Establishment of dual basic RPA detection method for Mycoplasma gallisepticum and Mycoplasma synoviae
Mycoplasma gallisepticum(MG)and Mycoplasma synoviae(MS)are the two most harmful mycoplasmas to poultry.This study aims to establish a dual detection method for MG and MS based on recombinase polymerase amplification(RPA).Several pairs of primers were designed with MG mgc2 gene and MS vlhA gene as detection targets,and the optimal primers were obtained through screening,and the reaction time and reaction temperature were optimized to determine the optimal reaction conditions.The reaction system of dual basic RPA was determined by optimizing the optimal primer ratios of MG and MS,and sensitivity,specificity and repeatability tests were conducted,and 120 clinical samples were tested by the method.The results showed that the established MG and MS dual basic RPA detection method could be amplified at 37 ℃ for 20 min,and the optimal primer ratio was 0.6∶1.4.When the recombinant plasmid was used as the template,the minimum detection limits of MG and MS were 3.75 copies/μ L and 3.46 copies/μL respectively.When genomic DNA was used as template,the minimum detection limits of MG and MS were 1.23 × 10-3 ng/μL and 5.68 × 10-3 ng/μL,respectively.The method had good specificity and stability.The results of 120 clinical samples tested by this method and the single PCR method recommended by OIE showed that the detection rate of this method was higher than that of OIE PCR method,and it was in good agreement with 0IE PCR method.The MG and MS dual RPA detection method established in this study has the advantages of simple operation,rapid response,high sensitivity,strong specificity and good stability,and can realize the differential diagnosis of clinical MG and MS infections.
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