Enhanced Two-Step LAMP-CRISPR Assay with an Engineered Zst Polymerase for Contamination-Free and Ultrasensitive DNA Detection,Analytical Chemistry

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Enhanced Two-Step LAMP-CRISPR Assay with an Engineered Zst Polymerase for Contamination-Free and Ultrasensitive DNA Detection
Analytical Chemistry ( IF 6.7 ) Pub Date : 2024-09-09 , DOI: 10.1021/acs.analchem.4c03965
Yi Xie _{1,

2} , Kai Jiang ₃ , Yuqian Zhang ₄ , Liqin Cao ₅ , Xiaokui Guo _{1,

2} , Jun Shi ₆ , Xiong Ding _{7,

8} , Kun Yin _{1,

2}

Affiliation

Current loop-mediated isothermal amplification (LAMP)-coupled clustered regularly interspaced short palindromic repeats (LAMP-CRISPR) biosensing in two-step or one-step formats has been applied to next-generation accurate molecular diagnosis. However, two-step LAMP-CRISPR assays intrinsically confront aerosol contamination, while one-step assays possess a compromised detection performance. To this end, we propose an enhanced two-step LAMP-CRISPR assay (ETL-CRISPR) with an engineered Zst polymerase to mediate ultrasensitive DNA detection and thoroughly eliminate aerosol contamination. Instead of supplementing any dTTP, the newly engineered Zst polymerase can efficiently polymerize four oligonucleotides (dATP, dCTP, dGTP, and dUTP), thereby enabling contamination-free and ultrasensitive ETL-CRISPR assay. By targeting the L1 gene of human papillomaviruses (HPV) 16 and the E7 gene of HPV18, our ETL-CRISPR assay achieves high specificity and single-copy level sensitivity within 1 h. Furthermore, we validated the assay by using 85 HPV clinical swab samples with an accuracy of 98.8%, which is comparable to the real-time quantitative polymerase chain reaction. Therefore, ETL-CRISPR provides a straightforward strategy for the contamination-free and ultrasensitive point-of-care diagnosis of clinical pathogens.

中文翻译：

使用工程 Zst 聚合酶增强两步 LAMP-CRISPR 检测，实现无污染和超灵敏 DNA 检测

当前两步或一步格式的环介导等温扩增（LAMP）耦合成簇规则间隔短回文重复序列（LAMP-CRISPR）生物传感已应用于下一代精确分子诊断。然而，两步式 LAMP-CRISPR 检测本质上会面临气溶胶污染，而一步式检测的检测性能会受到影响。为此，我们提出了一种增强的两步 LAMP-CRISPR 检测 (ETL-CRISPR)，采用工程Zst聚合酶来介导超灵敏 DNA 检测并彻底消除气溶胶污染。新设计的Zst聚合酶无需补充任何 dTTP，而是可以有效聚合四种寡核苷酸（dATP、dCTP、dGTP 和 dUTP），从而实现无污染且超灵敏的 ETL-CRISPR 测定。通过靶向人乳头瘤病毒 (HPV) 16 的 L1 基因和 HPV18 的 E7 基因，我们的 ETL-CRISPR 检测在 1 小时内实现了高特异性和单拷贝水平灵敏度。此外，我们使用 85 份 HPV 临床拭子样本验证了该检测方法，准确度为 98.8%，与实时定量聚合酶链反应相当。因此，ETL-CRISPR 为临床病原体的无污染和超灵敏的现场诊断提供了一种简单的策略。

更新日期：2024-09-09

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