMP technique that can rapidly amplify nucleic acids with high specificity and sensitivity under isothermal conditions has overcome some of the deficiencies of nucleic acid-based diagnostic tests and has made on-site diagnosis possible. To establish a flexible loop-mediated isothermal amplification (LAMP) assay for the rapid detection of orf virus, two pairs of primers, including outer primers F3/B3 and inner primers FIP/BIP, were designed to amplify the DNA polymerase gene. Optimal time and temperature conditions for LAMP were found to be 45 min and 62 °C, respectively. The LAMP assay was shown to be specific, with no cross-reactivity with sheeppox virus, goatpox virus, avian molluscum roup virus or vesicular stomatitis virus. Additionally, the sensitivity of the LAMP method was similar to that of real-time PCR and demonstrated greater sensitivity than a conventional polymerase chain reaction (PCR) assay. To assess the utility of LAMP in the detection of orf virus in clinical samples, a total of 35 samples collected from orf virus-infected sheep and goats were tested using the optimized LAMP assay, real-time PCR, and conventional PCR. Of the samples, 26 were found to be positive by LAMP, and 25 (74.3 %) were positive by real-time PCR, whereas only 18 (51.4 %) were positive by conventional PCR. Our results have shown that the LAMP assay developed in this study can be used for the rapid detection of orf virus.     

Rapid detection of bovine herpesvirus 1 in bovine semen by loop-mediated isothermal amplification (LAMP) assay

Bovine herpesvirus 1 (BoHV-1) is the most common viral pathogen found in bovine semen, causing numerous reproductive disorders leading to economic losses to the cattle industry. For rapid detection of BoHV-1 in bovine semen, in this study, we applied a loop-mediated isothermal amplification (LAMP) assay. The assay could be completed within 90 min, including total DNA isolation, target amplification, and visual interpretation of positive or negative results with the naked eye. The assay detected as little as 10 fg of BoHV-1 DNA per reaction. The analytical sensitivity of the assay was 0.2 TCID₅₀ BoHV-1 per reaction, which was 100 times more sensitive than conventional PCR and comparable to TaqMan real-time PCR. The applicability of the assay was assessed by analysing 118 semen samples collected from breeding bulls. On comparison with TaqMan real-time PCR, the LAMP assay had a diagnostic sensitivity of 97 %, specificity of 100 %, and accuracy of 99.2 % for detection of BoHV-1 in bovine semen. The LAMP assay developed in this study is a rapid, sensitive, and cost-effective alternative for detection of BoHV-1 in bovine semen.     

Rapid simultaneous detection and quantitation of infectious pancreatic necrosis virus (IPNV)

Infectious pancreatic necrosis virus (IPNV) is a pathogen of great concern in the salmon industry as well as in the environment. Taking advantage of the early immunofluorescent visualization of viral proteins in infected cells, a titration method was developed. At 16 h p.i., fluorescent foci were visualized with a monoclonal antibody against VP3-structural protein of the virus. The counting of each fluorescent cell allows the quantitation of infection foci; titres expressed in fluorescent foci/ml were equivalent to plaque forming units (PFU)/ml. With slight modifications, the same method used to detect the virus in field samples, can be applied to estimate virus contents. Some of the samples used during the assays were obtained from routine screening procedures. The titres recorded from positive samples correlated well with the clinical condition of the fish. With this method, rapid diagnosis and quantitation may simultaneously be performed with the same tissue extract.