The Establishment And Application Of Isothermal Amplification Techniques In Detecting WSSV And YHV

In recent years, aquaculture has developed rapidly. It has gradually become oneof the industry, which develops fast in China’s agriculture, and it occupies a pivotalposition in the total global aquaculture. However, as the scale of aquaculture hasgrown, various diseases followed. Shrimp farming industry as one of the pillarindustries in the world aquaculture industry, also plagued by many diseases. Thesediseases caused significant economic losses to the shrimp farming industry. Therefore,the establishment of special and sensitive and efficient pathogen detection technologyis imminent. Isothermal amplification detection technology as a new means ofdetection, has been widely recognized by people, and has been widely applied invarious fields.White spot syndrome virus (WSSV) is one of the main pathogens seriouslythreatening penaeid shrimp farming in the world. This study designed and synthesized5specific primers based on the conserved sequences of WSSV genome, optimized thereaction system and conditions, and finally established a cross priming amplification(CPA)-based detection method for WSSV (WSSV-CPA). The results indicated thatthe optimized reaction temperature and time of WSSV-CPA were63°C and60minrespectively. The detection limit of the WSSV-CPA assay was as low as10copies/Land shared same sensitivity with the WSSV-qPCR assay. Due to dispensing withexpensive thermal cycler, time-and cost-saving, and ease of use in field, it isanticipated that the WSSV-CPA method developed in this study will be instrumentalfor the diagnosis and surveillance of WSSV.Yellow head virus is a new virus that can cause a large number of death inshrimp, and a new genotype of yellow head virus was found in China.In this study，aone-step, real-time reverse-transcription loop-mediated isothermalamplification(rRT-LAMP) assay was developed to rapidly detect and diagnose yellowhead virus (YHV), and compared with polymerase chain reaction (PCR) method andreal-time PCR method in accuracy, sensitivity and specificity. The detection limit ofthe LAMP method was100copies/μL, and it was100times more sensitive than the conventional PCR technique. A set of six specific primers was successfully designedfrom an restriction fragment of both the Tailand and China YHV genome. Mg2+concentrations, dNTPs concentrations, the reaction temperature, and the reaction timeof LAMP were optimized to8mM,1.4mM,58C, and60min, respectively. Thespecificity of the method was validated by the absence of any cross-reaction withRNA samples of other shrimp viruses, followed by nucleotide sequencing of theamplified product. The rRT-LAMP method for the detection of yellow head virusaestablished in this study, which contain the novel genotype yellow head virus, laidthe foundation for the pathogenic mechanism of virus research, clinical earlydiagnosis and quantitative analysis of the degree of infection type yellow head virus.Finally, this paper conducted an epidemiological analysis on WSSV and the newgenotype of yellow head virus. Using the established WSSV qCPA method detectWSSV and the established YHV rRT-LAMP method detect YHV. Through theepidemiological survey and analysis, a basic understanding of these two kinds of virusdiseases of shrimp incidence trend. By conducting shrimp speciesand and breedingarea with virus disease relations, we conclude that WSSV is one of the most importantshrimp pathogen; The yellow head virus have appeared in our two provinces, andChina’s shrimp farming industry are under a huge potential threat.