猫疱疹病毒1型RPA-Cas12a-LFD检测方法的建立及初步应用

doi:10.11843/j.issn.0366-6964.2022.05.032

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (5): 1638-1643.doi: 10.11843/j.issn.0366-6964.2022.05.032

猫疱疹病毒1型RPA-Cas12a-LFD检测方法的建立及初步应用

黄坚^1,2, 刘韵佳¹, 杨晓农^1,2, 李妍^1,2*

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

收稿日期:2021-08-12 出版日期:2022-05-23 发布日期:2022-05-25
通讯作者: 李妍,主要从事临床兽医学研究,E-mail:lyvet2004@163.com
作者简介:黄坚(1984-),广西柳州人,讲师,博士,主要从事动物临床疾病诊疗和兽医临床微生物研究,E-mail:huangjian.1122@163.com;刘韵佳(1996-),四川成都人,硕士,主要从事动物疾病临床诊断研究,E-mail:846964301@qq.com。黄坚和刘韵佳为同等贡献作者
基金资助:
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)

RPA-Cas12a-LFD Based Nucleic Acid Detection for Feline Herpesvirus-1 and Preliminary Application

HUANG Jian^1,2, LIU Yunjia¹, YANG Xiaonong^1,2, LI Yan^1,2*

1. College of Animal Husbandry & Veterinary Sciences, Southwest Minzu University, Chengdu 610041, China;
2. Veterinary Teaching Hospital, Southwest Minzu University, Chengdu 610041, China

Received:2021-08-12 Online:2022-05-23 Published:2022-05-25

摘要/Abstract

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。

关键词: 猫疱疹病毒1型, RPA-Cas12a-LFD, 核酸检测, 应用

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.

Key words: feline herpesvirus-1, RPA-Cas12a-LFD, nucleic acid detection, application

中图分类号:

S852.659.6

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.

HUANG Jian, LIU Yunjia, YANG Xiaonong, LI Yan. RPA-Cas12a-LFD Based Nucleic Acid Detection for Feline Herpesvirus-1 and Preliminary Application[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(5): 1638-1643.

参考文献 17

[1]	LEWIN A C, KOLB A W, MCLELLAN G J, et al. Genomic, recombinational and phylogenetic characterization of global feline herpesvirus 1 isolates[J]. Virology, 2018, 518:385-397.
[2]	DIGANGI B A, GRAY L K, LEVY J K, et al. Detection of protective antibody titers against feline panleukopenia virus, feline herpesvirus-1, and feline calicivirus in shelter cats using a point-of-care ELISA[J]. J Feline Med Surg, 2011, 13(12):912-918.
[3]	DALL'ARA P, LABRIOLA C, SALA E, et al. Prevalence of serum antibody titres against feline panleukopenia, herpesvirus and calicivirus infections in stray cats of Milan, Italy[J]. Prev Vet Med, 2019, 167:32-38.
[4]	BERGMANN M, SPECK S, RIEGER A, et al. Antibody response to feline herpesvirus-1 vaccination in healthy adult cats[J]. J Feline Med Surg, 2020, 22(4):329-338.
[5]	HELPS C R, LAIT P, DAMHUIS A, et al. Factors associated with upper respiratory tract disease caused by feline herpesvirus, feline calicivirus, Chlamydophila felis and Bordetella bronchiseptica in cats:experience from 218 European catteries[J]. Vet Rec, 2005, 156(21):669-673.
[6]	FERNANDEZ M, MANZANILLA E G, LLORET A, et al. Prevalence of feline herpesvirus-1, feline calicivirus, Chlamydophila felis and Mycoplasma felis DNA and associated risk factors in cats in Spain with upper respiratory tract disease, conjunctivitis and/or gingivostomatitis[J]. J Feline Med Surg, 2017, 19(4):461-469.
[7]	LIU C H, LIU Y X, QIAN P, et al. Molecular and serological investigation of cat viral infectious diseases in China from 2016 to 2019[J]. Transbound Emerg Dis, 2020, 67(6):2329-2335.
[8]	SCHULZ C, HARTMANN K, MUELLER R S, et al. Sampling sites for detection of feline herpesvirus-1, feline calicivirus and Chlamydia felis in cats with feline upper respiratory tract disease[J]. J Feline Med Surg, 2015, 17(12):1012-1019.
[9]	HUSSEIN I T M, FIELD H J. Development of a quantitative real-time TaqMan PCR assay for testing the susceptibility of feline herpesvirus-1 to antiviral compounds[J]. J Virol Methods, 2008, 152(1-2):85-90.
[10]	MAZZEI M, VASCELLARI M, ZANARDELLO C, et al. Quantitative real time polymerase chain reaction (qRT-PCR) and RNAscope in situ hybridization (RNA-ISH) as effective tools to diagnose feline herpesvirus-1-associated dermatitis[J]. Vet Dermatol, 2019, 30(6):491-e147.
[11]	CHEN J, MA E B, HARRINGTON L, et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity[J]. Science, 2018, 360(6387):436-439.
[12]	BROUGHTON J P, DENG X D, YU G X, et al. CRISPR-Cas12-based detection of SARS-CoV-2[J]. Nat Biotechnol, 2020, 38(7):870-874.
[13]	HENZEL A, BRUM M C S, LAUTERT C, et al. Isolation and identification of feline calicivirus and feline herpesvirus in southern Brazil[J]. Braz J Microbiol, 2012, 43(2):560-568.
[14]	TOWNSEND W M, JACOBI S, TAI S H, et al. Ocular and neural distribution of feline herpesvirus-1 during active and latent experimental infection in cats[J]. BMC Vet Res, 2013, 9:185.
[15]	LIU M Z, HAN X H, YAO L Q, et al. Development and application of a simple recombinase polymerase amplification assay for rapid point-of-care detection of feline herpesvirus type 1[J]. Arch Virol, 2019, 164(1):195-200.
[16]	WANG J C, LIU L B, WANG J F, et al. Recombinase polymerase amplification assay-a simple, fast and cost-effective alternative to real time PCR for specific detection of Feline herpesvirus-1[J]. PLoS One, 2017, 12(1):e0166903.
[17]	TAN Y X, DONG G Y, XU H F, et al. Development of a cross-priming isothermal amplification assay based on the glycoprotein B gene for instant and rapid detection of feline herpesvirus type 1[J]. Arch Virol, 2020, 165(3):743-747.

猫疱疹病毒1型RPA-Cas12a-LFD检测方法的建立及初步应用

RPA-Cas12a-LFD Based Nucleic Acid Detection for Feline Herpesvirus-1 and Preliminary Application

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