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Dipstick-based rapid nucleic acids purification and CRISPR/Cas12a-mediated isothermal amplification for visual detection of African swine fever virus

文献类型： 外文期刊

作者： Qian, Siwenjie 1 ; Chen, Yanju 1 ; Peng, Cheng 2 ; Wang, Xiaofu 2 ; Wu, Hui 1 ; Che, Yang 3 ; Wang, Huanying 3 ; Xu, Junfeng 2 ; Wu, Jian 1 ;

作者机构： 1.Zhejiang Univ, Coll Biosyst Engn & Food Sci, Hangzhou 310058, Peoples R China

2.Zhejiang Acad Agr Sci, State Key Lab Managing Biot & Chem Threats Qual &, Hangzhou 310021, Peoples R China

3.Zhejiang Inst Microbiol, Key Lab Microbiol Technol & Bioinformat Zhejiang, Hangzhou 310012, Peoples R China

4.Minist Agr, Key Lab Site Proc Equipment Agr Prod, Hangzhou 310058, Peoples R China

关键词： Nucleic acid purification; Dipsticks; Loop-mediated isothermal amplification; CRISPR; Cas12a; African swine fever virus

期刊名称：TALANTA （ 影响因子：6.556； 五年影响因子：5.77 ）

ISSN： 0039-9140

年卷期： 2022 年 242 卷

页码：

收录情况： SCI

摘要： African swine fever virus (ASFV) can cause highly contagious and fatal disease among domestic pigs, resulting in considerable economic losses for swine breeders. There is a strong demand for accurate, rapid, and simple detection methods especially for on-site application. Nucleic acid testing is the most commonly used method for ASFVdetection. However, traditional nucleic acid purification step is time- and labor-consuming. The nucleic acid purification, amplification and amplicons detection rely on laboratory settings which limits the on-site detection. Here, we proposed a simple and cost-effective detection method that utilized filter paper to purify nucleic acids from swine blood and employed CRISPR/Cas12a-mediated loop-mediated isothermal amplification (LAMP) reaction to detect ASFV. The filter paper which was made into dipsticks could effectively purify nucleic acids from whole blood in 2 min. This simple and low-cost purification method avoided multiple pipetting steps and potential amplification inhibitors (e.g., ethanol) that were generally used in traditional nucleic acids extraction processes. After nucleic acid purification, the lyophilized LAMP reagent dissolved by elution solution was employed to perform isothermal amplification reaction on a portable heating block. The CRISPR/Cas12a system was designed to specifically detect amplicons. Assisted by a portable homemade device, the fluorescent signals produced by positive samples could be observed by the naked eye, while negative samples remained colorless. The whole detection procedure could be finished within 50 min with a detection limit of one copies/mu L. This established method provided a novel strategy for rapid visualized detection and showed great potential for on-site application.

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