Establishment of a Cas12a-Based Visual Detection Method Involving PMNT for the Colletotrichum gloeosporioides Species Complex

文献类型： 外文期刊

第一作者： Zheng, Liu

作者： Zheng, Liu;Zou, Xiaohua;Fang, Xianping;Zhang, Liqing;Zheng, Liu;Xu, Xiaofeng;Jiang, Wei;Wu, Xiao;Song, Lili;Han, Yongchao;Lian, Hongli

作者机构：

关键词： anthracnose; CRISPR-Cas12a; field diagnosis; PMNT; visual method

期刊名称：PLANT DISEASE （ 影响因子：4.4； 五年影响因子：4.8 ）

ISSN： 0191-2917

年卷期： 2025 年 109 卷 3 期

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收录情况： SCI

摘要： Strawberry anthracnose, caused by Colletotrichum spp., is a devastating disease that significantly reduces strawberry yield and quality. This study aimed to develop a simple diagnostic method to detect infection by the Colletotrichum gloeosporioides species complex (CGSC), the most predominant and virulent Colletotrichum species complex causing strawberry anthracnose in China. In this study, a Cas12aVIP diagnostic method was developed for the rapid detection of the CGSC in strawberry seedlings. This method targets the beta-tubulin gene and combines recombinase polymerase amplification (RPA), the CRISPR/Cas12a system, and a cationic-conjugated polythiophene derivative [poly(3-(3 '-N,N,N-triethylamino-1 '-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT)] mixed with single-stranded DNA. This method shows high sensitivity (10 copies per reaction) and no cross-reactivity against related pathogens. The entire procedure, from sample to result, can be completed within 50 min, including simplified DNA extraction (15 min), RPA reaction (37 degrees C for 20 min), CRISPR/Cas12a detection (37 degrees C for 10 min), and visual detection by the naked eye (1 to 2 min). Furthermore, the Cas12aVIP assay successfully detected the CGSC in naturally infected strawberry seedling samples in field conditions. Asymptomatic infected plants and plant residues have been identified as primary inoculum sources for the CGSC. This method enables visible detection without the need for expensive equipment or specialized technical skills, thereby offering an efficient and straightforward approach for detecting the CGSC in strawberries. The newly developed detection method can be used to promote healthier strawberry production.

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