ABSTRACT
White spot syndrome virus (WSSV) is a global threat to the prawn industry, and there is no simple method for field-based testing of this virus. We designed a padlock probe and primers to the capsid protein gene VP28 of WSSV, and established a hyperbranched rolling circle amplification (HRCA) assay and a corresponding strip-based test. The assay and the test strip both had similar high accuracy and specificity, and their sensitivity was about 10 copies/µL, which is 100 times higher than conventional PCR. In this study, 68 batches of prawns were tested for WSSV with the HRCA assay and test strip, and the results were compared with the PCR assay. The results indicated that both the assay and test strip had accuracy similar to each other and to the PCR results. However, the assay and strip were more sensitive and user-friendly than PCR. Establishment of this method will provide a rapid detection of WSSV and also a basis for field-based detection of animal disease.
Subject(s)
Nucleic Acid Amplification Techniques/methods , Pandalidae/virology , Reagent Strips , White spot syndrome virus 1/isolation & purification , Animals , Sensitivity and Specificity , White spot syndrome virus 1/geneticsABSTRACT
An assay was developed for the detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) based on real-time quantitative polymerase chain reaction (PCR). A pair of primers and a TaqMan probe were designed that are specific for the recognition of a conservative region in the IHHNV genome. The IHHNV real-time PCR assay had a detection limit of 9 DNA copies, with a dynamic range of detection between 9 x 106 and 9 DNA copies. The primer pairs and probe were specific to IHHNV and did not cross-react with shrimp genomic DNA or other shrimp viruses such as White Spot Syndrome Virus (WSSV), Monodon Baculovirus (MBV), and hepatopancreatic parvovirus (HPV). This assay has a broad application for basic and clinical investigations. For clinical samples, the real-time PCR assay detected all the positive samples screened by conventional PCR, which indicated the sensitivity of the real-time assay. The IHHNV real-time PCR assay with high sensitivity, specificity, wide range of detection ability, and simplicity is particularly useful for screening large numbers of specimens and measuring viral loads to monitor the broodstock.
Subject(s)
Chemistry Techniques, Analytical/methods , Crustacea/virology , Infectious hematopoietic necrosis virus/metabolism , Oligonucleotide Probes/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Animals , DNA/metabolism , DNA Primers/chemistry , Necrosis , Plasmids/metabolism , Polymerase Chain Reaction , Sensitivity and Specificity , Viral Load , Virus Diseases/metabolismABSTRACT
Constructing genetic linkage map is an essential tool to acknowledge genome in aquaculture species. This paper has reviewed the current status of genetic linkage map research, including mapping population, mapping method and molecular markers used to construct linkage map. Linkage map has great potential in marker assisted selection (MAS), gene locating and cloning, and comparative genome mapping. Genetic linkage map with high density and wide coverage of genome will allow cloning the genes which contribute to economically important traits. The ultimate aim of the constructing linkage map is the development of fast-growing, disease-resistant strains of the major aquaculture species.
