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論文題目
Harnessing enhanced CRISPR/Cas12a trans-cleavage activity with
extended reporters and reductants for early diagnosis of Helicobacter pylori,
the causative agent of peptic ulcers and stomach cancer
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作者
Jean de Dieu Habimana, Omar Mukama, Guiquan Chen, Mengjun Chen, Obed
Boadi Amissah, Lin Wang, Yujie Liu, Yirong Sun, Amy L. Li, Sihao Deng,
Jufang Huang, Xiao-xin Yan, Theobard Rutaganda, Dieudonne Mutangana, Lin-Ping Wu,
Rongqi Huang ,Zhiyuan Li
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論文出處
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刊物名稱
Biosensors and Bioelectronics
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年卷期頁(yè)
2022, 222, , 114939
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聯(lián)系作者
Zhiyuan Li
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影響因子
12.545
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摘要
Developing rapid and non-invasive diagnostics for Helicobacter pylori (HP) is imperative to prevent associated diseases such as stomach gastritis, ulcers, and cancers. Owing to HP strain heterogeneity, not all HP-infected individuals incur side effects. Cytotoxin-associated gene A (CagA), and vacuolating cytotoxin A (VacA) genes predominantly drive HP pathogenicity. Therefore, diagnosing CagA and VacA genotypes could alert active infection and decide suitable therapeutics. We report an enhanced LbCas12a trans-cleavage activity with extended reporters and reductants (CEXTRAR) for early detection of HP. We demonstrate that extended ssDNA reporter acts as an excellent signal amplifier, making it a potential alternative substrate for LbCas12a collateral activity. Through a systematic investigation of various buffer components, we demonstrate that reductants improve LbCas12a trans-cleavage activity. Overall, our novel reporter and optimal buffer increased the trans-cleavage activity to an order of 16-fold, achieving picomolar sensitivity (171 pM) without target pre-amplification. Integrated with loop-mediated isothermal amplification (LAMP), CEXTRAR successfully attained attomolar sensitivity for HP detection using real-time fluorescence (43 and 96 aM), in-tube fluorescence readouts (430 and 960 aM), and lateral flow (4.3 and 9.6 aM) for CagA and VacA, respectively. We also demonstrate a rapid 2-min Triton X-100 lysis for clinical sample analysis, which could provide clinicians with actionable information for rapid diagnosis. CEXTRAR could potentially spot the 13C urea breath test false-negatives. For the first time, our study unveils an experimental outlook to manipulate reporters and reconsider precise cysteine substitution via protein engineering for Cas variants with enhanced catalytic activities for use in diagnostics and genetic engineering.
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英文摘要
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外單位作者單位
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