甘宁黄土高原阿拉善黄鼠鼠疫疫源地鼠疫菌耐链霉素rpsL基因突变位点的检测

Detection of streptomycin resistant rpsL gene mutations in Yersinia pestis in Citellus alaschanicus plague foci of Ganning Loess Plateau

目的 通过检测甘宁黄土高原阿拉善黄鼠鼠疫疫源地鼠疫菌耐链霉素rpsL基因位点,掌握该地区鼠疫菌对链霉素的敏感性,以期为今后该地区鼠疫的治疗及临床用药提供参考依据。方法 根据鼠疫菌rpsL基因序列及我国发现的耐链霉素菌株(S19960127)突变位点分别设计非突变菌株FAM(128∶A)和突变菌株VIC(128∶G)两种MGB探针,通过TaqMan-MGB探针法对1962—1991年分离自甘宁黄土高原阿拉善黄鼠鼠疫自然疫源地的49株代表性鼠疫菌进行耐链霉素rpsL基因突变位点检测。结果 被试菌株对应检测rpsL(128∶A),49株FAM染料RFU峰值均为阳性,其RFU峰值>2 000,阴性<200;对应检测rpsL(128∶G)未检测到VIC染料RFU峰值阳性菌株,RFU峰值均<200,阳性对照>1 000,该疫源地分离的鼠疫菌未发现耐链霉素菌株。结论 甘宁黄土高原阿拉善黄鼠鼠疫疫源地49株鼠疫菌对链霉素均敏感。TaqMan-MGB探针荧光定量PCR体系具有较高灵敏性、特异性,可应用于鼠疫菌耐药性监测。

Objective To understand the sensitivity of Yersinia pestis to streptomycin by detecting the streptomycin resistant rpsL gene loci of Y.pestis in Citellus alaschanicus plague foci of Ganning Loess Plateau,so as to provide reference for the treatment and clinical medication of plague in this area in the future.Methods Two MGB probes,FAM(128∶A) for nonmutant strain and VIC(128∶G) for mutant strain,were designed according to the rpsL gene sequence of Y.pestis and the mutation site of streptomycin resistant strain(S19960127) found in China.TaqMan-MGB probe method was performed to detect the mutations of streptomycin resistant rpsL gene in 49 representative Y.pestis strains isolated from Citellus alaschanicus plague natural foci of Ganning Loess Plateau from 1962 to 1991.Results The corresponding rpsL(128∶A) of the tested strains was detected,and the peak values of FAM dye RFU of 49 strains were positive and bigger than 2 000,while the negative value was less than 200.No VIC dye RFU peak positive strain was detected in the rpsL(128∶G) of the tested strain,and the RFU peak values were less than 200,while the positive control was more than 1 000.No streptomycin resistant strains were found in Y.pestis isolated from this plague focus.Conclusion Forty-nine strains were sensitive to streptomycin.TaqMan-MGB probe method is of high sensitivity and specificity,and can be applied in monitoring of Y.pestis resistance.