幽门螺杆菌多重耐药株hefA基因表达的检测与分析

Expression of the hefA efflux pump gene in MDR Helicobacter pylori isolated from the Guiyang area

目的了解hefA外排基因表达与贵阳市幽门螺杆菌(Hp)多重耐药的关系。方法提取耐3种抗生素的多重耐药菌株(10株)、敏感菌株(6株)及质控菌株SS1的基因组DNA,PCR扩增hefA基因片段,PCR产物经2%琼脂糖凝胶电泳检测后测序,将测序基因片段序列输入NCBI中进行BLAST比对;利用TRIzol试剂提取上述细菌的RNA并逆转录成cDNA,以16sRNA为内参基因,通过实时荧光定量PCR(Real time PCR)检测hefA基因和内参基因的CT值,通过比较2-ΔΔCT判断多重耐药菌株和敏感菌株hefA基因表达的差异,以两小样本t检验进行统计学分析。结果 2%琼脂糖凝胶电泳显示,6株敏感菌和10株多耐药菌及SS1hefA基因PCR产物均约140bp,测序显示受试菌hefA基因序列与SS1菌株序列的一致性大于96%;RT-PCR检测hefA外排基因的表达,敏感菌株的2-ΔΔCT为0.895±0.540,多重耐药菌株的2-ΔΔCT为3.387±1.597,差异无统计学意义(t=9.399,P<0.05)。结论 hefA外排基因高表达是贵阳地区Hp多重耐药的机制之一。

Objective To ascertain expression of the hefA gene in multi-drug resistant（MDR）Helicobacter pylori isolated from Guiyang. Methods DNA of MDR strains（n=10）,susceptible strains（n=6,）and a quality control strain（SS1）was extracted.The hefA gene fragments of these strains were amplified with PCR and were then electrophoresed on an agarose gel.The PCR products were then sequenced by Genentech.The sequences were analyzed using BLAST on the NCBI website.The RNA of these strains was extracted with a TRIzol kit,and the RNA concentration and purity were determined using UV spectrophotometry and agarose gel electrophoresis.Reverse transcription PCR was carried on these RNA samples.The CT value of the hefA gene and a reference gene（16sRNA）was determined using real-time PCR.2-ΔΔCT was calculated for MDR strains and susceptible strains group and then compared using a t test. ResultsFragments of about 140 bp were amplified from all of the strains.The sequences of these fragments had similarity of 96%or higher to the hefA gene of H.pylori.2-ΔΔCT was 0.895±0.540 for susceptible strains and 3.387±1.597 for MDR strains.MDR strains had a higher 2-ΔΔCTthan that of susceptible strains（t test,t=9.399,P〈0.05）. Conclusion The higher level of expression of the hefA gene might be one of the mechanisms for the multi-drug resistance of clinical isolates of H.pylori from Guiyang.