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Purpose <p>This study aims to establish a platform and to achieve rapid, simultaneous detection of <i>Helicobacter pylori</i> along with its key virulence genes, cytotoxin-associated gene A <i>(cagA)</i> and vacuolating cytotoxin A <i>(vacA)</i>.</p> Methods <p>We developed a multiplex recombinase polymerase amplification combined with lateral flow strip (mRPA-LFS) assay for the rapid, simultaneous detection of <i>H. pylori</i> (targeting the <i>16&#xa0;S rRNA</i> gene) and its key virulence genes, <i>cagA</i> and <i>vacA</i>. The assay employs a nucleic acid tag (NAT)-bridging strategy for multiplex visualization without instrumentation. Performance was evaluated using recombinant plasmids and clinical saliva samples (<i>n</i> = 28), with <i>16&#xa0;S rRNA</i> detection compared against the urea breath test and genotyping results against quantitative PCR (qPCR).</p> Results <p>The mRPA-LFS assay completes detection within 30&#xa0;min at a constant temperature of 39&#xa0;°C. It demonstrated high sensitivity with limits of detection of 10¹, 10³, and 10² copies/µL for the <i>16&#xa0;S rRNA</i>, <i>cagA</i>, and <i>vacA</i> genes, respectively, and showed no cross-reactivity with nine common non-<i>H. pylori</i> pathogens. The detection results of the mRPA-LFS for <i>H. pylori</i> were highly consistent with those of qPCR (complete agreement for <i>16&#xa0;S rRNA</i> and <i>cagA</i> genes; sensitivity of 94.4% and specificity of 100% for <i>vacA</i>).</p> Conclusions <p>The established mRPA-LFS platform represents a significant advance toward point-of-care molecular typing of <i>H. pylori</i>, offering a simple, rapid, and multiplex strategy that is particularly suitable for frontline screening and clinical decision-making in low-resource environments.</p>

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Simultaneous point-of-care detection and virulence typing of Helicobacter pylori using multiplex recombinase polymerase amplification combined with lateral flow strip

  • 期刊论文

  • 发表日期：

    2026年4月18日

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  • Lele Song,
  • Lufei Yuan,
  • Zhoujie Ma,
  • Rong Jiang,
  • Jilu Shen

摘要

Purpose

This study aims to establish a platform and to achieve rapid, simultaneous detection of Helicobacter pylori along with its key virulence genes, cytotoxin-associated gene A (cagA) and vacuolating cytotoxin A (vacA).

Methods

We developed a multiplex recombinase polymerase amplification combined with lateral flow strip (mRPA-LFS) assay for the rapid, simultaneous detection of H. pylori (targeting the 16 S rRNA gene) and its key virulence genes, cagA and vacA. The assay employs a nucleic acid tag (NAT)-bridging strategy for multiplex visualization without instrumentation. Performance was evaluated using recombinant plasmids and clinical saliva samples (n = 28), with 16 S rRNA detection compared against the urea breath test and genotyping results against quantitative PCR (qPCR).

Results

The mRPA-LFS assay completes detection within 30 min at a constant temperature of 39 °C. It demonstrated high sensitivity with limits of detection of 10¹, 10³, and 10² copies/µL for the 16 S rRNA, cagA, and vacA genes, respectively, and showed no cross-reactivity with nine common non-H. pylori pathogens. The detection results of the mRPA-LFS for H. pylori were highly consistent with those of qPCR (complete agreement for 16 S rRNA and cagA genes; sensitivity of 94.4% and specificity of 100% for vacA).

Conclusions

The established mRPA-LFS platform represents a significant advance toward point-of-care molecular typing of H. pylori, offering a simple, rapid, and multiplex strategy that is particularly suitable for frontline screening and clinical decision-making in low-resource environments.