AUTHOR=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 

TITLE=Validation of a High-Throughput Multiplex Genetic Detection System for Helicobacter pylori Identification, Quantification, Virulence, and Resistance Analysis

JOURNAL=Frontiers in Microbiology

VOLUME=7

YEAR=2016

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2016.01401

DOI=10.3389/fmicb.2016.01401

ISSN=1664-302X

ABSTRACT=<p><italic>Helicobacter pylori</italic> (<italic>H. pylori</italic>) infection is closely related to various gastroduodenal diseases. Virulence factors and bacterial load of <italic>H. pylori</italic> 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 <italic>H. pylori</italic>. High-throughput Multiplex Genetic Detection System (HMGS) assay was established to simultaneously detect and analyze a set of genes for <italic>H. pylori</italic> 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 <italic>H. pylori</italic> 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 <italic>H. pylori</italic> 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 <italic>H. pylori</italic> identification. HMGS assay for <italic>H. pylori</italic> target genes detection were completely consistent with the corresponding genetic background. Mixed infection with different drug-resistant isolates of <italic>H. pylori</italic> could be distinguished by HMGS assay. HMGS assay could efficiently diagnose <italic>H. pylori</italic> infection in gastric biopsy specimens directly. HMGS assay is a rapid and high throughput method for the simultaneous identification and quantification of <italic>H. pylori</italic>, 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 <italic>H. pylori</italic> infection in gastric biopsy specimens directly.</p>