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基于CRISPR/Cas12a和重组酶聚合酶扩增的鸭圆环病毒快速现场核酸检测

CRISPR/Cas12a and recombinase polymerase amplification-based rapid on-site nucleic acid detection of duck circovirus.

作者信息

Liang Qi-Zhang, Chen Wei, Liu Rong-Chang, Fu Qiu-Ling, Fu Guang-Hua, Cheng Long-Fei, Chen Hong-Mei, Jiang Nan-Song, Zhu Ting, Huang Yu

机构信息

Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou, 350013, China.

College of Animal Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.

出版信息

Virol J. 2024 Dec 19;21(1):322. doi: 10.1186/s12985-024-02577-7.

DOI:10.1186/s12985-024-02577-7
PMID:39702333
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11661005/
Abstract

BACKGROUND

Duck circovirus (DuCV) infections commonly induce immunosuppression and secondary infections in ducks, resulting in significant economic losses in the duck breeding industry. Currently, effective vaccines and treatments for DuCV have been lacking. Therefore, rapid, specific, and sensitive detection methods are crucial for preventing and controlling DuCV.

METHODS

A lateral flow strip (LFS) detection method was developed using recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a). The RPA-CRISPR/Cas12a-LFS targeted the DuCV replication protein (Rep) and was operated at 37 ℃ and allowed for visual interpretation without requiring sophisticated equipment.

RESULTS

The results revealed that the reaction time of RPA-CRISPR/Cas12a-LFS is only 45 min. This method achieved a low detection limit of 2.6 gene copies. Importantly, this method demonstrated high specificity and no cross-reactivity with six other avian viruses. In a study involving 97 waterfowl samples, the Rep RPA-CRISPR/Cas12a-LFS showed 100% consistency and agreement with real-time quantitative polymerase chain reaction.

CONCLUSION

These findings underscored the potential of this user-friendly, rapid, sensitive, and accurate detection method for on-site DuCV detection.

摘要

背景

鸭圆环病毒（DuCV）感染通常会导致鸭免疫抑制和继发感染，给养鸭业造成重大经济损失。目前，缺乏针对DuCV的有效疫苗和治疗方法。因此，快速、特异且灵敏的检测方法对于预防和控制DuCV至关重要。

方法

利用重组酶聚合酶扩增（RPA）和规律成簇间隔短回文重复序列（CRISPR）/CRISPR相关蛋白12a（Cas12a）开发了一种侧向流动试纸条（LFS）检测方法。RPA-CRISPR/Cas12a-LFS靶向DuCV复制蛋白（Rep），在37℃下运行，无需复杂设备即可进行可视化解读。

结果

结果显示，RPA-CRISPR/Cas12a-LFS的反应时间仅为45分钟。该方法实现了2.6个基因拷贝的低检测限。重要的是，该方法显示出高特异性，与其他六种禽病毒无交叉反应。在一项涉及97份水禽样本的研究中，Rep RPA-CRISPR/Cas12a-LFS与实时定量聚合酶链反应显示出100%的一致性和相符性。

结论

这些发现强调了这种用户友好、快速、灵敏且准确的检测方法在现场检测DuCV方面的潜力。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec78/11661005/56f08da3ac6d/12985_2024_2577_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec78/11661005/4dbb6fce8d95/12985_2024_2577_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec78/11661005/3efa37942bb7/12985_2024_2577_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec78/11661005/3a6e786ed2a3/12985_2024_2577_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec78/11661005/158d8141be9b/12985_2024_2577_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec78/11661005/f531aa422ff6/12985_2024_2577_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec78/11661005/56f08da3ac6d/12985_2024_2577_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec78/11661005/4dbb6fce8d95/12985_2024_2577_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec78/11661005/3efa37942bb7/12985_2024_2577_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec78/11661005/3a6e786ed2a3/12985_2024_2577_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec78/11661005/158d8141be9b/12985_2024_2577_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec78/11661005/f531aa422ff6/12985_2024_2577_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec78/11661005/56f08da3ac6d/12985_2024_2577_Fig6_HTML.jpg

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