基于RPA-CRISPR/Cas12a技术快速检测犬猫皮肤癣菌方法的建立

doi:10.11843/j.issn.0366-6964.2023.11.024

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (11): 4702-4711.doi: 10.11843/j.issn.0366-6964.2023.11.024

基于RPA-CRISPR/Cas12a技术快速检测犬猫皮肤癣菌方法的建立

郜平平¹, 付金玉², 王丽仰¹, 史硕博^2*, 张跃平^1*, 张迪^1*

1. 中国农业大学动物医学院, 北京 100193;
2. 北京化工大学北京软物质科学与工程高精尖创新中心, 北京 100029

收稿日期:2023-01-06 出版日期:2023-11-23 发布日期:2023-11-26
通讯作者: 史硕博,主要从事代谢工程、系统生物学与合成生物学的基础与应用性研究,E-mail:shishuobo@mail.buct.edu.cn;张跃平,主要从事酵母合成生物学,真菌致病机理以及耐药性机制研究,E-mail:zhangyueping@cau.edu.cn;张迪,主要从事小动物皮肤病学研究,E-mail:dzhangdvm@cau.edu.cn
作者简介:郜平平(1998-),女,安徽淮北人,硕士生,主要从事犬猫皮肤病诊断方面的研究,E-mail:gaopingping@cau.edu.cn
基金资助:
国家自然科学基金(32100156)

Rapid RPA-CRISPR/Cas12a Detection Platform for Dermatophytes in Dogs and Cats

GAO Pingping¹, FU Jinyu², WANG Liyang¹, SHI Shuobo^2*, ZHANG Yueping^1*, ZHANG Di^1*

1. College of Veterinary Medicine, China Agricultural University, Beijing 100193, China;
2. Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, Beijing 100029, China

Received:2023-01-06 Online:2023-11-23 Published:2023-11-26

摘要/Abstract

摘要： 旨在建立重组酶聚合酶扩增技术(recombinase polymerase amplification, RPA)结合CRISPR/Cas12a技术的荧光检测方法,以快速、灵敏、可视化检测犬猫皮肤癣菌。以犬小孢子菌、石膏样小孢子菌及须毛癣菌为研究对象,针对真菌内转录间隔区,设计并合成特异性RPA引物和CRISPR RNA(crRNA),建立可同时或分别检测上述皮肤癣菌的荧光检测方法,并评价其检测灵敏度,通过检测临床样本评价本检测方法的敏感性和特异性。结果表明:RPA-CRISPR/Cas12a在37 ℃、总反应时间30 min的条件下,对三种皮肤癣菌的检测灵敏度可低至单拷贝。对24份临床样本进行检测,以真菌培养和菌落测序结果作为标准,使用可同时识别犬小孢子菌、石膏样小孢子菌及须毛癣菌的皮肤癣菌crRNA(dermatophyte crRNA,crRNA-DM)和可特异性识别犬小孢子菌的犬小孢子菌crRNA(Microsporum canis crRNA,crRNA-Mc)参与RPA-CRISPR/Cas12a反应时,敏感性和特异性均为100%。RPA-CRISPR/Cas12a技术可同时和分别检测犬小孢子菌、石膏样小孢子菌及须毛癣菌,所需时间短,结果可视化,敏感性和特异性高,无需昂贵仪器,适合临床快速诊断。

关键词: RPA, CRISPR/Cas12a, 犬猫皮肤癣菌, 快速检测

Abstract: This study was conducted to establish a fluorescence detection method of recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a for rapid, sensitive and visual detection of dermatophytes in dogs and cats. Taking Microsporum canis, Nannizia gypsea and Trichophyton mentagrophytes as research objects, specific RPA primers and CRISPR RNA (crRNA) were designed and synthesized in the internal transcribed spacer region. A fluorescence detection method for simultaneous and separate detection of the dermatophytes was established, and the limit of detection was evaluated. The sensitivity and specificity of the method were evaluated by detecting clinical samples. Under the condition of 37 ℃, the limit of detection (LOD) for three kinds of dermatophytes can be as low as a single copy in 30 min. Twenty-four clinical samples were tested, and the results of fungal culture and colony sequencing were taken as standard results. The sensitivity and specificity of this assay were 100% when using the dermatophyte crRNA (crRNA-DM) that can simultaneously detect M. canis, N. gypsum and T. mentagrophytes, and the Microsporum canis crRNA (crRNA-Mc) that can specifically detect M. canis in the RPA-CRISPR/Cas12a reaction. The RPA-CRISPR/Cas12a established in this study can detect M. canis, N. gypsum and T. mentagrophytes simultaneously and separately. This technique required short time, visualization, high sensitivity and specificity, did not need expensive instruments, and was suitable for rapid clinical diagnosis.

Key words: recombinase polymerase amplification, CRISPR/Cas12a, dermatophytes in dogs and cats, rapid detection

中图分类号:

S857.5

郜平平, 付金玉, 王丽仰, 史硕博, 张跃平, 张迪. 基于RPA-CRISPR/Cas12a技术快速检测犬猫皮肤癣菌方法的建立[J]. 畜牧兽医学报, 2023, 54(11): 4702-4711.

GAO Pingping, FU Jinyu, WANG Liyang, SHI Shuobo, ZHANG Yueping, ZHANG Di. Rapid RPA-CRISPR/Cas12a Detection Platform for Dermatophytes in Dogs and Cats[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4702-4711.

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基于RPA-CRISPR/Cas12a技术快速检测犬猫皮肤癣菌方法的建立

Rapid RPA-CRISPR/Cas12a Detection Platform for Dermatophytes in Dogs and Cats

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