RPA-CRISPR/Cas12a在病原微生物检测中的研究进展

Research progress of RPA-CRISPR/Cas12a in the detection of pathogenic microorganisms

摘要:

重组酶聚合酶扩增(recombinase polymerase amplification, RPA)可以在恒温条件下高效扩增靶标序列以快速达到可以检测的水平，具有检测灵敏度高、检测速度快和设备依赖程度低等优点，是应用较广泛的恒温扩增技术之一。然而，RPA的非特异扩增会导致假阳性等问题。成簇的规律间隔短回文重复序列相关蛋白12a (clustered regularly interspaced short palindromic repeats/associated protein 12a, CRISPR/Cas12a)具有特异性酶切靶标双链功能，并且其最适温度与RPA反应温度相近。近年来，研究者将RPA与CRISPR/Cas12a联合对目的基因进行双重特异性识别，极大地提高了检测特异性，同时也进一步提升了检测的灵敏度，展示出检测特异性强、灵敏度高、适用范围广和操作简单等诸多优点，尤其在病原微生物检测方面展示出较大的应用前景。本文就RPA-CRISPR/Cas12a的技术原理、产物检测策略和在病原微生物检测等方面的研究进展进行综述，以期为RPA-CRISPR/Cas12a进一步开发、利用以及在病原微生物检测方面的应用提供借鉴。

Abstract:

Recombinase polymerase amplification (RPA) can efficiently amplify the target sequence at constant temperatures and is praised for high sensitivity, short time consumption, and low equipment dependence. Therefore, RPA is a widely used isothermal amplification technology in recent years. However, non-specific amplification of RPA has limited its further development. The clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a) is capable of specifically cleaving the double strands and has similar optimal temperature to the PRA temperature. Therefore, researchers have combined RPA with CRISPR/Cas12a to improve the detection specificity. Moreover, this method demonstrates high sensitivity, a wide application range, and simple operation. Particularly, the RPA-CRISPR/ Cas12a-based approach shows a promising application prospect in the detection of pathogenic microorganisms. This paper introduces RPA-CRISPR/Cas12a regarding the principles, product detection strategies, and application, providing a reference for the further development and application of RPA-CRISPR/Cas12a in the detection of pathogenic microorganisms.