Key Laboratory of Biomass Chemical Engineering (Education Ministry), College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
Institute of Biological Engineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
Anal Chem. 2021 Dec 21;93(50):16965-16973. doi: 10.1021/acs.analchem.1c04649. Epub 2021 Dec 10.
Invasive fungi (IF) have become a significant problem affecting human health. However, the culture-based assay of IF, known as the most commonly used clinical diagnostic method, suffers from time consumption, complicated operation, and the requirement of trained operators, which may cause the delay diagnosis of the disease. In this report, a microfluidic ruler-readout and CRISPR Cas12a-responded hydrogel-integrated paper-based analytical device (μReaCH-PAD) was established for visible and quantitative point-of-care testing of IF. Using the genus-conserved fragments of 18s rRNA as the detection target, this platform relied on a CRISPR Cas12a system for target recognition, a DNA hydrogel coupled with a cascade of enzymatic reactions for signal amplification and transduction, and paper-based microfluidic chips for visual quantitative readout by naked eyes. The 18s rRNA fragments of or were employed as a model target and introduced with PAM sites for Cas12a-recognition during reverse transcription recombinase-aided amplification. Using μReaCH-PAD, as low as 10 CFU/mL and were visually identified by unaided eyes. The calculated detection limits were 4.90 and 4.13 CFU/mL (in 1 mL samples), respectively. The quantitative detection results can be obtained in the range from 10 to 10 CFU/mL with reasonable specificity and accuracy compared with qRT-PCR. Furthermore, μReaCH-PAD can analyze complex biological samples by , , and detection systems and identify specific genera of different IF by naked eyes, indicating a good agreement with the culture-based assay and the advantages over G-testing and GM-testing systems. With the benefits of high sensitivity, selectivity, quantitative readout, low cost, and ease of operation, μReaCH-PAD is expected to provide a portable detection tool of IF in resource-limited settings by untrained personnel and technical support for early diagnosis.
侵袭性真菌（IF）已成为影响人类健康的重大问题。然而，基于 IF 的培养检测，作为最常用的临床诊断方法，存在耗时、操作复杂以及需要经过培训的操作人员等问题，可能导致疾病的延误诊断。在本报告中，建立了一种微流控尺读数和 CRISPR Cas12a 响应的水凝胶集成纸基分析装置（μReaCH-PAD），用于 IF 的可见和定量即时检测。该平台以 18s rRNA 的属保守片段作为检测靶标，依赖于 CRISPR Cas12a 系统进行靶标识别、与级联酶反应偶联的 DNA 水凝胶用于信号放大和转导，以及纸基微流控芯片用于通过肉眼进行可视化定量读数。采用 18s rRNA 片段作为模型靶标，并在逆转录重组酶辅助扩增过程中引入 PAM 位点用于 Cas12a 识别。使用 μReaCH-PAD，可通过肉眼直观识别低至 10 CFU/mL 的 和 。计算得出的检测限分别为 4.90 和 4.13 CFU/mL（在 1 mL 样本中）。与 qRT-PCR 相比，定量检测结果可在 10 到 10 CFU/mL 的范围内获得，具有合理的特异性和准确性。此外，μReaCH-PAD 可以通过 、 、 检测系统分析复杂的生物样本，并通过肉眼识别不同 IF 的特定属，与基于培养的检测方法具有良好的一致性，并且优于 G 检测和 GM 检测系统。μReaCH-PAD 具有高灵敏度、选择性、定量读数、低成本和易于操作等优点，有望为资源有限环境中的 IF 提供便携检测工具，并为早期诊断提供无需经过培训的人员和技术支持。
