| 猫疱疹病毒1型RPA-Cas12a-LFD检测方法的建立及初步应用 |
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| 引用本文： | 黄坚,刘韵佳,杨晓农,李妍. 猫疱疹病毒1型RPA-Cas12a-LFD检测方法的建立及初步应用[J]. 畜牧兽医学报, 2022, 53(5): 1638-1643. DOI: 10.11843/j.issn.0366-6964.2022.05.032 |
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| 作者姓名： | 黄坚 刘韵佳 杨晓农 李妍 |
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| 作者单位： | 1. 西南民族大学畜牧兽医学院, 成都 610041;2. 西南民族大学教学动物医院, 成都 610041 |
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| 基金项目： | 西南民族大学中央高校基本科研业务费专项(2020NQN32);西南民族大学引进人才科研启动金资助项目(RQD2021098) |
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| 摘 要： | 猫疱疹病毒1型(feline herpesvirus-1,FHV-1) 为水痘病毒属的双链DNA包膜病毒,是引起猫上呼吸道感染的主要病原之一。本研究以重组聚合酶扩增(RPA)、Cas12a反式酶切反应和侧流层析试纸条(LFD)显示技术为基础,旨在建立靶向FHV-1 TK基因的RPA-Cas12a-LFD快速可视化检测方法。根据FHV-1 TK基因的保守片段序列设计RPA引物和合成crRNA的引物,并通过反应体系验证、RPA-Cas12a-LFD检测灵敏度和特异性分析,以及临床样本的符合检测,评价FHV-1 RPA-Cas12a-LFD方法的有效性。结果显示,建立的FHV-1 RPA-Cas12a-LFD方法可以特异性地检出FHV-1(与其他猫相关病原无交叉反应),灵敏度极高(最低检测限为2.35×10-1 copies·μL-1),检测时间短,结果可视化。该方法对20份表现明显症状的患猫呼吸道样本的FHV-1检出率(35%,7/20)高于现有的TB Green qPCR方法(25%,5/20),对其中5份阳性样本的检测符合率为100%。综上表明,成功建立的FHV-1 RPA-Cas12a-LFD检测方法的特异性好和敏感性极高,不需要特殊的检测设备,可以作为FHV-1现场快速检测的有效工具。
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| 关 键 词： | 猫疱疹病毒1型 RPA-Cas12a-LFD 核酸检测 应用 |
| 收稿时间： | 2021-08-12 |
RPA-Cas12a-LFD Based Nucleic Acid Detection for Feline Herpesvirus-1 and Preliminary Application |
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| HUANG Jian,LIU Yunjia,YANG Xiaonong,LI Yan. RPA-Cas12a-LFD Based Nucleic Acid Detection for Feline Herpesvirus-1 and Preliminary Application[J]. Chinese Journal of Animal and Veterinary Sciences, 2022, 53(5): 1638-1643. DOI: 10.11843/j.issn.0366-6964.2022.05.032 |
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| Authors: | HUANG Jian LIU Yunjia YANG Xiaonong LI Yan |
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| Affiliation: | 1. College of Animal Husbandry & Veterinary Sciences, Southwest Minzu University, Chengdu 610041, China;2. Veterinary Teaching Hospital, Southwest Minzu University, Chengdu 610041, China |
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| Abstract: | Feline herpesvirus-1 (FHV-1) is double-stranded DNA enveloped virus of Varicellovirus, is one of major causative pathogen for feline upper respiratory tract infection. Based on recombinase polymerase amplification (RPA), Cas12a trans-cleavage reaction and lateral flow dipstick (LFD), a rapid visulization method for FHV-1 detection (RPA-Cas12a-LFD) was developed and validated. RPA amplified primers and crRNA synthesized primers were designed by targeting the FHV-1 TK gene, and then validation of RPA-Cas12a-LFD reaction system, sensitivity and specificity tests, and consistensy analysis of TB Green qPCR and RPA-Cas12a-LFD for FHV-1 detection were performed respectively. The results showed that the RPA-Cas12a-LFD method could specifically detect FHV-1 without cross-reaction with other associated pathogens, with advantages of high sensitivity (detection limit was 2.35×10-1copies·μL-1), short detection time and visual readout. The detection rate of FHV-1 in 20 clinical samples (35%, 7/20) was higher than TB Green qPCR (25%, 5/20) and the coincidence rate of 5 positive samples was 100%. In conclusion, RPA-Cas12a-LFD method was of good specificity and ultrasensitivity for FHV-1 detection without special equipment, and could be a promising and reliable tool for rapid on-site diagnosis. |
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| Keywords: | feline herpesvirus-1 RPA-Cas12a-LFD nucleic acid detection application |
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