Duplex PCR And Double Real-time PCR For Detection Of African Swine Fever Virus And Classical Swine Fever Virus

African swine fever (ASF) and classical swine fever (CSF) are Office International des Epizooties (OIE) "List A" diseases. These diseases are of major importance for international trade of animals and animal products and the initial diagnosis of any of these diseases in a country is rapidly reported to the OIE. At present, there is no reports about ASF but CSF in our country. ASF and CSF are highly contagious viral diseases of swine that occur in peracute, acute, subacute, chronic, and persistant forms. Pathologically and clinically, CSF and ASF can be strikingly similar in presentation, especially the acute and subacute forms of ASF are characterized by hemorrhages and can be easily confused with those observed in CSF. Epizootics caused by highly virulent CSFV or ASFV isolates are characterized by extremely high morbidity, with case-fatality rates approaching 100%. In recent years, ASF epizootics have devastated domestic swine herds in a number of European countries such as Russina that near our country. Therefore, enforcement actions of prevention, control and eradication must be adopted.Molecular techniques known as the polymerase chain reaction (PCR) /reverse-transcriptase PCR (RT-PCR) and real-time fluorescence quantitative PCR have been used for the detection of ASFV and CSFV, respectively. These assays are characterized by a rapid turnaround time and analytic sensitivity comparable with virus isolation. In this study, a rapid and specific TaqMan-based, double PCR and real-time PCR for the detection of ASFV and CSFV was established. These two methods can be used to detect pork and pork products and have contributed to an healthy production of the stockbreeding. The nucleotide sequences of VP72 protein genes of ASFV Tengani 62 strains was available in Genebank, synthesized the plasmid ASFV-vp72 and Q-ASFV-vp72 as the template. Using the OIE recombinant two pairs of primers and one probe, the PCR and real-time PCR assay were developed with 254bp and 278bp. According to the nucleotide sequences of CSFV in Genebank, two pairs of primers and one probe were designed based on the 5'untranslated region to amplify 424bp and 120bp in PCR and real-time PCR respectively.Because the nucleic acid of ASFV is DNA and that of CSFV is RNA, so we first established a plasmid of CSFV with the target genes that can be used as the PCR and real-time PCR templates simultaneously. After the reaction conditions were optimized, a duplex PCR and duplex real-time PCR were established, that specificity, sensitivity and repeatability were all measured. The result indicated that the expected fragments were amplified only with the specific primers and relevant templates and no any products were amplified with other templates that showed this method was specific. The duplex PCR was able to detect the minimum 16ng/L ASFV and 22ng/L CSFV of the nucleic acid templates. The sensitivity of the real-time PCR assay was found to be 100 DNA copies ASFV and CSFV per reaction. And the method of real-time PCR is 100 times more sensitive than the method of conventional PCR. The experiment was repeated three times, and the results were consistent. It showed the duplex PCR had better repeatability.