Study On Detection Of Duck Hepatitis A Virus Types 1 And 3 Using Reverse Transcription Loop-Mediated Isothermal Amplification

Duck Viral Hepatitis(DVH) is an acute contagious disease caused by the Duck Hepatitis A Virus(DHAV), The main characteristics of the disease are acute, high mortality and hepatic lesion, DHAVs mainly infect ducklings within 3 weeks old and spread widely all over the world. In our country, the main epidemic viruses are DHAV-1 and DHAV-3, which cause a great harm to the duck-feeding industry. The cross-neutralization reaction does not occur between DHAV-1 and DHAV-3,so they are two different serotypes. The two viruses can infect duckling and cause similar clinical symptoms and pathological changes, which give great difficulties in diagnosis and treatment of the disease. There are many methods to detect DHAV, such as virus isolation and characterization, immunological tests, serological tests, RT-PCR detection, and so on. These methods are not suitable for the detection of the viruses in the basic level laboratory because they are complicated and time-consuming, or need expensive instrument. Loop-Mediated Isothermal Amplification(LAMP) is a new DNA amplification techniques invented by Notomi T, a Japanese scholar, in 2000. The method uses 4 different primers which are specifically designed to recognize 6 distinct regions on target genes. The LAMP reaction can be completed at a constant temperature 65℃ by using Bst DNA polymerase in ten minutes. It has the characteristics of high efficiency, specificity, fast, convenience, etc. Aimed to develop the RT-LAMP methods of DHAV-1 and DHAV-3, respectively, the vp1 genes of DHAV-1 and DHAV-3 were downloaded from Gen Bank and analyzed by using Seq Men tools of software DNAStar, respectively. The "consesus" sequences were got by selecting the base which appears most frequently as the default base, and then these two sequences were aligened by using DNAMAN tools and the quite different segments were selected as the target genes. The outer primers F3/B3, inner primers FIP/BIP and loop primers LF/LB were then designed according to the target genes of vp1 by using the software Primer Explorer V4, respectively. DHAV-1 and DHAV-3 VP1 genes were ligated to p GEM- T vector, respectively, to obtain the respective VP1 gene RNA sample solutions by vitro transcription. The reaction systems including the concentration of Mg2 +, d NTPs, betaine and primers and the reaction conditions of temperature and time were optimized using the RNA sample solution according to the brightness of the 2% agarose gel electrophoresis bands. The reaction results could be visualized by adding hydroxy naphthol blue(HNB) as indicator. Specific test results showed, DHAV-1 RT-LAMP and DHAV-3 RT-LAMP assays had no cross-reaction with each other and other duck pathogens. Sensitivity test results showed that the detection limit of DHAV-1 RT-LAMP assay was 100 copies/μL, which was 102-fold higher than that of RT-PCR; DHAV-3 RT-LAMP assay was 10 copies/μL, which was 103-fold higher than that of RT-PCR.The duck embryo viruses of DHAV with known DELD50 were subjected to10-fold serial dilutions for RT-LAMP tests, the results showed that the RT-LAMP assays were sensitive enough to detect 4.2 DELD50 of DHAV-1 and 1.1 DELD50 of DHAV-3, respectively. The samples collected from artificially infected ducklings and field ducklings were detected usingthe established DHAV-1 RT-LAMP and DHAV-3 RT-LAMP assays. The results showed that viral RNAs could be detected in serum and liver samples and the cross-reaction did not occur between two viral RNAs. The detection results of the sera collected from the different time after infection showed that viral RNAs could be detected in part samples at 16 h after infection, in all samples at 22 h after infection and in the liver samples from the dead ducklings, respectively. The viral RNAs could not be detected in fecal samples from the infected ducklings before 22 h or from the field ducklings. Preliminary application showed the established RT-LAMP assays could early and rapidly detect the viral RNAs in the serum and liver samples from the infected ducklings and could identify the DHAV-1 or DHAV-3, which provide an important basis for the diagnosis of DVH and the use of the corresponding vaccines and antisera.