Ratiometric Readout of Bacterial Infections via a Lyophilized CRISPR–Cas12a Sensor with Color-Changeable Bioluminescence,Analytical Chemistry

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Ratiometric Readout of Bacterial Infections via a Lyophilized CRISPR–Cas12a Sensor with Color-Changeable Bioluminescence
Analytical Chemistry ( IF 6.7 ) Pub Date : 2024-07-24 , DOI: 10.1021/acs.analchem.4c02114
Yu Wang ₁ , Xiaoning Xu ₁ , Jinqi Que ₁ , Xinyu Wang ₁ , Wei Ni ₂ , Yunhua Wu ₁ , Liu Yang ₂ , Yong Li _{1,

3}

Affiliation

The healthcare burden imposed by bacterial infections demands robust and accessible diagnostic methods that can be performed outside hospitals and centralized laboratories. Here, we report Pathogen Assay with Ratiometric Luminescence (PEARL), a sensitive and easy-to-operate platform for detecting pathogenic bacteria. The PEARL leveraged a color-changeable CRISPR–Cas12a sensor and recombinase polymerase amplification to elicit ratiometric bioluminescence responses to target inputs. This platform enabled robust and visualized identification of attomolar bacteria genome deoxyribonucleic acid according to the color changes of the reactions. In addition, the components of the color-changeable Cas12a sensor could be lyophilized for 3 month storage at ambient temperature and then be fully activated with the amplicons derived from crude bacterial lysates, reducing the requirements for cold-chain storage and tedious handling steps. We demonstrated that the PEARL assay is applicable for identifying the infections caused by Pseudomonas aeruginosa in different clinical specimens, including sputa, urines, and swabs derived from wounds. These results revealed the potential of PEARL to be used by untrained personnel, which will facilitate decentralized pathogen diagnosis in community- and resource-limited regions.

中文翻译：

通过具有可变色生物发光功能的冻干 CRISPR–Cas12a 传感器按比例读出细菌感染

细菌感染造成的医疗负担需要强大且易于使用的诊断方法，这些方法可以在医院和集中实验室之外进行。在这里，我们报告了比率发光病原体检测（PEARL），这是一种灵敏且易于操作的检测致病菌的平台。 PEARL 利用可变色的 CRISPR-Cas12a 传感器和重组酶聚合酶扩增来引发对目标输入的比例生物发光响应。该平台能够根据反应的颜色变化对阿托摩尔细菌基因组脱氧核糖核酸进行稳健且可视化的识别。此外，可变色Cas12a传感器的组件可以在环境温度下冻干保存3个月，然后用来自粗制细菌裂解物的扩增子完全激活，减少了对冷链存储和繁琐处理步骤的要求。我们证明，PEARL 检测适用于识别不同临床标本（包括痰液、尿液和伤口拭子）中铜绿假单胞菌引起的感染。这些结果揭示了 PEARL 可供未经培训的人员使用的潜力，这将有助于在社区和资源有限的地区进行分散的病原体诊断。

更新日期：2024-07-24

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