快速鉴定幽门螺杆菌与宿主基因型的方法学研究  

Study on rapid identification of Helicobacter pylori and host genotyping

作　　者：吕江峰 虞闰六 王栋梁 王敏 杨文波 任绪义 

机构地区：[1]杭州迪安医学检验中心,浙江杭州310030

出　　处：《中华医院感染学杂志》2011年第15期3117-3121,共5页Chinese Journal of Nosocomiology

摘　　要：目的探索采用焦磷酸测序技术进行幽门螺杆菌(Hp)鉴定与耐药以及宿主胃癌易感相关基因多态性分析的可行性,为临床提供一种快速的分析手段和诊疗依据。方法提取60例胃黏膜活检标本DNA(Hp阳性、阴性各30例),PCR扩增Hp 16S rDNA,23S rDNA以及人白细胞介素1B(IL-1B)基因特异性片段,对3个片段同时进行焦磷酸测序分析。结果 30例Hp阳性标本16S rDNA测序结果一致,均为5-′CGCGCAATCAGCGT-CAGTAATG-3′,该序列可作为标志性序列来鉴定Hp;对Hp阳性标本23S rDNA 2142及2143位点的多态性分析发现2142A→G突变,该突变通常会导致Hp对克拉霉素的耐药;对60例标本IL-1B基因-31和-511位点的多态性检测表明,-31位点有两种基因型,其中A/G基因型占80.0%,G/G 20.0%;-511位点有3种基因型,其中T/C基因型占60.0%,T/T基因型占26.7%,C/C基因型占13.3%。结论运用焦磷酸测序技术进行Hp鉴定、耐药性分析以及宿主胃癌易感性分析,具有准确、快速、操作方便、通量高等优势;除了胃黏膜活检标本,该方法亦可应用于其他标本如粪便、唾液、胃液等的Hp检测,同时可推广至其他菌种的鉴定和耐药分析,在弥补甚至替代传统的病原菌检测方面具有广阔的应用前景。OBJECTIVE To explore the feasibility of using Pyrosequencing technology for rapid identification,resistance analysis of H.pylori（Hp）,along with analysis of host gastric cancer susceptibility which allow the physician to predict within hours the likely outcome of an infection and to make informed decisions about optimal therapeutic regimen.METHODS The DNA of 60 gastric biopsy samples were extracted,and then PCR was processed to amplify the specific sequence of Hp 16S rDNA,23S rDNA and IL-1B with their corresponding primers.The sequence information after sequencing the three target sequences with pyrosequencing technology was analyzed.RESULTS The consistent sequence of 30 Hp positive samples,which was distinguished from the sequence of Hp negative samples was used as a signature to identify Hp.In the SNP analysis of Hp 23S rDNA position 2142,position 2143 and IL-1B position-31,position-511,we found the 2142 A→G mutation which led to the clarithromycin resistance,position-31 of IL-1B had two genotypes A/G and G/G,accounted for 80.0% and 20.0%,respectively,position-511 had three genotypes-T/C,T/T and C/C,accounted for 60%,26.7% and 13.3%,respectively.CONCLUSION PCR-Pyrosequencing technology,as a convenient,accurate and high-throughput method,can be used for Hp identification,antibiotic resistance analysis and host genotyping and these three tests can be processed in parallel.Besides,we believe that this method could also be applied to samples such as stool,saliva,gastric juice and could extend to the detection and drug resistance analysis of other pathogens.

关 键 词：幽门螺杆菌 焦磷酸测序 克拉霉素 耐药 胃癌 易感 单核苷酸多态性分析 

分 类 号：R378[医药卫生—病原生物学]