鉴别滑液囊支原体MS-Y田间株与MS-H疫苗株多重PCR方法的建立

Establishment of a multiplex PCR method for identification of Mycoplasma synoviae MS-Y field isolates and MS-H vaccine strains

为了建立一种快速准确鉴别滑液囊支原体MS-Y分离株和活疫苗MS-H的方法,根据滑液囊支原体ppm和deoD基因的2个碱基突变位点序列,运用错配扩增突变分析(MAMA)PCR方法,设计了2对特异性引物和1对通用引物,用于扩增目的基因,通过优化反应条件,建立一种能同时鉴别出MS-Y和MS-H的多重PCR检测方法,并检测了该方法的特异性和敏感性。结果显示,利用特异性引物的多重PCR方法能同时鉴别滑液囊支原体MS-H活疫苗株和滑液囊支原体MS-Y分离株的DNA,分别扩增出相应的特异目的条带,检测MS-H株的最低敏感度为4.21×10^(5)copies/μL,检测MS-Y株的最低敏感度为6.47×10^(5)copies/μL;而利用通用引物对检测滑液囊支原体DNA的最低浓度为2.18×10^(3)copies/μL,检测MS-H DNA的浓度下限均为1×10^(5)CCU/mL,检测MS-Y DNA的最低浓度为1×10^(3)CCU/mL。本研究建立了一种能同时快速鉴别滑液囊支原体MS-H活疫苗株和MS-Y分离株的多重PCR方法。

To establish a rapid and accurate method for the identification of Mycoplasma synoviae MS-Y isolate and live vaccine MS-H isolate,two pairs of specific primers and one pair of universal primers were designed by mismatch amplification mutation analysis(MAMA)PCR to amplify the target gene according to the two base mutation sequence sites of ppm and deoD genes of M.synoviae,and a multiplex PCR assay by optimizing the reaction conditions was established,which could be identified for MS-Y and MS-H by simultaneous amplification and gel electrophoresis,and the specificity and sensitivity of this method were detected.The results showed that the multiplex PCR method could specifically amplify the specific target bands from DNA of live M.synoviae vaccine MS-H strain and M.synoviae MS-Y isolate,respectively.The lowest sensitivity from MS-H strain was 4.21×10^(5)copies/μL and that of MS-Y isolate was 6.47×10^(5)copies/μL.The lowest detection concentration from M.synoviae DNA by universal primers was 2.18×10^(3)copies/μL,the lower limit of detection concentration to M.synoviae bacterial solution was 1×10^(5)CCU/mL,and the lowest detection concentration of bacterial solution by universal primers was 1×10^(3)CCU/mL.In this study,a multiplex PCR method was established to rapidly identificate live vaccine MS-H strain and M.synoviae MS-Y isolate.