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Development of a rapid visual detection technology for BmNPV based on CRISPR/Cas13a system

文献类型： 外文期刊

作者： Zhou, Xue-Min 1 ; Shen, Zhen-Yu 1 ; Wu, Yi-Xiang 1 ; Lin, Su 1 ; Wang, Meng-Dong 1 ; Xu, Tao 1 ; Wang, Lu-Lai 1 ; Sadiq, Samreen 1 ; Jiao, Xin-Hao 1 ; Wu, Ping 1 ;

作者机构： 1.Jiangsu Univ Sci & Technol, Sch Biotechnol, Zhenjiang, Peoples R China

2.Chinese Acad Agr Sci, Minist Agr & Rural Affairs, Sericultural Res Inst, Key Lab Silkworm & Mulberry Genet Improvement, Zhenjiang, Peoples R China

关键词： CRISPR/Cas13a; BmNPV; RPA; Rapid visual nucleic acid detection technology

期刊名称：JOURNAL OF INVERTEBRATE PATHOLOGY （ 影响因子：3.4； 五年影响因子：3.1 ）

ISSN： 0022-2011

年卷期： 2024 年 203 卷

页码：

收录情况： SCI

摘要： Pathogenic microorganism of silkworm are important factors that threaten the high-quality development of sericulture. Among them, Bombyx mori nucleopolyhedrovirus (BmNPV) caused diseases often lead to frequent outbreaks and high mortality, resulting in huge losses to sericultural industry. Current molecular detection methods for BmNPV require expensive equipment and sikilled technical personnel. As a result, the most commonly detection method for silkworm egg production enterprises involves observing the presence of polyhedra under a microscope. However, this method has low accuracy and sensitivity. There is an urgent need to develop a new detection technology with high sensitivity, high specificity, and applicability for silkworm farms, silkworm egg production enterprises and quarantine departments. In this study, we successfully established the CRISPR/Cas13a BmNPV visualized detection technology by combining Recombinase Polymerase Amplification (RPA) technology and CRISPR/Cas13a system. This technology is based on microplate lateral, flow test strips and portable fluorescence detector. The detection sensitivity can reach up to 1 copies/mu L for positive standard plasmid and 1 fg/mu L for BmNPV genome in 30-45 min, demonstrating high sensitivity. By detecting silkworm tissues infected with different pathogens, we determined that CRISPR/Cas13a detection technology has good specificity. In summary, the newly established nucleic acid detection technology for BmNPV is characterized by high sensitivity, high specificity, low cost and convenience for visualization. It can be applied in field detection and silkworm egg quality monitory system.

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