The Differentiation And Detection Of Virulent And Vaccine Strains Of Classical Swine Fever Virus By Loop-mediated Isothermal Amplification Assay

Classical swine fever (CSF) is a highly infectious disease affecting swine andboar, resulting in serious economic losses, characterized by fever, hemorrhage, andhigh mortality rates. CSF is caused by the classical swine fever virus(CSFV). CSFVis a member of the genus Pestivirus, belonging to the family Flaviviridae. CSFV is asmall, enveloped virus with a12.3kb positive, single-stranded RNA genome, whichcontains a large open reading frame encoding a polyprotein, which is cleaved intostructural and nonstructural proteins by cellular and viral proteases. Nonstructuralproteins contain eight proteins, that is P7、Npro、NS2、NS3、NS4A、NS4B、NS5A、NS5B. Structural proteins contain four proteins, that is Erns、 C、 E1、 E2.5′-untranslated regions (UTR)、3′-untranslated regions (UTR) and NS5B are highlyconserved among all of the virus isolates. The NS5B gene can be used for genotyping.Recently, Hog Cholera Lapinized Virus(HCLV) is often used to prevent and controlCSFV, the method of detecting CSFV wild-type infection and vaccination isincomplete, so it is hard to make a distinction between wild-type infection andvaccination.To develop a rapid and practical method to differentiate virulent and vaccinestrains of classical swine fever virus, a loop-mediated isothermal amplification assay（LAMP）was established with a set of primers based on the NS5A gene of CSFV.Eleven genomic sequences of pestiviruses, including5virulent strains of CSFV,4vaccine strains of CSFV, BVDV-1and BVDV-2were aligned with MegAlign.External primers F3and B3, internal primers FIP and BIP and loop primers LF andLB specific for HCLV and virulent strains of CSFV respectively were designed by theonline LAMP designing software Primer Explorer V4. The viral cDNA synthesizedby reverse transcription was amplified with Bst DNA polymerase at a constanttemperature of65℃, then a ladderlike pattern of products was visible on20g/Lagarose gel electrophoresis, and products could be visualized with rhodamine B dye.The results showed that no cross-reactivity was observed in the samples with other related viruses. Detecting limit of the LAMP assay was1000fold higher than that ofRT-PCR. In detecting20swine samples by LAMP assay and RT-PCR, thecoincidence rate of LAMP and RT-PCR was90.91%. Therefore, the assay was asensitive, simple, rapid and practical method for differentiation and detection ofvirulent and vaccine strains of classical swine fever virus. It played an important rolein controlling CSFV infection and detecting CSFV.