Establishment Of Dual RPA Rapid Detection Method For Mycoplasma Gallisepticum And Mycoplasma Synoviae

Mycoplasma gallisepticum(MG)and Mycoplasma synoviae(MS)are the two most harmful mycoplasmas in poultry farming.MG and MS were distributed worldwide without obvious seasonality.They can be transmitted horizontally and vertically,and can be transmitted to chickens of all ages.As long as the infection will be carried for life,it is difficult to eradicate.Therefore,early diagnosis of MG and MS was the key to prevent and control avian mycoplasma.In this study,based on the mgc2 gene of MG and the vlhA gene of MS,a rapid detection method for simultaneous detection of MG and MS was constructed.Recombinase polymerase amplification(RPA)was a new type of isothermal nucleic acid amplification technology,which has the advantages of simple operation,short reaction time,high sensitivity,strong specificity,and no need for precious instruments.It was the most promising nucleic acid rapid diagnosis technology.The basic RPA rapid detection method of MG and MS was established,and the LFD-RPA detection method was established by combining with the lateral flow chromatography test strip(LFD).The technique is simple,rapid,specific and allows visual observation of results to further improve the efficiency of detection of MG and MS infections.1.Using MG mgc2 gene and MS vlhA gene as detection targets,several pairs of RPA primers were designed,and the best primers were screened to establish a single basic RPA detection method for MG and MS.The reaction time and temperature were optimized to determine the optimal reaction conditions,and the sensitivity,specificity and repeatability test were carried out.The results showed that the established MG and MS single basic RPA detection method could complete the amplification at 37℃for 20 min.The minimum detection limit of MG was 3.75 copies/μL,and the minimum detection limit of MS was 3.46 copies/μL.When genomic DNA was used as a template,the minimum detection limit of MG was 1.23×10-4 ng/μL,and the minimum detection limit of MS was 5.68×10-4 ng/μL.It has good specificity and stability.2.On the basis of MG and MS single RPA,a dual basic RPA detection method was established.The reaction system of dual basic RPA was determined by optimizing the optimal primer ratios of MG and MS,and the sensitivity,specificity and repeatability test were carried out.The results showed that the optimal primer ratios of RPA-MG-F2/R2 and RPA-MS-F1/R1 was 0.6:1.4 μL.When the recombinant plasmid was used as the template,the minimum detection limits of MG and MS were 3.75 copies/μL and 3.46 copies/μL respectively,which were consistent with the sensitivity of single RPA.When genomic DNA was used as the template,the minimum detection limit of MG and MS were 1.23×10-3 ng/μL and 5.68×10-3 ng/μL respectively,which were 10 times lower than the sensitivity of single RPA,and it had good specificity and stability.The method was used to test 80 clinical samples and the PCR method recommended by OIE.The results showed that the positive detection rate of this method was higher than the OIE PCR method,and it was highly consistent with OIE PCR method.3.On the basis of MG and MS dual RPA,a dual LFD-RPA detection method was established.The reaction time and temperature were optimized to determine the optimal reaction conditions,and the sensitivity,specificity and repeatability test were carried out.The results showed that the established dual LFD-RPA detection method could complete the amplification in 5-10 min at 37℃ and the results were visible to the naked eye.The minimum detection limit of MG and MS were consistent with the dual basic RPA,with good specificity and stability.In summary,based on the mgc2 gene of MG and the vlhA gene of MS,this experiment successfully established MG and MS single basic RPA,dual basic RPA,and dual LFD-RPA detection methods.The three detection methods have the advantages of simple operation,rapid response,high sensitivity,strong specificity,and good stability.Based on the comparison of its convenience and visualization,the LFD-RPA method is more suitable for the detection of single infection or mixed infection of MG and MS at the grassroots level,which can provide technical support for the early diagnosis and prevention and control of MG and MS.