快速检测犬瘟热病毒的实时荧光RPA方法的建立
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摘要
针对犬瘟热病毒(canine distemper virus,CDV)基因保守序列,设计特异性引物和荧光探针,并进行了敏感性和特异性试验,建立了CDV实时重组聚合酶等温扩增(real-time recombinase polymerase amplification,real-time RPA)检测方法。结果表明,该实时荧光RPA方法在39℃恒温反应25 min能特异性扩增目的基因,与其他犬病毒以及CDV同属的麻疹病毒均未出现交叉反应;该检测方法具有与RT-PCR一致的敏感性;通过对CDV阳性犬的组织样品、体表样品和不同时期分离鉴定的临床样本的检测,证实建立的实时荧光RPA方法具有良好的稳定性和可靠性。与现有的核酸检测方法相比,该实时荧光RPA检测方法在核酸上样25 min内即可判读结果,可满足CDV快速检疫的需要。
Using pairs of special primers and probes for conserved sequence of canine distemper virus(CDV), the real-time recombinase polymerase amplification assay(RPA) was developed after tests of sensitivity and specificity.The results showed that the new method could detect target gene specifically at 39℃ within 25 min and there was no cross-reaction with other canine virus and measles virus.The new method had high sensitivity,which was in accordance with RT-PCR.The excellent stability of the new method was confirmed by detection of CDV infected positive and negative samples and clinical samples seperated at different time.Compared with other method, the real-time RPA method could finish detection within 25 min after nucleic acid isolated and meet the requirement of rapid detection of CDV.
Using pairs of special primers and probes for conserved sequence of canine distemper virus(CDV), the real-time recombinase polymerase amplification assay(RPA) was developed after tests of sensitivity and specificity.The results showed that the new method could detect target gene specifically at 39℃ within 25 min and there was no cross-reaction with other canine virus and measles virus.The new method had high sensitivity,which was in accordance with RT-PCR.The excellent stability of the new method was confirmed by detection of CDV infected positive and negative samples and clinical samples seperated at different time.Compared with other method, the real-time RPA method could finish detection within 25 min after nucleic acid isolated and meet the requirement of rapid detection of CDV.
引文
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[14]曾杨茹,王娅,涂蕊,等.鉴别犬瘟热病毒野毒株与疫苗株的SYBR Green I real-time PCR方法的建立[J].中国兽医科学,2017,47(04):435-441.ZENG Yang-ru,WANG Ya,TU Rui,et al.Establishment of real-time PCR for differentiation of wildtype from vaccine isolates of canine distemper virus[J].Chinese Veterinary Science,2017,47(04):435-441.(in Chinese)
[15]全传松,李博韬,姜骞,等.犬瘟热病毒TaqMan实时荧光定量RT-PCR检测方法的建立及应用[J].中国兽医科学,2014,44(06):589-592.QUAN Chuan-song,LI Bo-tao,JIANG Qian,et al.Development and applications of TaqMan real-time RT-PCR for detection of canine distemper virus[J].Chinese Veterinary Science,2014,44(06):589-592.(in Chinese)
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[17] XIA X,YU Y,WEIDMANN M,et al.Rapid detection of shrimp white spot syndrome virus by real time,isothermal recombinase polymerase amplification assay[J].Plos One,2014,9(8):e104667.
[18] WANG J,YUAN W,HAN Q,et al.Reverse transcription recombinase polymerase amplication assay for the rapid detection of type 2 porcine reproductive and respiratory syndrome virus[J].Journal of Virological Methods,2017,243:55-60.
[19] WANG J,LIU L,LI R,et al.Rapid and sensitive detection of canine parvovirus type 2 by recombinase polymerase amplication[J].Archives of Virology,2016,161:1015-1018.
[2] MARTINEZ-GUTIERREZ M,RUIZ-SAENZ J.Diversity of susceptible hosts in canine distemper virus infection:a systematic review and data synthesis[J].BMC Veterinary Research,2016,12:78-88.
[3] BEINEKE A,BAUMG RTNER W,WOHLSEIN P.Cross-species transmission of canine distemper virus-an update[J].One Health,2015,1:49-59.
[4] SAKAI K,YOSHIKAWA T,SEKI F,et al.Canine distemper virus associated with a lethal outbreak in monkeys can readily adapt to use human receptors[J].Journal of Virology,2013,87,(12):7170-7175.
[5] BIERINGER M,HAN J W,KENDL S,et al.Experimental adaptation of wild-type canine distemper virus(CDV)to the human entry receptor CD150[J].Plos One,2013,8(3):e57488.
[6] SEKULIN A,HAFNER-MARXB A,KOLODZIEJEKA J,et al.Emergence of canine distemper in Bavarian wildlife associated with a specific amino acid exchange in the haemagglutinin protein[J].Veterinary Journal,2011,187(3):399-401.
[7] ATHANASIOU LV,KANTERE M C,KYRIAKIS C S,et al.Evaluation of a direct immunofluorescent assay and/or conjunctival cytology for detection of canine distemper virus antigen[J].Viral Immunology,2018,31(3):272-275.
[8] NEMETH N M,OESTERLE P T,CAMPBELL G D,et al.Comparison of reverse-transcription real-time PCR and immunohistochemistry for the detection of canine distemper virus infection in raccoons in Ontario,Canada[J]. Journal of Veterinary Diagnostic Investigation,2018,30(2):319-323.
[9] LI Z,ZHANG Y,WANG H,et al.Sandwich-dot enzymelinked immunosorbent assay for the detection of canine distemper virus[J].Canadian Journal of Veterinary Research,2013,77(4):303-308.
[10] ROMANUTTI C,GALLO CALDER N M,KELLER L,et al.RT-PCR and sequence analysis of the full-length fusion protein of canine distemper virus from domestic dogs[J].Journal of Virology Methods,2016,228:79-83.
[11] FISCHER C D,IKUTA N,CANAL C W,et al.Detection and differentiation of field and vaccine strains of canine distemper virus using reverse transcription followed by nested real time PCR(RT-nqPCR)and RFLP analysis[J].Journal of Virological Methods,2013,194(1-2):39-45.
[12]胡嘉欣,温永俊,霍志云,等.犬瘟热病毒RT-LAMP检测方法的建立及初步应用[J].中国畜牧兽医,2012,39(8):45-48.HU Jia-xin,WEN Yong-jun,HUO Zhi-yun,et al.Establishment and preliminary application development of a Loop-mediated isothermal amplification assay for detection of canine distemper virus[J].Chinese Animal Hu sbandry and Veterinary Medicine,2012,39(8):45-48.(in Chinese)
[13] WILKES R P,TSAI Y L,LEE P Y,et al.Rapid and sensitive detection of canine distemper virus by one-tube reverse transcription-insulated isothermal polymerase chain reaction[J].BMC Veterinary Research,2014,10:213-220.
[14]曾杨茹,王娅,涂蕊,等.鉴别犬瘟热病毒野毒株与疫苗株的SYBR Green I real-time PCR方法的建立[J].中国兽医科学,2017,47(04):435-441.ZENG Yang-ru,WANG Ya,TU Rui,et al.Establishment of real-time PCR for differentiation of wildtype from vaccine isolates of canine distemper virus[J].Chinese Veterinary Science,2017,47(04):435-441.(in Chinese)
[15]全传松,李博韬,姜骞,等.犬瘟热病毒TaqMan实时荧光定量RT-PCR检测方法的建立及应用[J].中国兽医科学,2014,44(06):589-592.QUAN Chuan-song,LI Bo-tao,JIANG Qian,et al.Development and applications of TaqMan real-time RT-PCR for detection of canine distemper virus[J].Chinese Veterinary Science,2014,44(06):589-592.(in Chinese)
[16] TRAN L,NUNAN L M,RENMAN R M,et al.Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp[J].Disease of Aquatic Organisms,2013,105:45-55.
[17] XIA X,YU Y,WEIDMANN M,et al.Rapid detection of shrimp white spot syndrome virus by real time,isothermal recombinase polymerase amplification assay[J].Plos One,2014,9(8):e104667.
[18] WANG J,YUAN W,HAN Q,et al.Reverse transcription recombinase polymerase amplication assay for the rapid detection of type 2 porcine reproductive and respiratory syndrome virus[J].Journal of Virological Methods,2017,243:55-60.
[19] WANG J,LIU L,LI R,et al.Rapid and sensitive detection of canine parvovirus type 2 by recombinase polymerase amplication[J].Archives of Virology,2016,161:1015-1018.