Establishment Of Single Cross Priming Isothermal Amplification For The Visual Detection Of Koi Herpesvirus

Koi herpesvirus disease(KHVD) is the most severe viral disease in farmed common carp, Cyprinus capio L. all over the world. It has been declared as a notifiable disease by the World Organisation for Animal Health(OIE) and classified into the second category of epidemic animal diseases of our country. The Koi herpesvirus(KHV) is highly contagious and virulent, and only infects koi carp, common carp and its common variants. Many detecting methods for KHV have been developed depending on expensive equipments and professional technicians, or are time consuming, which cannot meet the requirements for KHV on-site diagnosis. Therefore, the development of a simple, sensitive and rapid method is significance for the diagnosis, prevention and control of the disease. In this study, the epidemiology and detection methods of koi herpesvirus disease, as well as the principle and applications of cross priming isothermal amplification technology, etc. were introduced in detail. In this study, a visual detection method for KHV has been developed by using the single cross priming isothermal amplification method and nucleic acid test strip detection combined with the colliadal gold labled. The contents and results of the study are listed as follows:1. Primers design and the construction of a viral DNA recombinant plasmid. According to the conserved sequence of K HV DN A polymerase gene(Sph), a set of single cross amplification primers were designed, including cross primer 2a1 s, detecting primers 2a(2a-Bio) and 3a(3a-FIT), displacement primers 4s and 5a. KHV was propagated in the Koi-Fin cell line and the genomic DNA of the virus was extracted as the template for PCR. The partial fragment of KHV Sph gene was amplified using primer 4a and 5a, then it was cloned into p MD®-19 T vector constructing recombinant plasmid p MD®-19T-Sph. The recombinant plasmid p MD®-19T-Sph was used as the standard DN A template in the following experiments.2. A basic reaction system and reaction conditions of single cross priming amplification were established according to the previo usly study. In order to obtain the optimized CPA reaction conditions, the concentration and proportion of each primer, Mg2+, Betaine and the dosage of Bst DNA polymerase and the reaction temperature and time were optimized. The final concentrations of each component in the optimal reaction condition are as follows: 1×Thermo Pol® Reaction Buffer, cross primer 2a1 s 1.0 μmol/L, displacement primers 2a, 3a 0.5 μmol/L, respectively, displacement primers 4s, 5a 0.6 μmol/L, respectively, the concentration of Mg2+ 8.0 mmol/L, d N TPs 1.2 mmol/L, Betaine 0.7 mol/L, the dosage of Bst DN A polymerase 5.6 U, DNA template 1 μL, nuclease- free water added to a total volume of 25 μL. The optimal amplification temperature is 63 ℃, the optimal reaction time is 60 min and the amplified products showed DNA ladder analyzed by agarose gel electrophoresis.3. The specificity and sensitivity test of KHV-SCPA. The genomic DNA of Koi herpes virus(KHV), Cyprinid herpesvirus II(Cy HV-2), Anguillae herpesvirus(Ang HV), Chinese giant salamander iridovirus(GSIV), White spot syndrome virus(WSSV) and Aeromonas hydrophila(Ah) were extracted a nd used in the specificity test of K HV-SCPA. The result showed that KHV could be specifically detected by the KHV-SCPA. Compared with the conventional PCR detection method, the sensitivity of KHV-SCPA method is about 1,000 times higher than that of conventional PCR. Combing with the nucleic acid test strip, the KHV-SCPA reaction products could be visually detected in 3~5 min.In summary, the established KHV-SCPA method could realized DNA amplification under 63 ℃ within 60 min. It could specifically detect KHV with a sensitivity of 100 copies/μ l viral DNA, which was about 1,000 times higher than that of the conventional PCR method. In addition, combing with the nucleic acid test strip detection technology, the visual detection of reaction product was realized within 3~5 min. The visible KHV-SCPA detection method does not require expensive equipment and skilled technicians, and it could be applied in the on-site diagnosis, which was significant for the effective prevention and control of Koi herpesvirus disease.