Establishment Of Conventional RT-PCR Assay For The Detection Of Southern African Territories Foot And Mouth Disease Viruses

Foot and mouth disease(FMD), caused by foot and mouth disease virus(FMDV), is an acute systemic vesicular disease of Artiodactyls. FMDV is an RNA virus and is divided into seven serotypes namely: A, O, Asial, C and Southern African Territories(SAT) 1, 2, 3. SAT FMDV has a strict region and is limited to South Africa. However, it has been reported that SAT-FMDV has been detected in Middle East in recent years, making up a potential threat to the animal husbandry in China. So it is necessary to establish a simple, rapid, specific method to diagnose SAT FMDV, serving as a strategic reserve technique.The study was aimed to establish a conventional RT-PCR assay for the detection of SAT FMDV. VP1 coding sequence, namely 1D gene, is the basis of FMDV classification while the nonstructural protein coding sequence 2B is conserved. Therefore, 1D2A2 B sequences served as the target gene for the detection method of SAT FMDV in this assay. So far, there are no SAT FMD outbreaks in China. To fully guarantee the safety and satisfy the need of establishing conventional RT-PCR method, 8 SAT1 FMDV, 10 SAT2 FMDV and 3 SAT3 FMDV representative strains were selected based on the comparative analysis of FMDV SAT genes. Then the 1D2A2 B gene of these representative viruses was synthesized and corresponding recombinant plasmids were constructed which were used in following experiments.Primers are always the key part in the establishment of RT-PCR. AS known, 1D gene is the most various genes among SAT FMDV(70%-85%), so multiplex primers are optimal choice. 3’ region of 1D gene was selected to design specific upstream primer and reverse primer located in the 2B gene that is quite conserved in all FMDV serotypes. Finally the multiplex primers composed with seven upstream primers and one reverse primer, namely SAT-D8 primers, was determined after testing.Thus the conventional PCR method was preliminarily established using SAT-D8 primers and recombinant plasmids mentioned above in DNA level. Special products of 257 bp were produced by PCR of all the 21 recombinant plasmids. SAT-FMDV 1D2A2 B RNA genes were obtained by transcription in vitro. The assay was further tested at RNA level. The results show that the SAT-D8 primers are able to detect the transcripts of the synthesized sequences. The method had no cross reaction with FMDV(A、O、Asia) and BVDV、ORFV、SPPV/GTPV、SVDV、BTV、PRRSV、CSFV、PRV、PPV、PCV. The sensitivity can be limited to 102copies/μL by the level of DNA while 103-104copies/μL by the level of RNA transcripts.3 inactivated antigens SAT1/2/3 obtained from Pirbright laboratory of Britain and 125 tissues and serum derived from sheep, cows and pigs of China served as the field samples which were tested by the SAT-D8 RT-PCR assay after extracting RNA. The results showed that the expected PCR product of inactivated antigen SAT1/2/3 appeared while the other 125 field samples were negative. Upon the RT-PCR method has been preliminarily established, RT-PCR kits for the detection of SAT FMDV were assembled and stored in- 20 ℃ refrigerator to test the stability. To date, test result of 6th month shows that the stability was quite good. The study need more field samples, especially SAT positive samples to be validated and the stability of kit should be further tested in longer time.The study successfully established the RT-PCR assay for the detection of SAT-FMDV with a good specificity and sensitivity. Furthermore, the assembled kit had a strong stability in the condition of- 20 ℃ for six months, providing a basis for the study of RT-PCR kit for the detection of SAT FMDV.