边缘无浆体探针法荧光定量PCR检测方法的建立与应用OA

This study aimed to establish a quantitative real-time polymerase chain reaction(qPCR)assay with high specificity and sensitivity for the detection of Anaplasma marginale.Specific primers and probes were designed based on the msp1β gene sequence of Anaplasma marginale retrieved from GenBank.Reaction conditions and procedures were optimized to screen the optimal primer and probe concentrations as well as the annealing temperature.Subsequently,sensitivity,specificity and repeatability tests were performed,and clinical samples were further detected.The results showed that the established qPCR assay exhibited a good linear relationship,with a correlation coefficient(R²)of 0.999 2 and a slope of-3.284.The limit of detection(LOD)was 3.92 copies·μL-1,which was 100 times more sensitive than the conventional PCR assay.This assay could only amplify the target gene of Anaplasma marginale,and no amplification was observed for 6 other blood pathogens,including Anaplasma bovis and Neospora caninum.Both the intra-assay and inter-assay coefficients of variation were less than 0.7%,showing good repeatability.When 374 clinical bovine blood samples were tested,the positive rate detected by the established qPCR assay was 5.41%higher than that detected by the conventional PCR assay.In conclusion,the qPCR method established in this study exhibits high sensitivity,strong specificity,and good reproducibility,providing reliable technical support for the early diagnosis,epidemiological investigation,prevention and control practices of Anaplasma marginale disease.