Preliminary Establishment And Evaluation Of A Visual RT-LAMP Detection Method For Foot-and-mouth Disease Virus

Fast and simple nucleic acid detection methods to detect foot-and-mouth disease virus(FMDV)would play vital role in epidemic prevention and control.The widely used fluorescent PCR method in laboratories is highly dependent on specific equipment and professionals which make it unsuitable for the pen-side diagnosis.Therefore,it is of great significance to establish a new nucleic acid detection method.The loop-mediated isothermal amplification(LAMP)method has some unique advantages such as easy operation,simple equipment,rapidity,high sensitivity and specificity which make this method suitable for the pen-side test.In this study,the RT-LAMP analysis was done by introducing indicator dyes into the reaction system and the newly-developed FMDV visual RT-LAMP assay provided convenience for the pen-side test for foot-and-mouth disease.Firstly,the genomes of seven serotypes of FMDV were analyzed using the highly conserved 3D gene encoding polymerase as the target region for RT-LAMP amplification.Through 3D sequence homology analysis,39 3D gene(3'-terminal end 500bp)sequences with extra 20 bp T7 promoter located upstream were selected,chemically synthesized and cloned into pUC57 vector with Ampicillin resistance.After the recombinant plasmids(3D-T7pUC57)were correctly identified,they were used in the follow-up tests.FMDV being an RNA virus,the corresponding 3D-T7pUC57 RNA transcripts were prepared and used to establish the visual RT-LAMP assay.For this assay,a systematic screening of RT-LAMP method of operation was performed with optimal primer concentration,reaction time,reaction temperature and chromogenic agents(hydroxy naphthol blue,Calcein,SYBR green I,neutral red,phenol red and bromothymol blue),with which visual RT-LAMP detection method for FMDV was established.The total reaction volume of RT-LAMP was 20?L containing 15.2?L of RT-LAMP buffer mixture,1.5?L Bst AMV enzyme mixture,0.04?L(100?M)each of F3 and B3 primers,0.5?L(100?M)each of FIP and BIP inner primers,0.11?L(100?M)each of loop primers Floop and Bloop as well as 1?L(500?M)of neutral red and 1?L template.The reaction time and temperature was 55 min and 65°C respectively.The change in the color of the reaction tubes to pink indicated positive result while orange indicated negative result.The FMDV visual RT-LAMP assay established here did not react with BVDV,ORFV,SPPV/GTPV,SVDV,BTV,PRRSV,CSFV,PRV,PPV and PCV₃,showing good analytical specificity.The serially ten-fold dilutions of RNA transcripts were used as the templates for analytical sensitivity detection and the results were compared with the detection method recommended by OIE.The result showed that the detection limit of RT-LAMP was 10⁴ copies/?L and the RT-q PCR and RT-PCR methods were 10³ and10⁴copies/?L respectively.39 3D RNA transcripts representing 7 FMDV serotypes and 44 clinical samples kept in National FMD Reference Lab were detected by the FMDV visual RT-LAMP and compared with RT-q PCR and RT-PCR.The results showed that the negative and positive detection rates of the RT-LAM were 100%and 95.7%.The results of six clinical samples by 10 times dilution showed that the detection limits of RT-LAMP and RT-PCR were the same,which was equivalent to the virus content when the CT value of RT-q PCR was 32.5.In this study,a visual RT-LAMP assay with good specificity and sensitivity for FMDV was successfully established.The convenient mode of operation,simple equipment(constant temperature hot block,such as metal bath)and visually observable test results make it suitable for pen-side FMDV diagnosis.This newly-developed FMDV visual RT-LAMP assay has a good prospective application.