School of Food Science and Pharmaceutical Engineering, School of Life Sciences, Nanjing Normal University, Nanjing 210023, China.
Jiangsu Key Laboratory of Marine Biological Resources and Environment, Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Jiangsu Ocean University, Lianyungang 222005, China.
J Agric Food Chem. 2023 Apr 26;71(16):6490-6498. doi: 10.1021/acs.jafc.2c08689. Epub 2023 Apr 4.
Acute hepatopancreatic necrosis disease (AHPND) is one of the most devastating diseases in aquaculture, causing significant economic losses in seafood supplies worldwide. Early detection is critical for its prevention, which requires reliable and fast-responding diagnosis tools with point-of-care testing (POCT) capacity. Recombinase polymerase amplification (RPA) has been combined with CRISPR/Cas12a for AHPND diagnosis with a two-step procedure, but the operation is inconvenient and has the risk of carryover contamination. Here, we develop an RPA-CRISPR one-pot assay that integrates RPA and CRISPR/Cas12a cleavage into simultaneous reactions. Using the special design of crRNA, which is based on suboptimal protospacer adjacent motifs (PAM), RPA and Cas12a are made compatible in one pot. The assay is highly specific with a good sensitivity of 10 copies/reaction. This study provides a new choice for AHPND diagnosis with a POCT facility and sets a good example for developing RPA-CRISPR one-pot molecular diagnosis assays.
急性肝胰腺坏死病（AHPND）是水产养殖中最具破坏性的疾病之一，导致全球海产品供应的重大经济损失。早期检测对于预防该病至关重要，这需要具有即时检测（POCT）能力的可靠和快速响应的诊断工具。重组聚合酶扩增（RPA）与 CRISPR/Cas12a 结合，通过两步法用于 AHPND 诊断，但操作不方便，有携带污染的风险。在这里，我们开发了一种 RPA-CRISPR 一锅法检测，将 RPA 和 CRISPR/Cas12a 切割整合到同时进行的反应中。通过基于次优原间隔基序（PAM）的 crRNA 的特殊设计，使 RPA 和 Cas12a 在一个管中兼容。该检测具有良好的特异性和灵敏度，可达 10 个拷贝/反应。本研究为具有 POCT 设备的 AHPND 诊断提供了新的选择，并为开发 RPA-CRISPR 一锅法分子诊断检测方法树立了良好的范例。
