Establishment Of Loop-mediated Isothermal Amplificatiaon For Detecting Of Pseudorabies Virus And Japanese Encephalitis Virus

Pseudorabies virus （PRV） is a member of the subfamily Alphaherpesvirinae,which causes a disease with a worldwide distribution in swine. It can cause fever,itching and encephalomyelitis in a variety of domestic and wild animals. Japaneseencephalitis virus （JEV）, transmitted by mosquitoes, is a significant cause of epidemicencephalitis in variety of animals. Both of these virus can cause abortion of pregnantsows, dead fetus or mummified and orchitis or infertility in boar. Thus, it can causesignificant economic losses in the swine industry. Isolation and identification ofviruses, Enzyme-Linked Immunosorbent Assay （ELISA）, Neutralization Tests andPolymerase Chain Reaction （PCR） are the most commonly diagnostic methods fordetecting PRV and JEV. Although these methods play a huge role in the practice, it istime consuming, more expensive, low sensitivity and requires specialized equipment.The purpose of this study is to establish a detection method, which is simple, rapid,high sensitivity and specificity.In this study, six PRV-LAMP primers were designed based on sequence of DBPPRV. The reaction system included2.5μL of10×Thermopol reaction buffer,2.0μL ofdNTP （10mmol/L）,1.5μL of Mg²⁺（50mmol/L）,6.0μL of mixed primer （FIP andBIP:40μmol/L; F3and B3:20μmol/L; LF and LB:5μmol/L; diluted primers weremixed with equal amount）,1.0μL of a Bst DNA polymerase （8U）,2.0μL of cDNAtemplate, plus9.0μL double distilled water. Six JEV-LAMP primers were designedbased on JEV’s of prM protein sequence. The reaction system included2.5μL of10×Thermopol reaction buffer,2.5μL of dNTP （10mmol/L）,2.0μL of Mg²⁺（50mmol/L）,6.0μL of mixed primer （FIP and BIP:40μmol/L; F3and B3:20μmol/L; LF and LB:5μmol/L,; diluted primer were mixed with equal amount）,1.0μL of a Bst DNApolymerase （8U）,2.0μL of cDNA template, plus9.0μL of double distilled water.In this paper, we established two LAMP methods for the detection of PRV and JEV. The sensitivity of PRV-LAMP （63℃for40min） is100times higher than thePCR, without any cross-reaction to porcine circovirus2（PCV₂）, procine reproductiveand respiratory syndrome virus （PRRVS）, swine fever virus （CSFV）. The sensitivity ofJEV-LAMP （61℃for50min） is10times higher than PCR, without any cross-reaction to PRV, PCV₂, PRRSV. Because of the combination of LAMP methods andfluorescent dye rhodamine B, the amplification result can be read with naked eyesaccording to the color change （blue for positive, purple for negative）. To sum up, weestablished a couple of LAMP methods, which is simple, rapid, high sensitivity,specificity and requires no expensive equipment. A preliminary application of thesediagnostic methods represents a reliable diagnostic result.