猪瘟病毒重组酶聚合酶扩增检测方法的建立及应用
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摘要
本研究基于猪瘟病毒(CSFV)5'UTR保守序列,设计特异性引物,建立了可快速检测CSFV的反转录重组酶聚合酶扩增检测方法(reverse-transcription recombinase polymerase amplification,RT-RPA)。该方法在40℃下恒温20 min即可完成反应;且特异性强,与其他瘟病毒和猪的重要病原无任何交叉反应。以体外转录的CSFV RNA为模板,该方法的检出下限为10~2copies。采用RT-RPA方法和实时荧光RT-PCR(real-time RT-PCR)方法同时对57份临床样品进行CSFV检测,结果,RT-RPA方法的阳性检出率为56.14%(32/57),略低于real-time RT-PCR方法的阳性检出率(64.19%,37/57)。以real-time RT-PCR作为参考方法,所建立的RT-RPA方法的诊断特异性为100%,诊断灵敏度为89.19%。上述结果表明,本研究建立的CSFV RT-RPA检测方法操作简单、反应快速,对没有装备荧光PCR仪地区的猪瘟诊断防控具有重要意义。
A reverse-transcription recombinase polymerase amplification assay(RT-RPA) was developed to detect CSFV using primers specific for the 5′UTR of the viral genome.In the result,the amplification was performed at 40 ℃ for 20 min,and there was no cross-reaction with other pestiviruses and important viruses in swine tested. Using the in vitro transcribed CSFV RNA as template,the detection limit of the assay was 1×10~2 copies. The assay performance was further evaluated by testing 57 clinical samples by RT-RPA and the real-time RT-PCR assay.CSFV RNA positive rate was 56.14%(32/57) by RT-RPA and 64.19%(37/57) by the real-time RT-PCR.Compared with the real-time RT-PCR,the RT-RPA assay showed diagnostic specificity of 100% and diagnostic sensitivity of 89.19%.In conclusion,the developed RT-RPA assay provides a useful alternative for simple,rapid and reliable detection of CSFV in under-equipped diagnostic laboratories,which is of great importance for the CSF control in the resource-limited settings.
A reverse-transcription recombinase polymerase amplification assay(RT-RPA) was developed to detect CSFV using primers specific for the 5′UTR of the viral genome.In the result,the amplification was performed at 40 ℃ for 20 min,and there was no cross-reaction with other pestiviruses and important viruses in swine tested. Using the in vitro transcribed CSFV RNA as template,the detection limit of the assay was 1×10~2 copies. The assay performance was further evaluated by testing 57 clinical samples by RT-RPA and the real-time RT-PCR assay.CSFV RNA positive rate was 56.14%(32/57) by RT-RPA and 64.19%(37/57) by the real-time RT-PCR.Compared with the real-time RT-PCR,the RT-RPA assay showed diagnostic specificity of 100% and diagnostic sensitivity of 89.19%.In conclusion,the developed RT-RPA assay provides a useful alternative for simple,rapid and reliable detection of CSFV in under-equipped diagnostic laboratories,which is of great importance for the CSF control in the resource-limited settings.
引文
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[2]BAUERMANN F V,RIDPATH J F,WEIBLEN R,et al.HoBilike viruses:an emerging group of pestiviruses[J].Journal of Veterinary Diagnostic Investigation,2013,25(1):6-15.
[3]CLAVIJO A,ZHOU E M,VYDELINGUM S,et al.Development and evaluation of a novel antigen capture assay for the detection of classical swine fever virus antigens[J].Veterinary Microbiology,1998,60(2-4):155-168.
[4]HANDEL K,KEHLER H,HILLS K,et al.Comparison of reverse transcriptase-polymerase chain reaction,virus isolation,and immunoperoxidase assays for detecting pigs infected with low,moderate,and high virulent strains of classical swine fever virus[J].Joural Veterinary Diagnostic Investigation Official Publication of the American Association of Veterinary Laboratory Diagnosticians Inc,2004,16(2):132-138.
[5]GREISER-WILKE I,BLOME S,MOENNING V.Diagnostic methods for detection of classical swine fever virusstatus quo and new developments[J].Animal Science Abroad,2007,25(30):5524-5530.
[6]DEWULF J,KOENEN F,MINTIENS K,et al.Analytical performance of several classical swine fever laboratory diagnostic techniques on live animals for detection of infection[J].Journal of Virological Methods,2004,119(2):137-143.
[7]PATON D J,MCGOLDRICK A,BENSAUDE E,et al.Classical swine fever virus:a second ring test to evaluate RT-PCR detection methods[J].Veterinary Microbiology,2000,73(2):159-174.
[8]王淑娟,刘梅芬,闫若潜,等.猪瘟病毒实时荧光定量PCR检测方法的建立与应用[J].中国畜牧兽医,2017,44(7):2112-2118.WANG Shu-juan,LIU Mei-fen,YAN Ruo-qian,et al.Establishment and application of real-time quantitative PCR assay for detection of classical swine fever virus[J].Chinese Animal Husbandry and Veterinary Medicine,2017,44(7):2112-2118.(in Chinese)
[9]PEREZ L J,DIAZ d A H,TARRADAS J,et al.Development and validation of a novel SYBR green real-time RT-PCR assay for the detection of classical swine fever virus evaluated on different real-time PCRplatforms[J].Journal of Virological Methods,2011,174(1-2):53-59.
[10]GB/T 27540-2011,猪瘟病毒实时荧光RT-PCR检测方法[S].GB/T 27540-2011,Method of the real-time RT-PCRfor the detection of classical swine fever virus[S].(in Chinese)
[11]LABARRE P,HAWKINS K R,GERLACH J,et al.A simple,inexpensive device for nucleic acid amplification without electricity-toward instrument-free molecular diagnostics in low-resource settings[J].Advances in Difference Equations,2014(1):1-12.
[12]LUNG O,PASICK J,FISHER M,et al.Insulated isothermal reverse transcriptase PCR(ii RT-PCR)for rapid and sensitive detection of classical swine fever virus[J].Transboundary and Emerging Diseases,2016,63(5):e395.
[13]CHOWDRY V K,LUO Y,WIDEN F,et al.Development of a loop-mediated isothermal amplification assay combined with a lateral flow dipstick for rapid and simple detection of classical swine fever virus in the field[J].Journal of Virological Methods,2014,197(3):14-18.
[14]CHEN H T,ZHANG J,MA L N,et al.Rapid pre-clinical detection of classical swine fever by reverse transcription loop-mediated isothermal amplification[J].Molecular and Cellular Probes,2009,23(2):71-74.
[15]YIN S,SHANG Y,ZHOU G,et al.Development and evaluation of rapid detection of classical swine fever virus by reverse transcription loop-mediated isothermal amplification(RT-LAMP)[J].Journal of Biotechnology,2010,146(4):147-150.
[16]DAHER R K,STEWART G,BOISSINOT M,et al.Recombinase polymerase amplification for diagnostic applications[J].Clinical Chemistry,2016,62(7):947-958.
[17]PIEPENBURG O,WILLIAMS C,STEMPLE D N,et al.DNA detection using recombination proteins-art.no.e204[J].Plos Biology,2006,4(7):1115-1121.
[18]WAHED A E W,EI-DEEB A,EI-THOLOTH M,et al.A portable reverse transcription recombinase polymerase amplification assay for rapid detection of footand-mouth disease[J].PLo S One,2013,8(8):e71642.
[19]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.
[20]WANG J C,ZHANG R X,WANG J F,et al.Real-time reverse transcription recombinase polymerase amplification assay for rapid detection of porcine epidemic diarrhea virus[J].Journal of Virological,2018,253(1):49-52.
[21]LOWINGS P,IBATA G,NEEDHAM J,et al.Classical swine fever virus diversity and evolution[J].Journal of General Virology,1996,77(6):1311-1321.
[22]PATON D J,MCGOLDRICK A,GREISER-WILKE I,et al.Genetic typing of classical swine fever virus[J].Veterinary Microbiology,2000,73(2):137-157.
[23]SUN S Q,YIN S H,GUO H C,et al.Genetic typing of classical swine fever virus isolates from China[J].Transboundary and Emerging Diseases,2013,60(4):370-375.
[24]张志,李晓成.我国猪瘟流行现状和防控建议[J].中国动物检疫,2015,32(8):8-12.ZHANG Zhi,LI Xiao-cheng.The current status of classical swine fever in china and suggestions for its control[J].China Animal Health Inspection,2015,32(8):8-12.(in Chinese)
[25]DAHER R K,STAWART 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.
[26]AEBISCHER A,WERNIKE K,HOFFMANN B,et al.Rapid genome detection of schmallenberg virus and bovine viral diarrhea virus by use of isothermal amplification methods and high-speed real-time reverse transcriptase PCR[J].Journal of Clinical Microbiology,2014,52(6):1883-1892.
[27]DUKES J P,KING D P,ALEXANDERSEN S.Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus[J].Archives of Virology,2006,151(6):1093-1106.