丝状支原体丝状亚种SC生物型PCR检测方法的建议

Development of Polymerase Chain Reaction Assay for Detection of Mycoplasma Mycoides subsp.Mycoides S.C.

建立了一个可以识别丝状支原体丝状亚种SC生物型 (MmmSC)的PCR方法。根据丝状支原体丝状亚种SC生物型(MmmSC)相关核苷酸序列 ,设计合成了MC1、MC2和SC1、SC2两对引物。MC1、MC2是一对簇特异性引物 ,用于鉴别丝状支原体族 6个成员 ,对MmmSC、MmmLC扩增出与预期结果相符的 4 6 2bp片段 ;而SC1、SC2是针对MmmSC的一对特异性引物 ,只能对MmmSC扩增出 2 77bp的片段 ,经过VspI、Bsp14 3I和Dral三种限制性内切酶鉴定与预期结果相符 ,而不能扩增出MmmLC型Y_goat代表株 ,说明具有非常好的特异性。对MmmSC进行的敏感性试验显示SC1、SC2引物能够检测到 10 0个CFU ,具有非常高的敏感性。

A polymerase chain reaction(PCR)assay for the detection of Mycoplasma mycoides subsp.mycoides SC(MmmSC)has been developed.According to MmmSC related nucleic acid sequence,two pairs of primers,MC1,MC2 and SC1,SC2,were designed and synthesized in this study.The MC primers,are specific for mycoides cluster,allowed amplification of a 462bp fragment from 6 mycoplasmas of the mycoides cluster assayed.The SC primers,are specific for MmmSC,selectively amplified a 277bp sequence from 5 MmmSC strain tested,with three specific internal restriction sites for the endonucleases Vsp I,Dra I and Bsp143I allowing confirmation of the identification.No amplification occurred with MuuLC(Y_Goat strain)assayed.The sensitivity assessed by direct agarose gel analysis for the PCR assay with SC primer was 100CFU.