Establishment Of Universal Rapid Detection Method For Foot And Mouth Disease Virus Based On Reverse Transcription Loop Mediated Isothermal Amplification(RT-LAMP)

FMD is one kind of animal epidemic diseases infected by FMDV.FMDV mainly infect cloven-hoofed animals,and swines,cows and sheep are the most easily infected animals.Humans and other animals are seldom infected.The lethality of FMDV is quite higher,the death rate of calves sometimes is as high as 100%,and the majority of calves are often sudden death without any infectious symptoms,what's more,adult individuals can be cured.FMD can constitute a great threat to feeding the livestock breeding industry and human health.It's one of the animal infectious diseases reported by the OIE.FMDV is RNA virus,which can mutate faster and brought huge difficulty to the development of FMD vaccine.Pathogen detection,serological testing and molecular biology detection are common traditional detection methods of FMDV,which require high technology,long detection cycle and are limited in the amplification for the detection of grass-roots organizations and spot detection.Reverse transcription loop mediated isothermal amplification(RT-LAMP)is a novel technology.The operation of the technology is simple and the test isquick.The whole method can complete in one hour with the help of a one water bath.Based on this principle,we explored and established a rapid detection method of RT-LAMP for FMDV.The main results were as follows:1)Primers design: By compare with the whole genome sequence of the three serotype of FMDV,we designed forty RT-LAMP primers with the help of primer explorer V5.0.Accroding to the primer designing principles,we selected three primers and finally we established the RT-LAMP detection method for the detection of FMDV.2)Optimization of the method: In order to improve the efficiency of RT-LAMP detection,the reaction system and reaction conditions were optimized.The RT-LAMP final reaction system of 25 ?L was as follows : the concentration of outer primer(F3 / B3)was 12 m M;the concentration of internal primer(FIP / BIP)was 40 m M;the concentration of loop primer(LF / LB)was 35 m M;the concentration of magnesium ions was 8 m M;the concentration of betaine was 0.24 M;the concentration of d NTP was 1.8 m M;the concentration of Bst DNA large polymerase was 6.4 U / ?L;the concentration of AMV reverse transcriptase enzyme was 0.16 U /?L;the amount of 10 × Bst DNA large fragment polymerase Buffer was 2.5 ?L;the amount of dye was 1?L;the amount of template was 2 ?L.Finally,making up the rest system with sterile double distilled water.LAMP reaction program: 63 ?,60min;80 ?,10 min.3)Sensibility test: The sensitivity of the RT-LAMP method was tested by using the seven different FMD samples from the Lanzhou Veterinary Research Institute(LVRI)of the Chinese Academy of Agricultural Sciences.Comparing with the detection of LVRI detection kit,the ? Rn values were higher than that of LVRI detection kit.As a result,the sensibility of RT-LAMP method was higher than LVRI detection kit.What's more,the RT-LAMP detection method in this literature was more sensitive to Mya-98,Pan Asia and Sea-97 G2.4)Sensitivity test: The detection method established in this study can detect three serotypes of FMD,and the sensitivity of O type was the highest.The sensitivity test of FMDV was detected by fluorescence method and nucleic acid dyemethod with the 10 times the gradient dilutions of type O,type A and type Asian?.The RT-LAMP detection limit of type O,type A and type Asian?were 10-6,10-4,and10-2.Compared with other FMDV detection methods,such as the RT-q PCR detection method of OIE,FMDV detection kit of LVRI and the RT-PCR detection method of NVDC,the sensitivity of type O was the same as that of the NVDC detection method and 1000 times higher than that of OIE detection method,and 100 times higher than that of LVRI detection kit.The sensitivity of type A was the same as that of NVDC detection method and 100 times higher than that of the OIE detection method and the LVRI detection kit.The sensitivity of type Asia?were lower than traditional detection methods.5)Specificity test: The specificity of the detection method established in this study was strong.The nucleic acid of type O,type A,type Asian?of FMDV,CSFV,PCV,PPV,PRV,PRRSV,Hps,BVDV were used as the template to study the specificity of the test.Type O,type A,type Asian?of FMDV were the positive and the positive colour was blue.Non-FMDV virus were the negative and the negative colour was lavender.In this study,a rapid RT-LAMP rapid detection method for FMDV was established.The RT-LAMP rapid detection method has good sensibility,high sensitivity and specificity.It is suitable for the detection of FMDV type in our country.What's more,this detection method is significant for the rapid detection of FMDV on site.