RPA技术及其在动物病原检测中的应用

doi:10.16431/j.cnki.1671-7236.2021.02.008

摘要/Abstract

摘要： 分子检测技术是进行病原检测、鉴定和疾病诊断的重要手段。重组酶聚合酶扩增技术（recombinase polymerase amplification，RPA）是一种新型核酸扩增技术，主要在能结合单链核酸（寡核苷酸引物）的重组酶、单链DNA结合蛋白（SSB）和链置换DNA聚合酶3种酶的作用下等温扩增DNA或RNA，具有操作便捷、特异性强、灵敏度高和反应迅速等优点，可实现对疾病的现场诊断。作者简述了RPA技术的反应体系组成、反应原理、引物和探针的设计以及扩增产物的检测方法等，介绍了RPA技术在动物重要病毒性、细菌性及寄生虫病原检测中的应用，分析了该技术的局限性，并对其发展前景进行了展望。

关键词: 重组酶聚合酶扩增技术; 动物病原; 检测; 应用

Abstract: Molecular detection technology is an important way of pathogen detection,identification and disease diagnosis.Recombinase polymerase amplification (RPA) is a new type of nucleic acid amplification technology.It can amplify DNA or RNA at a constant temperature with a recombinase that is capable of pairing oligonucleotide primers, single-stranded DNA binding (SSB) protein and strand-displacing polymerase.It has advantages of convenient operation,strong specificity,high sensitivity and rapid reaction,which can realize the field diagnosis of diseases.In this paper,the composition of reaction system,reaction principle,the design of primers and probes,and the detection methods of amplified products of RPA technology were introduced.The application of RPA technology in the detection of important viral,bacterial and parasitic pathogens in animals was introduced,the limitations of RPA technology were analyzed,and its development prospect was prospected.

Key words: recombinase polymerase amplification; animal pathogen; detection; application

中图分类号:

S852.6

石美奕, 杨发龙. RPA技术及其在动物病原检测中的应用[J]. 中国畜牧兽医, 2021, 48(2): 467-476.

SHI Meiyi, YANG Falong. RPA and Its Application in Detection of Animal Pathogens[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(2): 467-476.

参考文献

[1] SAIKI R K,BUGAWAN T L,HORN G T,et al.Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes[J].Nature,1986,324(6093):163-166.
[2] YAN L,ZHOU J,ZHENG Y,et al.Isothermal amplified detection of DNA and RNA[J].Molecular Biosystems,2014,10(5):970-1003.
[3] LI J,MACDONALD J,STETTEN F V.Correction:Review:A comprehensive summary of a decade development of the recombinase polymerase amplifi-cation[J].The Analyst,2020,145(5):1950-1960.
[4] YONESAKI T,RYO Y,MINAGAWA T,et al.Purification and some of the functions of the products of bacteriophage T4 recombination genes,uvsX and uvsY[J].European Journal of Biochemistry,1985,148(1):127-134.
[5] JAMES A,MACDONALD J.Recombinase polymerase amplification:Emergence as a critical molecular technology for rapid,low-resource diagnostics[J].Expert Review of Molecular Diagnostics,2015,15(11):1475-1489.
[6] DAHER R K,STEWART G,BOISSINOT M,et al.Influence of sequence mismatches on the specificity of recombinase polymerase amplification technology[J].Molecular and Cellular Probes,2015,29(2):116-121.
[7] 姜一曈,王昭华,鑫婷,等.基于重组酶聚合酶扩增技术的犬细小病毒快速检测方法的建立[J].中国预防兽医学报,2018,40(10):926-929. JIANG Y T,WANG Z H,XING T,et al.Development of the recombinase polymerase amplification assay for rapid detection of canine parvovirus[J].Chinese Journal of Preventive Veterinary Medicine,2018,40(10):926-929.(in Chinese)
[8] 樊晓旭,赵永刚,李林,等.重组酶聚合酶扩增技术在疾病快速检测中的研究进展[J].中国动物检疫,2016,33(8):72-77. FAN X X,ZHAO Y G,LI L,et al.Research progress of recombinant enzyme polymerase amplification (RPA) in rapid detection of diseases[J].China Animal Health Inspection,2016,33(8):72-77.(in Chinese)
[9] 郭正洋,刘钟栋,刘小青,等.重组酶聚合酶扩增技术的研究进展[J].食品科技,2018,43(9):55-59. GUO Z Y,LIU Z D,LIU X Q,et al.Research progress of recombinase polymerase amplification[J].Food Science and Technology,2018,43(9):55-59.(in Chinese)
[10] WAHED A A E,EL-DEEB A,EL-THOLOTH M,et al.A portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot-and-mouth disease virus[J].PLoS One,2013,8(8):e71642.
[11] 刘立兵,王金凤,张若曦,等.口蹄疫病毒实时荧光RT-RPA快速检测方法的建立[J].中国预防兽医学报,2019,41(2):168-173. LIU L B,WANG J F,ZHANG R X,et al.Development of the Real-time reverse transcription recombinase polymerase amplification for rapid detection of foot-and-mouth disease virus[J].Chinese Journal of Preventive Veterinary Medicine,2019,41(2):168-173.(in Chinese)
[12] 王建昌,王金凤,刘立兵,等.非洲猪瘟病毒RPA等温检测方法的建立[J].中国动物检疫,2016,33(7):78-81. WANG J C,WANG J F,LIU L B,et al.Rapid and sensitive detection of african swine fever virus by recombinase polymerase amplification[J].China Animal Health Inspection,2016,33(7):78-81.(in Chinese)
[13] YEHIA N,ARAFA A S,WAHED A A E,et al.Development of reverse transcription recombinase polymerase amplification assay for avian influenza H5N1 HA gene detection[J].Journal of Virological Methods,2015,223:45-49.
[14] YANG Y,QIN X D,SONG Y M,et al.Development of Real-time and lateral flow strip reverse transcription recombinase polymerase amplification assays for rapid detection of peste des petits ruminants virus[J].Virology Journal,2017,14(1):24.
[15] 李园丽.重组酶聚合酶扩增(RPA)技术在小反刍兽疫病毒快速检测中的应用[D].沈阳:沈阳农业大学,2018. LI Y L.Application of recombinase polymerase amplification in rapid detection of peste des petites ruminant virus[D].Shenyang:Shenyang Agricultural University,2018.(in Chinese)
[16] 王金凤,张若曦,刘立兵,等.猪瘟病毒重组酶聚合酶扩增检测方法的建立及应用[J].中国兽医科学,2019,49(6):687-693. WANG J F,ZHANG R X,LIU L B.et al.Rapid and simple detection of classical swine fever virus by reverse transcription recombinase polymerase amplification assay[J].Chinese Veterinary Science,2019,49(6):687-693.(in Chinese)
[17] YANG Y,QIN X D,ZHANG W,et al.Rapid and specific detection of porcine parvovirus by isothermal recombinase polymerase amplification assays[J].Molecular and Cellular Probes,2016,30(5):300-305.
[18] YANG Y,QIN X D,WANG G X,et al.Development of an isothermoal amplification-based assay for rapid visual detection of an Orf virus[J].Virology Journal,2016,13(1):46.
[19] AMER H M,WAHED A A E,SHALABY M A,et al.A new approach for diagnosis of bovine coronavirus using a reverse transcription recombinase polymerase amplification assay[J].Journal of Virological Methods,2013,193(2):337-340.
[20] YANG Y,QIN X D,SUN Y J,et al.Rapid detection of highly pathogenic porcine reproductive and respiratory syndrome virus by a fluorescent probe-based isothermal recombinase polymerase amplifi-cation assay[J].Virus Genes,2016,52(6):883-886.
[21] WANG J C,WANG J F,LIU L B,et al.Rapid detection of porcine circovirus 2 by recombinase polymerase amplification[J].Journal of Veterinary Diagnostic Investigation,2016,28(5):574-578.
[22] 肖帅,刘新生,方玉珍,等.猪德尔塔冠状病毒重组聚合酶扩增检测方法的建立与应用[J].动物医学进展,2019,40(3):9-14. XIAO S,LIU X S,FANG Y Z,et al.Establishment and application of RPA detection method for porcine deltacoronavirus[J].Progress in Veterinary Medicine,2019,40(3):9-14.(in Chinese)
[23] 樊晓旭,宋翥远,赵永刚,等.塞尼卡谷病毒重组酶聚合酶扩增-侧流层析试纸条检测方法的建立[J].中国预防兽医学报,2018,40(5):406-410. FAN X X,SONG Z Y,ZHAO Y G,et al.Establishment of recombinase polymerase amplification-lateral flow dipstick for the detection of Seneca Valley virus[J].Chinese Journal of Preventive Veterinary Medicine,2018,40(5):406-410.(in Chinese)
[24] EULER M,WANG Y J,NENTWICH O,et al.Recombinase polymerase amplification assay for rapid detection of Rift Valley fever virus[J].Journal of Clinical Virology,2012,54(4):308-312.
[25] 杨俊,聂福平,周庆,等.绵羊痘病毒和山羊痘病毒实时荧光重组酶聚合酶检测方法的建立[J].中国兽医科学,2019,49(5):544-550. YANG J,NIE F P,ZHOU Q,et al.Development of Real-time RPA assay for the detection of SPPV and GTPV[J].Chinese Veterinary Science,2019,49(5):544-550.(in Chinese)
[26] 侯佩莉,王洪梅,何成强,等.牛病毒性腹泻病毒RPA-LFD快速检测方法的建立[A].中国畜牧兽医学会生物技术学分会暨中国免疫学会兽医免疫分会第十二次学术研讨会论文集[C].2016. HOU P L,WANG H M,HE C Q,et al.Establishment of rapid detection method of bovine viral diarrhea virus RPA-LFD[A].Proceedings of the Twelfth Academic Symposium of the Chinese Society of Animal Science and Veterinary Medicine and the Twelfth Academic Symposium of the Chinese Society of Veterinary Medicine[C].2016.(in Chinese)
[27] 侯佩莉,王洪梅,宋玲玲,等.牛传染性鼻气管炎病毒RPA-LFD快速检测方法的建立[A].中国畜牧兽医学会生物技术学分会暨中国免疫学会兽医免疫分会第十二次学术研讨会论文集[C].2016. HOU P L,WANG H M,SONG L L,et al.Establishment of rapid detection method of bovine infectious rhinotracheitis virus RPA-LFD[A].Proceedings of the Twelfth Academic Symposium of the Chinese Society of Animal Science and Veterinary Medicine and the Twelfth Academic Symposium of the Chinese Society of Veterinary Medicine[C].2016.(in Chinese)
[28] EL-WAHED A A,WEIDMANN M,HUFERT F.Diagnostics-in-a-suitcase:Development of a portable and rapid assay for the detection of the emerging avian influenza A (H7N9) virus[J].Journal of Clinical Virology,2015,69:16-21.
[29] 马国和,高冬生,王增,等.鸡传染性喉气管炎病毒RPA-LFD检测方法的建立[J].中国预防兽医学报,2018,40(12):1128-1132. MA G H,GAO D S,WANG Z,et al.Development of a recombinase polymerase amplification assay combined with lateral flow dipstick for detection of the infectious laryngotracheitis virus[J].Chinese Journal of Preventive Veterinary Medicine,2018,40(12):1128-1132.(in Chinese)
[30] WANG J C,LIU L B,LI R W,et al.Rapid and sensitive detection of canine parvovirus type 2 by recombinase polymerase amplification[J].Springer Vienna,2016,161(4):1015-1018.
[31] 李春华,魏晓锋,熊炜,等.犬副流感病毒实时荧光RPA检测方法的建立[J].中国兽医科学,2019,49(10):1233-1238. LI C H,WEI X F,XIONG W,et al.Establishment of Real-time recombinase polymerase amplification assay for the detection of canine parainfluenza virus[J].Chinese Veterinary Science,2019,49(10):1233-1238.(in Chinese)
[32] REN H,YANG M,ZHANG G X,et al.Development of a rapid recombinase polymerase amplification assay for detection of Brucella in blood samples[J].Molecular and Cellular Probes,2016,30(2):122-124.
[33] 许笑,王艳华,MUMAR ZAFAR KHAN,等.鸡源产气荚膜梭菌重组酶聚合酶扩增检测方法的建立[J].中国兽医科学,2019,49(12):1475-1483. XU X,WANG Y H,MUMAR Z K,et al.Recombinase polymerase amplification:A detection method for the detection of Clostridium perfringens from chicken[J].Chinese Veterinary Science,2019,49(12):1475-1483.(in Chinese)
[34] 彭遥.类鼻疽、土拉热和鼠疫病原体的LF-RPA检测技术的建立[D].北京:中国疾病预防控制中心,2019. PENG Y.Development of LF-RPA assay for detecting pathogens of melioidosis,tularemia and plague[D].Beijing:Chinese Center for Disease Control and Prevention,2019.(in Chinese)
[35] EULER M,WANG Y,HEIDENREICH D,et al.Development of a panel of recombinase polymerase amplification assays for detection of biothreat agents[J].Journal of Clinical Microbiology,2013,51(4):1110-1117.
[36] 赵巧雅,马秀丽,刘存霞,等.兔源肺炎克雷伯氏菌重组酶聚合酶扩增检测方法的建立[J].中国动物检疫,2019,36(7):82-85. ZHAO Q Y,MA X L,LIU C X,et al.Establishment of a RPA assay for detecting Klebsiella pneumoniae of rabbit origin[J].China Animal Health Inspection,2019,36(7):82-85.(in Chinese)
[37] LI T T,WANG J L,ZHANG N Z,et al.Rapid and visual detection of Trichinella spp.using a lateral flow strip-based recombinase polymerase amplification (LF-RPA) assay[J].Frontiers in Cellular and Infection Microbiology,2019,9:1.
[38] 吴耀东.隐孢子虫和弓形虫重组酶聚合酶扩增检测方法的建立及应用[D].北京:中国农业科学院,2017. WU Y D.Development and application recombinase polymerase amplification assay for detection of Cryptosporidium spp.and Toxoplasma gondii[D].Beijing:Chinese Academy of Agricultural Sciences,2017.(in Chinese)
[39] 景纪春,赵姗姗,闫亚群,等.吕氏泰勒虫18S rRNA基因重组酶聚合酶扩增技术的建立与初步应用[A].2018年全国养羊生产与学术研讨会论文集[C].2018. JING J C,ZHAO S S,YAN Y Q,et al.Establishment and preliminary application of 18S rRNA gene recombinase polymerase amplification technique of Theileria luwenshuni[A].Proceedings of the 2018 National Sheep Breeding and Symposium[C].2018.(in Chinese)
[40] SUN K,XING W W,YU X L,et al.Recombinase polymerase amplification combined with a lateral flow dipstick for rapid and visual detection of Schistosoma japonicum[J].Parasites & Vectors,2016,9(1):476.
[41] POULTON K,WEBSTER B.Development of a lateral flow recombinase polymerase assay for the diagnosis of Schistosoma mansoni infections[J].Analytical Biochemistry,2018,546:65-71.
[42] 翟肖辉.牛支原体重组酶聚合酶扩增(RPA)检测方法的建立和样品DNA提取方法的研究[D].北京:中国农业科学院,2019. ZHAI X H.Establishment of recombinase polymerase amplification (RPA) for detection of Mycoplasma bovis and the method for sample DNA extraction[D].Beijing:Chinese Academy of Agricultural Sciences,2019.(in Chinese)
[43] LILJANDER A,YU M,O'BRIEN E,et al.Field-applicable recombinase polymerase amplification assay for rapid detection of Mycoplasma capricolum subsp.capripneumoniae[J].Journal of Clinical Microbio-logy,2015,53(9):2810-2815.
[44] LIU L B,LI R W,ZHANG R X,et al.Rapid and sensitive detection of Mycoplasma hyopneumoniae by recombinase polymerase amplification assay[J].Journal of Microbiological Methods,2019,159:56-61.
[45] WANG J F,LI R W,SUN X X,et al.Development and validation of the isothermal recombinase polymerase amplification assays for rapid detection of Mycoplasma ovipneumoniae in sheep[J].BMC Veterinary Research,2020,16(1):172.