Zhang Yanmei, Zhao Fuju, Kong Mimi, Wang Shiwen, Nan Li, Hu Binjie, Olszewski Michal A, Miao Yingxin, Ji Danian, Jiang Wenrong, Fang Yi, Zhang Jinghao, Chen Fei, Xiang Ping, Wu Yong, Zhao Hu
Department of Laboratory Medicine, Huadong Hospital affiliated to Fudan UniversityShanghai, China; Key Laboratory of Clinical Geriatric MedicineShanghai, China; Research Center on Aging and Medicine, Fudan UniversityShanghai, China.
Department of Laboratory Medicine, Huadong Hospital affiliated to Fudan University Shanghai, China.
Front Microbiol. 2016 Sep 7;7:1401. doi: 10.3389/fmicb.2016.01401. eCollection 2016.
Helicobacter pylori (H. pylori) infection is closely related to various gastroduodenal diseases. Virulence factors and bacterial load of H. pylori are associated with clinical outcomes, and drug-resistance severely impacts the clinical efficacy of eradication treatment. Existing detection methods are low-throughput, time-consuming and labor intensive. Therefore, a rapid and high-throughput method is needed for clinical diagnosis, treatment, and monitoring for H. pylori. High-throughput Multiplex Genetic Detection System (HMGS) assay was established to simultaneously detect and analyze a set of genes for H. pylori identification, quantification, virulence, and drug resistance by optimizing the singlet-PCR and multiple primers assay. Twenty-one pairs of chimeric primers consisted of conserved and specific gene sequences of H. pylori tagged with universal sequence at the 5' end were designed. Singlet-PCR assay and multiple primers assay were developed to optimize the HMGS. The specificity of HMGS assay was evaluated using standard H. pylori strains and bacterial controls. Six clinical isolates with known genetic background of target genes were detected to assess the accuracy of HMGS assay. Artificial mixed pathogen DNA templates were used to evaluate the ability to distinguish mixed infections using HMGS assay. Furthermore, gastric biopsy specimens with corresponding isolated strains were used to assess the capability of HMGS assay in detecting biopsy specimens directly. HMGS assay was specific for H. pylori identification. HMGS assay for H. pylori target genes detection were completely consistent with the corresponding genetic background. Mixed infection with different drug-resistant isolates of H. pylori could be distinguished by HMGS assay. HMGS assay could efficiently diagnose H. pylori infection in gastric biopsy specimens directly. HMGS assay is a rapid and high throughput method for the simultaneous identification and quantification of H. pylori, analysis of virulence and drug resistance in both isolated strains and biopsy specimens. It could also be used to distinguish the mixed infection with different resistant genotype strains. Furthermore, HMGS could detect H. pylori infection in gastric biopsy specimens directly.
幽门螺杆菌（H. pylori）感染与多种胃十二指肠疾病密切相关。幽门螺杆菌的毒力因子和细菌载量与临床结局相关，而耐药性严重影响根除治疗的临床疗效。现有的检测方法通量低、耗时且 labor intensive。因此，临床诊断、治疗和监测幽门螺杆菌需要一种快速且高通量的方法。通过优化单重PCR和多重引物检测，建立了高通量多重基因检测系统（HMGS）检测法，用于同时检测和分析一组用于幽门螺杆菌鉴定、定量、毒力和耐药性的基因。设计了21对嵌合引物，其由在5'端标记有通用序列的幽门螺杆菌保守和特异性基因序列组成。开发了单重PCR检测法和多重引物检测法以优化HMGS。使用标准幽门螺杆菌菌株和细菌对照评估HMGS检测法的特异性。检测了6株具有已知目标基因遗传背景的临床分离株，以评估HMGS检测法的准确性。使用人工混合病原体DNA模板评估HMGS检测法区分混合感染的能力。此外，使用具有相应分离菌株的胃活检标本评估HMGS检测法直接检测活检标本的能力。HMGS检测法对幽门螺杆菌鉴定具有特异性。用于检测幽门螺杆菌目标基因的HMGS检测法与相应的遗传背景完全一致。HMGS检测法可区分幽门螺杆菌不同耐药分离株的混合感染。HMGS检测法可直接有效诊断胃活检标本中的幽门螺杆菌感染。HMGS检测法是一种快速且高通量的方法，可同时鉴定和定量幽门螺杆菌，分析分离菌株和活检标本中的毒力和耐药性。它还可用于区分不同耐药基因型菌株的混合感染。此外，HMGS可直接检测胃活检标本中的幽门螺杆菌感染。
