鸽源禽毛滴虫套式 PCR检测方法的建立与初步应用OA

To provide an efficient and accurate technical approach for the early diagnosis of pigeon-derived trichomoniasis,this study applied nested polymerase chain reaction(PCR)technology to the detection of pigeon-derived Trichomonas gallinae.Based on the 18S rRNA gene sequence,two pairs of specific primers(A1/S1 and F2/R2)were designed.Recombinant positive plasmids were constructed,and reaction conditions were optimized to establish a nested PCR assay for the detection of pigeon-derived T.gallinae.The specificity and sensitivity of the method were evaluated,and its reliability was systematically verified through parasite revival,artificial infection experiments,and clinical sample testing.The results showed that the recombinant positive plasmid was successfully constructed and sequencing confirmed it as a specific sequence of pigeon-derived T.gallinae.The optimal primer concentrations for both the outer and inner primers were 0.08 µmol/L,and the optimal annealing temperatures were 58 and 56℃,respectively.The nested PCR assay showed no cross-reactivity with common pathogens,including Escherichia coli,Salmonella,Candida albicans,Eimeria,and Toxoplasma gondii,demonstrating high specificity.The assay also exhibited extremely high sensitivity,with a minimum detection limit of 0.25 copies/µL.In artificial infection experiments,the nested PCR assay detected infection much earlier than in vitro culture and conventional microscopic examination.In clinical sample testing,the positive detection rate of conventional PCR was 73.21%(41/56),whereas the positive rate of nested PCR reached 96.43%(54/56).These results indicate that the nested PCR assay established in this study possesses high specificity and sensitivity and has strong practical application value.