| Foot-and-mouth disease(FMD)is an acute,highly infectious,economically devastating and frequently occurring significant disease of cloven-hoofed animals that is caused by infection with the foot-and-mouth virus(FMDV).It is a positive-sense,single-stranded RNA virus that belongs to the Aphthovirus genus,Picornaviridae family.There are seven main different serotypes of FMDV,namely A,O,C,Asia-1,SAT 1,SAT 2,and SAT 3.In addition,numerous variants and subtypes have been further evolved from each serotype.It outbreaks have significant economic impact and is a threat to the livestock industry worldwide.Thus,this study identify P1 and 3A genes sequence difference of different hosts-adapted strains type O FMDV,studied the trend of genetic variation,finding the genetic mutation of P1 and 3A genes and using the uniform design method to development of two-temperature RT-PCR for detection.(1)In this text,FMDV type O was inoculated into bovine,neonatal rat,swine and BHK-21 cell line to obtain corresponding adapted virus strains.The RNA of FMDV in each adapted virus was taken as template for reverse transcription and amplification of P1(1A,1B,1C,1D)and 3A genes,and these genes was cloned into the vector pMD19-T,screened positive colonies and cultured,sequenced and analysis of these gene sequences.The results were as follows: picked up a single colony of bacteria for cultured after transformation,the positive culture colonies of 1A(VP4),1B(VP2),1C(VP3),1D(VP1)and 3A genes were amplified by PCR and electrophoresis detected,the bands of 399 bp,683 bp,780 bp,672 bp and 543 bp were obtained with the expected bands of 1A,(VP4),1B(VP2),1C(VP3),1D(VP1)and 3A genes have been inserted into the pMD19-T vector.The nucleotide sequence similarity of P1 genes were 99.6%~99.8% and the amino acid sequence similarity was 99.3%~99.9% in the different hosts-adapted virus strains,and the nucleotide and amino acid sequences were not deleted,the main antigenic sites were conserved.The P1 genes of the bovine,swine,neonatal rat,and BHK-21 cells adapted to the virus strains had some variation,according to the degree of variation was VP1,VP2>VP3>VP4;VP4was most the conserved region;the 3A genes nucleotide sequence similarity between99.6%~100% and amino acid sequence similarity in 99.3%~100%,nucleotide similarity is high and no deletion.3A genes were more stable and less variation and accorded to the degree of variation was swine adapted strain>BHK-21 cells adapted strain >bowine adapted strain>neonatal rat adapted strain.There is greater variability in the gene of VP1,however,the main antigenic sitesof VP1 were conserved;VP2 mutations were located in its antigenic epitope;Based on the results of the least genetic variation of the neonatal rat adapted strain,the storage of the original virus of FMDV is more favorable.(2)A pair of specific primers were designed according to conserved sequence of P1,3A genes of FMDV type O strains.Applied the reaction parameter optimized by using the uniform design method to development of two-temperature RT-PCR for detection of FMDV type O strains and the specificity and sensitivity test were carried out.The results of sensitivity and specificity showed that two-temperature RT-PCR was only specific for type O FMDV without amplification of the other viruses;the amplified fragment was same with the expected length(339 bp).The cloning and sequencing results revealed that the sequence of amplified fragment had 100%simililarity to the target sequence,and the minimum detection quantity was 1.665 pg/?L,which the effective detection rate was consistent with the three step RT-PCR sensitivity results.The animals were tested for 54 pigs tests using this method,positive identification results and three-step PCR consistent.Compared with the three step PCR,it could save 20 minutes.These results indicated that the developmented two-temperature RT-PCR for detection of FMDV type O strains was a kind of accurate,rapid,specific and sensitive detection method. |
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