Establishment And Application Of Real-time Duplex Fluorescence PCR Assay For Detection Of Mycoplasma Gallisepticum And Mycoplasma Synoviae

Mycoplasma gallisepticum and Mycoplasma synoviae are the two most harmful mycoplasma in intensive poultry cultivation.On the one hand,mycoplasma infection is chronic and has no typical symptoms for differential diagnosis.On the other hand,at present,there is no effective treatment to completely eliminate mycoplasma in chicken flocks,and the vaccine cannot provide sufficient protection to chicken flocks,but has the risk of disease.Mycoplasma infects chickens and then spreads rapidly and circulates continuously.This seriously reduces the resistance of chickens,so that chickens can have concurrent or secondary infections of other pathogens,aggravating symptoms and even outbreaks of diseases,and further form greater losses.The current prevention and control programs of mycoplasma mainly rely on pathogen detection,isolation and purification of infected chickens.There is no doubt that the development of rapid and sensitive detection protocols for early diagnosis of mycoplasma infection is the foundation for prevention and control of mycoplasma,containment of disease outbreaks and reduction of economic losses.This study focuses on the clinical needs for efficient detection of mycoplasma.A MG-MS duplex fluorescence quantitative PCR(MG-MS q PCR)assay was established to detect the conserved genes of Mycoplasma gallisepticum and Mycoplasma synoviae.The specificity,sensitivity and repeatability of the established method were analyzied,and the coincidence rate of the assay for detecting mycoplasma with known background was verified.Then the established detection assay was applied to clinic.The main research contents and results are as follows:1)Establishment of MG-MS q PCR assayIn this study,the gene mg C2 encoding Mycoplasma gallisepticum adherent protein and the gene vlh A encoding Mycoplasma synoviae adherent protein were used as the detection targets according to OIE standard.Mg C2 and vlh A sequences published in Gen Bank database were compared,and primers and probes were designed by selecting highly conserved and specific regions.Then the MG-MS q PCR assay was established.The concentration of primers,probes and annealing temperature of the reaction program were adjusted,and the reaction condition with the minimum Ct value and the maximum fluorescence intensity was selected.The positive control plasmid was diluted tenfold and amplified with optimized conditions as a template.The minimum detection limit of the method was obtained,and the standard curve was drawn according to the amplification results.The results showed that the minimum detection limit of MG was 250 copies/reaction,the correlation coefficient(R~2)of the standard curve was 0.999,and the amplification efficiency(E%)was 73%.The minimum detection limit for MS was 250 copies/reaction,the correlation coefficient(R~2)of the standard curve was 0.993,and the amplification efficiency(E%)was 78%.The genomes of MG and MS pure cultures with known color change units were extracted and diluted tenfold.Mg-MS q PCR detection and OIE standard MG and MS ordinary PCR detection were performed respectively.The sensitivity of Mg-MS q PCR was 100times higher than OIE standard MG PCR and 100 times higher than OIE standard MS PCR,showing good sensitivity.The genomes of MG,MS,IBDV,IBV,MDV,AIV,ALV,CIAV,Pasteurella,Salmonella,E.coli were used as templates for specificity test.The results showed that the method only had specific amplification for MG and MS.There was no amplification to other pathogenic genomes and the specificity was good.Positive control plasmids with three concentrations were used as templates for three times of MG-MS q PCR detection.During each amplification,the templates with each concentration were repeated for three times.The dispersion coefficients of repeatability tests for different batches and different concentrations were all less than4%,indicating good repeatability.The established MG-MS q PCR assay and tripartite mechanism detection method were used to detect the clinical samples for comparative analysis.The results showed that the coincidence rate of positive samples was 94.73%,and that of negative samples was 100%.15 MG positive nasopharyngeal swabs,6 MS positive joint capsule samples,and 40 nasopharyngeal swabs and joint capsule swabs collected from SPF chickens were detected.The results were consistent with each other,and the coincidence rate was 100%.The above indicated that the established MG-MS q PCR method had good sensitivity,specificity and repeatability,and could correctly distinguish negative and positive samples.2)Preliminary application of MG-MS q PCR assayA total of 181 nasopharyngeal swabs and anal swabs were collected from a local chicken farm in Hubei Province.MG and MS were detected by MG-MS q PCR assay.The results showed that MG detection rate was 37%(67/181)and MS detection rate was 50%(91/181).Five samples with positive results were randomly selected from181 samples for mycoplasma detection with commercial kits,and the results were all positive.This indicated that the detection results are highly accurate.In conclusion,the established MG-MS q PCR assay is sensitive,specific and efficient,and has high accuracy in detecting clinical samples.It is a tool for clinical detection,epidemiological investigation and mycoplasma eradication and purification,and provides a feasible alternative for rapid clinical detection of mycoplasma.