马伊氏锥虫PFR基因PCR检测方法的建立和应用OA

To establish a convenient and accurate polymerase chain reaction(PCR)detection method for Trypanosoma evansi(T.evansi)infection in horses for clinical diagnosis and epidemiological investigation in Xinjiang Uygur Autonomous Region,specific primers were designed based on the T.evansi paraflagellar rod(PFR)gene.A positive plasmid was constructed,and a PCR detection system was optimized.The specificity and sensitivity of the established method were validated,followed by testing of 150 equine blood samples.The PCR results were compared with traditional blood smear microscopy,and eight positive samples were randomly selected for population similarity and genetic diversity analysis.The results showed that the optimized PCR method successfully amplified a specific T.evansi band of approximately 799 bp.The assay exhibited high specificity and sensitivity,with a minimum detectable concentration of 3.46×10-16 g/μL.Among 150 clinical samples,the PCR detection rate was 31.33%(47/150),significantly higher than that of microscopy(8.00%,12/150).Phylogenetic analysis of the PFR gene sequences revealed that T.evansi isolates from this study formed an independent branch but clustered closely with Trypanosoma brucei(T.brucei),Trypanosoma cruzi,Trypanosoma rangeli,and Trypanosoma conorhini,showing close genetic relationships with T.evansi isolates from India and T.brucei isolates from the United States,the United Kingdom,and Switzerland.These results indicate that the established PCR detection method is highly specific and sensitive,suitable for detecting T.evansi infection in horses in Xinjiang,and provides valuable data for regional epidemiological surveillance.