Isolation And Identification Of Porcine Encephalomyocarditis Virus And Development Of Rapid Detection By A RT-LAMP Method

Porcine encephalomyocarditis is an acute infectious disease of swine caused by encephalomyocarditis virus(EMCV). This disease is clinically characterized by encephalitis, myocarditis and reproductive failure in sows. The death rate was 100%. It was a zoonosis, not only brought serious economic losses for pig feeds but also endangered human health.In this study, EMCV has been isolated from the clinical tissue samples of farms in Hebei, by cell separation, RT-PCR, TCID50 and physicochemical characteristic. The result showed: Cytopathogenic effects (CPE) (circle shrinking, disintegration) were showing after three generations of blind passages; the fragment length of RT-PCR consistented with the expected length(286 bp); results of physicochemical characteristic tests indicated that the EMCV strains were not able to endure acid and not sensitive to chloroform and trypsin either. Exposed under 50℃for 1 h, the viruses could be inactivated .Loop mediated isothermal amplification (LAMP) technique is a novel nucleic acid amplification method which relies on autocycling strand displacement DNA synthesis performed by the Bst DNA polymerase large fragment. It is an isothermal reaction uses a single temperature and the result can be directly judged through the colorimetric detection of nucleic acid dye. A set of primers, which composed of outer primers, inner primers, was designed for a published EMCV consensus sequencesequence data(6232 bp-6441 bp). The experiments results showed that The LAMP mixture was made in 25μL of reaction mixture containing 2.5μL each inner primer (FIP and BIP), 0.5μL each outer primer (F3 and B3), 3μL dNTPs (2.5mM), 2.5μL MgCl2(25mM), 2.5μL 10×Bst DNA Buffer, 1.5μL Bst DNA polymerase large fragment, 2μL RNA templates and 6μL DEPC H2O; the optimal reaction time and temperature were determined to be 50 min at 63℃. RT-LAMP turned out to be more sensitive than conventional RT-PCR; no cross reactivity was observed from the samples of other related viruses; the repeatability and stability of RT-LAMP was high.In summary, porcine encephalomyocarditis virus (BD2) was successfully isolated, which provides a diseased source for studing detection method of EMCV; and RT-LAMP method to detect encephalomyocarditis virus (EMCV) was developed.