猪流行性腹泻病毒RAA-CRISPR/Cas13a检测方法的建立与初步应用

doi:10.11843/j.issn.0366-6964.2023.09.037

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (9): 3991-3997.doi: 10.11843/j.issn.0366-6964.2023.09.037

猪流行性腹泻病毒RAA-CRISPR/Cas13a检测方法的建立与初步应用

刘华^1,2, 殷冬冬³, 邵颖¹, 宋祥军¹, 王振宇¹, 潘孝成³, 涂健¹, 何长生², 朱良强^2*, 祁克宗^1*

1. 安徽农业大学, 合肥 230036;
2. 安徽省动物疫病预防与控制中心, 合肥 230091;
3. 安徽省农业科学院畜牧兽医研究所, 合肥 230031

收稿日期:2023-02-24 发布日期:2023-09-22
通讯作者: 祁克宗,主要从事动物疫病病理与生物防制研究,E-mail:qkz@ahau.edu.cn;朱良强,主要从事动物疫病防控研究,E-mail:vetzhu@126.com
作者简介:刘华(1982-),男,安徽宁国人,高级兽医师,博士生,主要从事动物病原生物学研究,Tel:0551-62930645,E-mail:vetliuhua@163.com
基金资助:
国家自然科学基金面上项目(31972642);安徽省高校协同创新项目(GXXT-2019-035)

Detection of Porcine Epidemic Diarrhea Virus by Recombinase Aided Amplification Combined with CRISPR/Cas13a

LIU Hua^1,2, YIN Dongdong³, SHAO Ying¹, SONG Xiangjun¹, WANG Zhenyu¹, PAN Xiaocheng³, TU Jian¹, HE Changsheng², ZHU Liangqiang^2*, QI Kezong^1*

1. Anhui Agricultural University, Hefei 230036, China;
2. Anhui Animal Disease Prevention and Control Center, Hefei 230091, China;
3. Institute of Animal Husbandry and Veterinary Medicine, Anhui Academy of Agricultural Sciences, Hefei 230031, China

Received:2023-02-24 Published:2023-09-22

摘要/Abstract

摘要： 将重组酶辅助扩增技术(recombinase aided amplification,RAA)与规律间隔性成簇短回文重复序列相关 Cas13a 蛋白(CRISPR-Cas13a)技术相结合,即RAA-Cas13a,建议建立高效、灵敏、特异的猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)检测方法。针对PEDV N基因保守区设计RAA 特异性引物和 CRISPR RNA(crRNA),利用 RAA 技术扩增样本核酸,并进行 CRISPR-Cas13a荧光检测,以RT-qPCR为对照方法,评价该方法的灵敏度、特异性及与RT-qPCR法的一致性。结果表明,该方法最低可检测至10¹ copies·μL^-1,且与猪圆环病毒1型、猪圆环病毒2型、猪圆环病毒3型、猪繁殖与呼吸综合征病毒、猪瘟病毒及猪伪狂犬病病毒等常见猪源病原核酸检测无交叉反应。采用RAA-Cas13a检测40份临床样本与RT-qPCR方法阳性符合率为100%,阴性符合率为84.6%,总符合率为95%,Kappa值为0.881。本研究建立的RAA-Cas13a方法灵敏度高、特异性强,为PEDV的临床诊断和流行病学监测提供了可靠的技术手段。

关键词: 猪流行性腹泻病毒, CRISPR/Cas13a, 重组酶辅助扩增, N基因, 检测

Abstract: The aim of this paper is to establish a rapid, sensitive, and specific detection method for porcine epidemic diarrhea virus (PEDV) by recombinase aided amplification (RAA) combined with the Clustered Regularly Interspaced Short Palindromic Repeats associated protein 13a (CRISPR-Cas13a), called RAA-Cas13a. The specific primer for RAA and CRISPR RNA (crRNA) of the conserved region of the PEDV N gene were designed. The sample nucleic acids were amplified by RAA, whose amplification products were then detected with CRISPR-Cas13a and use RT-qPCR as a control method to evaluate the sensitivity, specificity, and consistency of this method. The results showed that the method could detect as low as 10¹ copies·μL^-1 and had no cross-reactivity with pig-derived pathogens such as porcine circovirus type 1, porcine circovirus type 2, porcine circovirus type 3, porcine reproductive and respiratory syndrome virus, swine fever virus and pseudorabies virus. The positive coincidence rate of 40 clinical samples detected by RAA-Cas13a and RT-qPCR method was 100%, the negative coincidence rate was 84.6%, the total coincidence rate was 95%, and the Kappa value was 0.881. The established RAA-Cas13a detection method has high sensitivity and strong specificity, providing a reliable technical means for the clinical diagnosis and epidemiological monitoring of PEDV.

Key words: porcine epidemic diarrhea virus, CRISPR/Cas13a, recombinase aided amplification, N gene, detection

中图分类号:

S852.659.6

刘华, 殷冬冬, 邵颖, 宋祥军, 王振宇, 潘孝成, 涂健, 何长生, 朱良强, 祁克宗. 猪流行性腹泻病毒RAA-CRISPR/Cas13a检测方法的建立与初步应用[J]. 畜牧兽医学报, 2023, 54(9): 3991-3997.

LIU Hua, YIN Dongdong, SHAO Ying, SONG Xiangjun, WANG Zhenyu, PAN Xiaocheng, TU Jian, HE Changsheng, ZHU Liangqiang, QI Kezong. Detection of Porcine Epidemic Diarrhea Virus by Recombinase Aided Amplification Combined with CRISPR/Cas13a[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3991-3997.

参考文献

[1] HAVE P, MOVING V, SVANSSON V, et al. Coronavirus infection in mink (Mustela vision). Serological evidence of infection with a coronavirus related to transmissible gastroenteritis virus and porcine epidemic diarrhea virus[J]. Vet Microbiol, 1992, 31(1):1-10.
[2] WOOD E N. An apparently new syndrome of porcine epidemic diarrhoea[J]. Vet Rec, 1977, 100(12):243-244.
[3] SUN R Q, CAI R J, CHEN Y Q, et al. Outbreak of porcine epidemic diarrhea in suckling piglets, China[J]. Emerg Infect Dis, 2012, 18(1):161-163.
[4] SONG D, PARK B. Porcine epidemic diarrhoea virus:a comprehensive review of molecular epidemiology, diagnosis, and vaccines[J]. Virus Genes, 2012, 44(2):167-175.
[5] KOCHERHANS R, BRIDGEN A, ACKERMANN M, et al. Completion of the porcine epidemic diarrhoea coronavirus (PEDV) genome sequence[J]. Virus Genes, 2001, 23(2):137-144.
[6] LIU J, LI L M, HAN J Q, et al. A TaqMan probe-based real-time PCR to differentiate porcine epidemic diarrhea virus virulent strains from attenuated vaccine strains[J]. Mol Cell Probes, 2019, 45:37-42.
[7] YU X W, SHI L, LV X P, et al. Development of a real-time reverse transcription loop-mediated isothermal amplification method for the rapid detection of porcine epidemic diarrhea virus[J]. Virol J, 2015, 12:76.
[8] KELLNER M J, KOOB J G, GOOTENBERG J S, et al. SHERLOCK:nucleic acid detection with CRISPR nucleases[J]. Nat Protoc, 2019, 14(10):2986-3012.
[9] MYHRVOLD C, FREIJE C A, GOOTENBERG J S, et al. Field-deployable viral diagnostics using CRISPR-Cas13[J]. Science, 2018, 360(6387):444-448.
[10] LI J, MACDONALD J, VON STETTEN F. Review:a comprehensive summary of a decade development of the recombinase polymerase amplification[J]. Analyst, 2019, 144(1):31-67.
[11] CHANG Y F, DENG Y, LI T Y, et al. Visual detection of porcine reproductive and respiratory syndrome virus using CRISPR-Cas13a[J]. Transbound Emerg Dis, 2020, 67(2):564-571.
[12] LIU Y F, XU H P, LIU C, et al. CRISPR-Cas13a nanomachine based simple technology for Avian Influenza a (H7N9) virus on-site detection[J]. J Biomed Nanotechnol, 2019, 15(4):790-798.
[13] QIN P W, PARK M, ALFSON K J, et al. Rapid and fully microfluidic ebola virus detection with CRISPR-Cas13a[J]. ACS Sens, 2019, 4(4):1048-1054.
[14] 陈浩, 鞠永政, 王文文, 等. 猪流行性腹泻病毒SYBR Green I荧光定量RT-PCR检测方法的建立与应用[J]. 中国动物检疫, 2022, 39(9):110-114.
CHEN H, JU Y Z, WANG W W, et al. Establishment and application of SYBR Green I fluorescent quantitative RT-PCR for detecting porcine epidemic diarrhea virus[J]. China Animal Health Inspection, 2022, 39(9):110-114. (in Chinese)
[15] WANG D, FANG L R, XIAO S B. Porcine epidemic diarrhea in China[J]. Virus Res, 2016, 226:7-13.
[16] LIANG W, ZHOU D N, GENG C, et al. Isolation and evolutionary analyses of porcine epidemic diarrhea virus in Asia[J]. PeerJ, 2020, 8:e10114.
[17] ABUDAYYEH O O, GOOTENBERG J S, ESSLETZBICHLER P, et al. RNA targeting with CRISPR-Cas13[J]. Nature, 2017, 550(7675):280-284.
[18] WANG Z L, LI X R, SHANG Y J, et al. Rapid differentiation of PEDV wild-type strains and classical attenuated vaccine strains by fluorescent probe-based reverse transcription recombinase polymerase amplification assay[J]. BMC Vet Res, 2020, 16(1):208.
[19] 翟刚, 顾文源, 刘涛, 等. 猪流行性腹泻病毒TaqMan检测方法的建立及基于S基因的遗传变异分析[J]. 畜牧兽医学报, 2023, 54(2):847-854.
ZHAI G, GU W Y, LIU T, et al. Establishment of TaqMan detection method of porcine epidemic diarrhea virus and analysis of genetic variation based on S gene[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2):847-854. (in Chinese)
[20] 冉伟, 田宇, 李梓健, 等. 基于ORF3基因检测猪流行性腹泻病毒荧光定量RT-PCR方法的建立与应用[J]. 中国预防兽医学报, 2021, 43(4):394-398.
RAN W, TIAN Y, LI Z J, et al. Development and preliminary application of ORF3-based fluorescent quantitative RT-PCR method for detection of porcine epidemic diarrhea virus[J]. Chinese Journal of Preventive Veterinary Medicine, 2021, 43(4):394-398. (in Chinese)

猪流行性腹泻病毒RAA-CRISPR/Cas13a检测方法的建立与初步应用

Detection of Porcine Epidemic Diarrhea Virus by Recombinase Aided Amplification Combined with CRISPR/Cas13a

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	杨恒, 李占鸿, 宋子昂, 高林, 李卓然, 廖德芳, 肖雷, 李华春. 帕利亚姆病毒实时荧光定量RT-PCR检测方法的建立与应用[J]. 畜牧兽医学报, 2024, 55(1): 395-400.
[2]	周建浩, 王东方, 刘影, 王淑娟, 马震原, 谢彩华, 赵雪丽, 杨海波, 冯桂丹, 康台生, 胡煜锋, 李博文, 闫若潜. 猪流行性腹泻病毒微滴式数字PCR定量检测方法的建立及初步应用[J]. 畜牧兽医学报, 2024, 55(1): 413-418.
[3]	杨芷翊, 王新凯, 史玉婷, 付思源, 张钰炘, 曹琛福, 贾伟新. 基于RT-RAA的禽流感H5亚型核酸CRISPR-Cas13a检测方法的建立[J]. 畜牧兽医学报, 2023, 54(9): 3803-3811.
[4]	赵永攀, 郑芳芳, 尹俊卿, 杜谦, 童德文, 黄勇. 七种猪常见病毒核酸磁-纳米微粒可视化快速检测技术的建立与应用[J]. 畜牧兽医学报, 2023, 54(9): 3848-3862.
[5]	姜玲玲, 牛小霞, 刘强, 张刚, 王璞, 李勇. 宁夏地区肉牛腹泻相关病毒感染状况的分析[J]. 畜牧兽医学报, 2023, 54(9): 3863-3871.
[6]	王姿月, 张子卉, 吴文学, 彭辰. 猴痘的诊断、预防和治疗[J]. 畜牧兽医学报, 2023, 54(8): 3195-3205.
[7]	张萍, 庄林林, 张笛, 董永毅, 盛中伟, 王成明, 徐步, 窦新红, 龚建森. 沙门菌分子检测方法的研究进展[J]. 畜牧兽医学报, 2023, 54(8): 3217-3229.
[8]	陈宏建, 曹艳, 樊杰, 甘荣萱, 宋文博, 喻盛炜, 杨婷, 赵艳霞, 魏春燕, 谢锐, 华琳, 彭忠, 吴斌. 2020—2022年湖北省生猪屠宰场伪狂犬病病毒的分离鉴定及遗传进化分析[J]. 畜牧兽医学报, 2023, 54(7): 2972-2981.
[9]	李昭燕, 高江, 郭时惠, 赵茹茜, 马文强. 猫过敏原检测方法与控制措施的研究进展[J]. 畜牧兽医学报, 2023, 54(6): 2272-2279.
[10]	张森豪, 王雪莹, 蔡李萌, 解伟纯, 匡虹迪, 王晓娜, 李佳璇, 崔文, 姜艳平, 周晗, 单智夫, 王丽, 乔薪瑗, 李一经, 唐丽杰. 猪流行性腹泻病毒重组酶聚合酶扩增快速诊断方法的建立和应用[J]. 畜牧兽医学报, 2023, 54(6): 2498-2508.
[11]	周广青, 刘晓庆, 史喜绢, 杨大鹏, 袁莉刚, 常惠芸. 口蹄疫病毒抗体SA-ELISA快速检测方法的建立[J]. 畜牧兽医学报, 2023, 54(5): 2020-2029.
[12]	陈杨, 孟林春, 郭梦娇, 张成成, 薄宗义, 楚电峰, 曹永忠, 吴艳涛, 张小荣. 检测鸡毒支原体抗体的间接ELISA方法和HI试验方法的建立及初步应用[J]. 畜牧兽医学报, 2023, 54(5): 2062-2072.
[13]	吕荞, 赵钟毅, 尹德玮, 刘宇梦, 汪伟, 郑敏, 胡诗悦, 赵宸辰, 张昕宇, 雷晓哓, 陆景仪, 孙文超, 兰添. 猪传染性胃肠炎病毒荧光RT-RAA方法的建立及初步应用[J]. 畜牧兽医学报, 2023, 54(5): 2208-2214.
[14]	阿比克哈莫, 汤承, 杨晨, 杨发龙. 嵴病毒的研究进展[J]. 畜牧兽医学报, 2023, 54(3): 900-913.
[15]	翟刚, 顾文源, 刘涛, 刘莹, 张帅, 范京惠, 左玉柱. 猪流行性腹泻病毒TaqMan检测方法的建立及基于S基因的遗传变异分析[J]. 畜牧兽医学报, 2023, 54(2): 847-854.