基于非洲猪瘟病毒MGF100-1L基因的荧光定量PCR方法的建立与评价OA

African swine fever(ASF),caused by African swine fever virus(ASFV),is a highly contagious disease with extremely high mortality.This study aimed to develop a specific and sensitive real-time PCR(qPCR)assay for detecting ASFV.The MGF100-1L gene(GenBank No.MK333180.1),belonging to the multigene families(MGFs)of ASFV,was analyzed to design and screen specific primers and probes targeting conserved regions.A recombinant plasmid containing the target gene fragment was constructed as a standard for optimizing the qPCR reaction system.The established ASFV qPCR method was evaluated for specificity,sensitivity,reproducibility,and agreement.A total of 90 clinical samples were tested and compared with the method recommended by the World Organisation for Animal Health(WOAH).Results showed that the standard curve linear equation was Y=-3.304X+38.793 with a correlation coefficient of 0.99,indicating good linearity.The limit of detection(LOD)was 0.87 copies/µL,demonstrating sensitivity comparable to the WOAH method.Specificity analysis confirmed no cross-reactivity with Classical swine fever virus(CSFV),Pseudorabies virus(PRV),Porcine parvovirus(PPV),Porcine circovirus type 2(PCV2),or Foot-and-mouth disease virus(FMDV).In clinical sample testing,the method showed high agreement with the WOAH method(Kappa=0.903,P＜0.01)and significantly improved detection rates for weakly positive samples(4 additional samples detected compared to the WOAH method).This study successfully established a qPCR detection system based on the ASFV MGF100-1L gene,exhibiting excellent specificity,sensitivity,and high consistency with existing standard methods.This method provides a more sensitive and reliable technical tool for clinical ASFV detection,particularly for early diagnosis and control.