Homemade isothermal amplification-initiated Cas14a assay for rapid quantitative detection of aquatic RNA virus gene with no PAM

IF 4.4 1区农林科学 Q1 FISHERIES

Aquaculture Pub Date : 2025-01-30 Epub Date: 2024-09-19 DOI:10.1016/j.aquaculture.2024.741661

Xiufen Yang , Lin Lu , Yanchi Luo , Qingwei Wang , Jianing Wang , Yihua Ren , Yuwei Wu , Masoud Negahdary , Khaydar E. Yunusov , Sherzod A. Yuldoshov , Abdushkur A. Sarimsakov , Haimei Mao , Yi Wan , Fengge Song

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引用次数: 0

Abstract

The red-spotted grouper nervous necrosis virus (RGNNV) is the most widely distributed nervous necrosis virus (NNV) and causes a significant risk to aquatic food safety. Though a few CRISPR-based aquatic pathogen detection methods have been reported recently, the absence of the PAM site of RGNNV specific gene RNA2 impedes its CRISPR-based diagnostic. Herein, we developed a rapid and accurate homemade isothermal amplification-initiated Cas14a assay (HiaCas14a) for RGNNV detection. The introduction of the PAM site into RT-LAMP specific amplicons of RGNNV RNA2 unlocked its PAM restriction. Furthermore, fish genomic RNA purified by magnetic beads and polymerases used in RT-LAMP prepared by ourselves obviously gave the system an enhanced convenience and a reduced cost. HiaCas14a detected 63.4 aM RNA with excellent specificity and sensitivity quantitatively, and it demonstrated a high accuracy of field sample validation results compared with RT-qPCR. The method provided an accurate, low-cost, and sensitive tool for aquatic pathogen detection in resource-poor areas.

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自制等温扩增启动 Cas14a 检测法，用于快速定量检测无 PAM 的水生 RNA 病毒基因

红斑石斑鱼神经坏死病毒（RGNNV）是分布最广的神经坏死病毒（NNV），对水产食品安全构成重大威胁。虽然最近有一些基于CRISPR的水生病原体检测方法的报道，但由于RGNNV特异基因RNA2的PAM位点缺失，阻碍了其基于CRISPR的诊断。在此，我们开发了一种快速、准确的自制等温扩增启动 Cas14a 检测方法（HiaCas14a），用于检测 RGNNV。在 RGNNV RNA2 的 RT-LAMP 特异性扩增子中引入 PAM 位点，解开了其 PAM 限制。此外，磁珠纯化的鱼类基因组 RNA 和自己制备的 RT-LAMP 中使用的聚合酶明显提高了系统的便利性，降低了成本。HiaCas14a 能定量检测出 63.4 aM 的 RNA，特异性和灵敏度都很高，与 RT-qPCR 相比，现场样本验证结果的准确性也很高。该方法为资源贫乏地区的水生病原体检测提供了一种准确、低成本和灵敏的工具。

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来源期刊

Aquaculture 农林科学-海洋与淡水生物学

CiteScore

8.60

自引率

17.80%

发文量

1246

审稿时长

56 days

期刊介绍： Aquaculture is an international journal for the exploration, improvement and management of all freshwater and marine food resources. It publishes novel and innovative research of world-wide interest on farming of aquatic organisms, which includes finfish, mollusks, crustaceans and aquatic plants for human consumption. Research on ornamentals is not a focus of the Journal. Aquaculture only publishes papers with a clear relevance to improving aquaculture practices or a potential application.