Rapid visual detection of Helicobacter pylori and vacA subtypes by Dual-Target RAA-LFD assay

IF 3.2 3区医学 Q2 MEDICAL LABORATORY TECHNOLOGY

Clinica Chimica Acta Pub Date : 2024-08-15 DOI:10.1016/j.cca.2024.119927

Sijie Yin , Yanghe Liu , Xinyi Yang , Nasifu Lubanga , Ping Tai , Mengqiu Xiong , Boyue Fan , Xincheng Yang , Zhenlin Nie , Qingsong Zhang , Bangshun He

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引用次数: 0

Abstract

Background

Helicobacter pylori (H. pylori) infects over 50% of the global population and is a significant risk factor for gastric cancer. The pathogenicity of H. pylori is primarily attributed to virulence factors such as vacA. Timely and accurate identification, along with genotyping of H. pylori virulence genes, are essential for effective clinical management and controlling its prevalence.

Methods

In this study, we developed a dual-target RAA-LFD assay for the rapid, visual detection of H. pylori genes (16s rRNA, ureA, vacA m1/m2), using recombinase aided amplification (RAA) combined with lateral flow dipstick (LFD) methods. Both 16s rRNA and ureA were selected as identification genes to ensure reliable detection accuracy.

Results

A RAA-LFD assay was developed to achieve dual-target amplification at a stable 37 °C within 20 min, followed by visualization using the lateral flow dipstick (LFD). The whole process, from amplification to results, took less than 30 min. The 95 % limit of detection (LOD) for 16 s rRNA and ureA, vacA m1, vacA m2 were determined as 3.8 × 10^-2 ng/μL, 5.8 × 10^-2 ng/μL and 1.4 × 10^-2 ng/μL, respectively. No cross-reaction was observed in the detection of common pathogens including Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis, showing the assay’s high specificity. In the evaluation of the clinical performance of the RAA-LFD assay. A total of 44 gastric juice samples were analyzed, immunofluorescence staining (IFS) and quantitative polymerase chain reaction (qPCR) were used as reference methods. The RAA-LFD results for the 16s rRNA and ureA genes showed complete agreement with qPCR findings, accurately identifying H. pylori infection as confirmed by IFS in 10 out of the 44 patients. Furthermore, the assay successfully genotyped vacA m1/m2 among the positive samples, showing complete agreement with qPCR results and achieving a kappa (κ) value of 1.00.

Conclusion

The dual-target RAA-LFD assay developed in this study provides a rapid and reliable method for detecting and genotyping H. pylori within 30 min, minimizing dependency on sophisticated laboratory equipment and specialized personnel. Clinical validation confirms its efficacy as a promising tool for effectively control of its prevalence and aiding in the precise treatment of H. pylori-associated diseases.

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通过双靶标 RAA-LFD 检测法快速目测幽门螺杆菌和 vacA 亚型。

背景：幽门螺杆菌（H. pylori）感染了全球 50%以上的人口，是胃癌的重要危险因素。幽门螺杆菌的致病性主要归因于vacA等毒力因子。及时准确地识别幽门螺杆菌毒力基因并对其进行基因分型，对于有效的临床管理和控制其流行至关重要：在这项研究中，我们利用重组酶辅助扩增（RAA）结合侧流点液法（LFD）开发了一种双靶 RAA-LFD 检测方法，用于快速、直观地检测幽门螺杆菌基因（16 s rRNA、ureA、vacA m1/m2）。选择 16 s rRNA 和 ureA 作为鉴定基因，以确保可靠的检测精度：结果：开发出一种 RAA-LFD 检测方法，可在 20 分钟内实现 37 ℃ 稳定温度下的双目标扩增，然后使用侧向流动点样器（LFD）进行检测。从扩增到结果的整个过程不到 30 分钟。16 s rRNA 和 ureA、vacA m1、vacA m2 的 95 % 检测限（LOD）分别为 3.8 × 10-2 ng/μL、5.8 × 10-2 ng/μL 和 1.4 × 10-2 ng/μL。在检测大肠埃希菌、肺炎克雷伯菌、粪肠球菌、金黄色葡萄球菌、铜绿假单胞菌和枯草芽孢杆菌等常见病原体时未发现交叉反应，显示了该检测方法的高特异性。在 RAA-LFD 检测法的临床性能评估中。共分析了 44 份胃液样本，并使用免疫荧光染色法（IFS）和定量聚合酶链反应法（qPCR）作为参照方法。RAA-LFD 检测 16 s rRNA 和 ureA 基因的结果与 qPCR 检测结果完全一致，44 例患者中有 10 例经 IFS 证实感染了幽门螺杆菌。此外，该检测方法还成功地对阳性样本中的 vacA m1/m2 进行了基因分型，结果与 qPCR 结果完全一致，卡帕（κ）值达到 1.00：本研究开发的 RAA-LFD 双靶标检测法是一种快速可靠的方法，可在 30 分钟内检测幽门螺杆菌并进行基因分型，最大程度地减少了对精密实验室设备和专业人员的依赖。临床验证证实了该方法的有效性，是有效控制幽门螺杆菌感染率和帮助精确治疗幽门螺杆菌相关疾病的有效工具。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Clinica Chimica Acta 医学-医学实验技术

CiteScore

10.10

自引率

2.00%

发文量

1268

审稿时长

23 days

期刊介绍： The Official Journal of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Clinica Chimica Acta is a high-quality journal which publishes original Research Communications in the field of clinical chemistry and laboratory medicine, defined as the diagnostic application of chemistry, biochemistry, immunochemistry, biochemical aspects of hematology, toxicology, and molecular biology to the study of human disease in body fluids and cells. The objective of the journal is to publish novel information leading to a better understanding of biological mechanisms of human diseases, their prevention, diagnosis, and patient management. Reports of an applied clinical character are also welcome. Papers concerned with normal metabolic processes or with constituents of normal cells or body fluids, such as reports of experimental or clinical studies in animals, are only considered when they are clearly and directly relevant to human disease. Evaluation of commercial products have a low priority for publication, unless they are novel or represent a technological breakthrough. Studies dealing with effects of drugs and natural products and studies dealing with the redox status in various diseases are not within the journal''s scope. Development and evaluation of novel analytical methodologies where applicable to diagnostic clinical chemistry and laboratory medicine, including point-of-care testing, and topics on laboratory management and informatics will also be considered. Studies focused on emerging diagnostic technologies and (big) data analysis procedures including digitalization, mobile Health, and artificial Intelligence applied to Laboratory Medicine are also of interest.