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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 investigate the relationship between the expression of hefA efflux gene and the multidrug resistance of Helicobacter pylori (Hp) in Guiyang. Methods The genomic DNA of multi-drug resistant strains (10 strains), sensitive strains (6 strains) and control strain SS1 were extracted from three kinds of antibiotics. The hefA gene fragment was amplified by PCR. After PCR products were detected by 2% agarose gel electrophoresis The sequences of the sequenced gene fragments were input into NCBI for BLAST alignment. The RNA of these bacteria was extracted by TRIzol reagent and reverse transcribed into cDNA. The 16sRNA was used as an internal reference gene to detect hefA gene and internal reference by real time PCR The CT value of the gene was compared. The differences of hefA gene expression between the multidrug-resistant and susceptible strains were compared by 2-ΔΔCT. Two-sample t-test was used for statistical analysis. Results The 2% agarose gel electrophoresis showed that the PCR products of 6 strains of susceptible bacteria and 10 strains of multidrug-resistant bacteria and SS1hefA gene were about 140bp. Sequencing showed that the sequence of hefA gene of the tested bacteria was more than 96% identical to the sequence of SS1 strain. RT The expression of hefA efflux gene was detected by PCR. The 2-ΔΔCT of the sensitive strains was 0.895 ± 0.540 and the 2-ΔΔCT of the multidrug-resistant strains was 3.387 ± 1.597, with no significant difference (t = 9.399, P <0.05). Conclusion High expression of hefA efflux gene is one of the mechanisms of Hp multi-drug resistance in Guiyang.