A multiplex real-time PCR assay for simultaneous detection of classical swine fever virus,African swine fever virus,and atypical porcine pestivirus  

作　　者：SONG Xiang-peng XIA Ying-ju XU Lu ZHAO Jun-jie WANG Zhen ZHAO Qi-zu LIU Ye-bing ZHANG Qian-yi WANG Qin 

机构地区：[1]China Institute of Veterinary Drug Control/National Reference Laboratory for Classical Swine Fever,Beijing 100081,P.R.China

出　　处：《Journal of Integrative Agriculture》2023年第2期559-567,共9页农业科学学报（英文版）

基　　金：supported by the National Natural Science Foundation of China (31872484) to Zhang Qianyi;the Non-profit Key Program of Veterinary Drug Industry from China Institute of Veterinary Drug Control (GY202011) to Xia Yingju。

摘　　要：With the implementation of the C-strain vaccine,classical swine fever(CSF) has been under control in China,which is currently in a chronic atypical epidemic situation.African swine fever(ASF) emerged in China in 2018 and spread quickly across the country.It is presently occurring sporadically due to the lack of commercial vaccines and farmers’ increased awareness of biosafety.Atypical porcine pestivirus(APPV) was first detected in Guangdong Province,China,in 2016,which mainly harms piglets and has a local epidemic situation in southern China.These three diseases have similar clinical symptoms in pig herds,which cause considerable losses to the pig industry.They are difficult to be distinguished only by clinical diagnosis.Therefore,developing an early and accurate simultaneous detection and differential diagnosis of the diseases induced by these viruses is essential.In this study,three pairs of specific primers and Taq-man probes were designed from highly conserved genomic regions of CSFV(5’ UTR),African swine fever virus(ASFV)(B646L),and APPV(5’ UTR),followed by the optimization of reaction conditions to establish a multiplex real-time PCR detection assay.The results showed that the method did not cross-react with other swine pathogens(porcine circovirus type 2(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV),foot-and-mouth disease virus(FMDV),pseudorabies virus(PRV),porcine parvovirus(PPV),and bovine viral diarrhea virus BVDV).The sensitivity results showed that CSFV,ASFV,and APPV could be detected as low as 1 copy μL–1;the repeatability results showed that the intra-assay and interassay coefficient of variation of ASFV,CSFV,and APPV was less than 1%.Twenty-two virus samples were detected by the multiplex real-time PCR,compared with national standard diagnostic and patented method assay for CSF(GB/T 27540–2011),ASF(GB/T 18648–2020),and APPV(CN108611442A),respectively.The sensitivity of this triple real-time PCR for CSFV,ASFV,and APPV was almost the same,and the compliance results wer

关 键 词：classical swine fever virus African swine fever virus atypical porcine pestivirus real-time PCR 

分 类 号：S852.65[农业科学—基础兽医学]