AUTHOR=Chi Wenjing , Wang Su , Liu Tao , Jiang Wenrong , Ding Li , Miao Yingxin , Yang Feng , Zhang Jinghao , Ji Danian , Xiao Zili , Zhu Haowei , Wu Yong , Bao Zhijun , Zhao Hu , Wang Shiwen 

TITLE=A rapid and high-throughput multiplex genetic detection assay for detection, semi-quantification and virulence genotyping of Helicobacter pylori in non-invasive oral samples

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=13

YEAR=2023

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2023.1267288

DOI=10.3389/fcimb.2023.1267288

ISSN=2235-2988

ABSTRACT=<sec><title>Aim</title><p>This study established a high-throughput multiplex genetic detection assay (HMGA) for rapid identification, semi-quantification and virulence analysis of <italic>Helicobacter pylori</italic> directly from the clinical non-invasive oral samples.</p></sec><sec><title>Methods</title><p>The gastric mucosa and oral samples were collected from 242 patients in Shanghai from 2021 to 2022. All the samples were detected by routine clinical tests for <italic>H. pylori</italic> and Sanger sequenced for inconsistent results. A new multiplex PCR assay providing results within 4 hours was designed and optimized involving fluorescent dye-labeled specific primers targeted <italic>16S rRNA</italic> gene, semi-quantitative gene <italic>ureC</italic> and 10 virulence genes of <italic>H. pylori</italic>. Semi-quantification was carried out by simulating the serial 10-fold dilutions of positive oral samples, and the <italic>H. pylori</italic> loads in different clinical samples were further compared. The mixed plasmids of virulence genes <italic>vacA s1</italic>, <italic>vacA m1</italic> and <italic>vacA m2</italic> were used to evaluate the performance on different genotypes. The consistency of 10 virulence genes in gastric mucosa, saliva, mouthwash and dental plaque of <italic>H. pylori</italic>-positive patients was compared.</p></sec><sec><title>Results</title><p>The non-invasive HMGA was highly specific for detection of all 12 targets of <italic>H. pylori</italic> and human internal reference gene <italic>β-globin</italic>, and the sensitivity to all target genes could reach 10 copies/μL. Compared with routine clinical tests and sequencing, non-invasive HMGA has a high level (&gt;0.98) of sensitivity, specificity, accuracy, PPV, NPV and kappa coefficient for direct detection of <italic>H. pylori</italic> in oral samples. Moreover, by detecting peak area levels of <italic>ureC</italic>, it was confirmed that the <italic>H. pylori</italic> loads in gastric mucosa were significantly higher than those of the three kinds of oral samples (<italic>p</italic>&lt;0.05). We also found that 45.0% (91/202) of patients had different <italic>H. pylori</italic> virulence genes in different oral samples. The concordance of positive detection rates of each virulence gene between saliva and gastric mucosa was more than 78% (<italic>p</italic>&lt;0.05).</p></sec><sec><title>Conclusion</title><p>The non-invasive HMGA proved to be a reliable method for the rapid <italic>H. pylori</italic> identification, semi-quantification and detection of 10 virulence genes directly in oral samples, providing a new idea for non-invasive detection of <italic>H. pylori</italic>.</p></sec>