Establishment And Preliminary Application Of Rapid Differential Diagnosis Of Mycoplasma Gallinaceum

Mycoplasma gallisepticum infection restricts the development of poultry industry in China,which reduces the carcass quality of broiler chickens,reduces the egg production of laying hens,increases the input of drugs,and opens the invasion portal for other pathogen infections as a basic infection pathogen,aggravating the disease,Caused serious economic losses to the poultry industry.Purification is considered to be the most effective measure to eliminate Mycoplasma gallisepticum.Fast and effective detection technology can provide technical support for purification.However,F36 live vaccine immunization is currently the main preventive measure in China,and the presence of vaccine strain F will affect the detection.As a result,a detection method capable of identifying a diagnostic vaccine strain F strain and other strains can provide technical support for purification.To this end,the research carried out the following two aspects:By analyzing the gene sequence of the attenuated vaccine strain F published in NCBI,the specific gene MGF＿3486 of Mycoplasma agalactiae strain A and the MGA＿0798 gene of Mycoplasma gallisepticum were selected as target genes,and the appropriate gene sequence was designed and synthesized.A set of LAMP primers for the MG attenuated vaccine strain F strain,a set of LAMP primers common to other strains other than the F strain,using hydroxynaphthol blue(HNB)as a color developer,by reacting the reaction conditions and the reagents in the reaction system.The concentration was optimized,and a visual LAMP detection method was established to distinguish the attenuated vaccine strain of Mycoplasma gallisepticum strain F and other strains.The reaction temperature was 63.6 °C and the reaction time was 60 min.The results show that the method meets the intended experimental purpose,and the detection limit is 10 times that of PCR,which can reach 10 fg/μL.By analyzing the gene sequences of Mycoplasma gallisepticum Rlow strain and Mycoplasma synoviae,the Rlow strain MGA＿0319 gene and the ATP-dependent heat shock protease gene of Mycoplasma synoviae were selected as target genes,and the two genes were combined and designed.Four pairs of PCR primers were specific primers of mycoplasma attenuated vaccine strain F,universal primers of other strains other than F strain,universal primers for MG,and universal primers for MS.The amplified bands of each primer were 750 bp,585 bp,279 bp,respectively 421 bp,distinguished by strip size.MG and MS quadruple PCR detection methods were established by stepwise establishment of multiplex PCR reaction method and optimization of reaction conditions.MG and MS were identified while MG and MS were identified,and the reaction conditions were determined.optimization.The results showed that the specificity or versatility of each primer was good,and the quadruple PCR annealing temperature was 51.5 ° C.The sensitivity of the quadruplex PCR detection was equivalent to that of ordinary single-plex PCR under the appropriate primer ratio,and it could reach 100 fg/μL.3,The results of detection of artificially experimental infection and clinical tracheal swabs showed the two methods could effectively identify and differentiate MG attenuated vaccine strain F with other strains,and the quadruple-PCR could further simultaneously identify MS and MG.In this study,LAMP and multiplex PCR methods were successfully established to discriminate MG attenuated strain F from other strains,and characterized with high specificity and sensitivity,which had potentially convenient for wide use in the futuer.