■幽门螺杆菌引起的感染(H.幽门螺杆菌)影响约50%的全球人口。是慢性胃炎和胃癌的主要致病身分。此外,对克拉霉素等抗生素的耐药性会降低根除率。目前,迫切需要一种快速的,易于执行,以及对幽门螺杆菌和克拉霉素耐药性的高灵敏度检测方法。
■我们使用FAM/地高辛标记的引物通过重组酶辅助扩增(RAA)扩增特定的幽门螺杆菌23SrRNA片段,和抗性突变使用CRISPR/Cas13a系统结合侧流条进行区分。使用qPCR分析了28个唾液样本,基因测序和该方法的检测效率评价。
■我们开发了一种同时检测幽门螺杆菌和克拉霉素抗性突变的方法,称为敏感的幽门螺杆菌易读双重检测(SHIELD)。结果表明,引起克拉霉素抗性的A2142G和A2143G突变体DNA均可与野生型区分,浓度为50拷贝/μL,未观察到与其他5种常见胃肠道细菌的交叉反应。用于检测28份唾液样本中的幽门螺杆菌,与qPCR相比,该方法的阳性预测值为100%(19/19)。为了检测克拉霉素耐药性,与基因测序相比,该方法的阳性预测值为84.6%(11/13)。
■SHIELD检测在检测幽门螺杆菌和克拉霉素抗性突变方面显示出高灵敏度和特异性。它可能是快速检测幽门螺杆菌的潜在措施,大规模筛查和指导临床用药。
UNASSIGNED: Infection caused by Helicobacter pylori (H. pylori) affects approximately 50% of the global population. It is a major pathogenic factor for chronic gastritis and gastric cancer. Besides, the resistance to antibiotics such as clarithromycin could reduce the eradication rate. Currently, there is an urgent need for a swift, easy to perform, and highly sensitive detection method for H. pylori and clarithromycin resistance.
UNASSIGNED: We used FAM/Digoxin labeled primers to amplify specific H. pylori 23S rRNA fragments by Recombinase Aided Amplification (RAA), and resistance mutations were distinguished using CRISPR/Cas13a system combined with lateral flow strip. Twenty-eight saliva samples were analyzed using qPCR, gene sequencing and this method to evaluate the detection efficiency.
UNASSIGNED: We developed a simultaneous detection method for H. pylori and clarithromycin resistance mutations named sensitive H. pylori easy-read dual detection (SHIELD). The results showed both A2142G and A2143G mutant DNAs causing clarithromycin resistance could be distinguished from the wild type with a concentration of 50 copies/μL, and no cross-reaction with other 5 common gastrointestinal bacteria was observed. For the detection of H. pylori in 28 saliva samples, the positive predictive value of this method was 100% (19/19) in comparison with qPCR. For detecting clarithromycin resistance, the positive predictive value of this method was 84.6% (11/13) compared with gene sequencing.
UNASSIGNED: SHIELD assay showed high sensitivity and specificity in detecting H. pylori and clarithromycin resistance mutations. It could be a potential measure in the rapid detection of H. pylori, large-scale screening and guiding clinical medication.