Establishment And Application Of RT-PCR And RT-LAMP For Differential Diagnosis Of Grass Carp Reovirus

Grass carp Reovirus (GCRV) has been assigned to an acute infectious Aquareovirus genus in the family of Reoviridae which lead to hemorrhagic disease and extremely high mortality rate in grass carp, one of the most mainly promising species for aquaculture in China. Grass carp hemorrhage disease is mainly clinically characterized on body surface, as well as the tissues of mouth, eye, gill, liver and spleen. It resulted in an almost90%mortality, not only brought serious economic losses for aquiculture but also endangered human health.According to multiple sequence comparison of highly pathogenic GCRV strainVP7gene published in GenBank, the primers of RT-PCR and RT-LAMP were designed. A fragment of345bp containing the region of GCRV-VP7between225bp and569bp could be respectively amplified by the two methods. The reaction conditions were optimized including the amount of template, Mg2+, dNTPs, DNA polymerase, the concentration and rate of primers, and Tm etc. The experiments results showed that the LAMP mixture was made in25μl of reaction mixture containing40μM each inner primer (FIP and BIP),5μM each outer primer (F3and B3),8μl dNTPs (2.5mM),10mM MgCl2,2.5μl10×Bst DNA Buffer,1μl Bst DNA polymerase(8U/μl) and1ul templates; the optimal reaction time and temperature were determined to be40min at63℃. Conditions for RT-PCR (25ul) amplification were5min at95℃, followed by35cycles of incubation at94℃,54℃, and72℃for45s each, followed by one cycle of final extension at72℃for10min. Sensitivity was tested using10-fold serial dilutions of template. Specificity test was carried out by choosing (Aeromonas veronii),(Aeromonas hydrophila) and (giant salamander iridovirus, GSIV) as the positive control. The results indicated that the sensitivity and specificity of RT-LAMP was almost equal to that of RT-PCR. Detected48suspected materials that collected from various regions of Sichuan Province by RT-PCR method, the result showed18samples were positive while the same as by RT-LAMP. The result of comparison test were no significant difference, which indicated that the detection method was high accuracy. Furthermore, for LAMP method is carried out under isothermal conditions with no special apparatus, which makes it more economical and practical than PCR. It is promising to facilitate the gene analysis applied in clinical laboratory and field surveillance. Meantime, it also has potentials for rapid test to detect the GCRV.