牛传染性鼻气管炎病毒恒温隔绝式荧光PCR检测方法的建立
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摘要
为建立检测牛传染性鼻气管炎病毒(IBRV)的恒温隔绝式荧光PCR (iiPCR)方法,本研究采用文献报道的引物和探针,通过优化反应体系,首次建立了检测IBRV核酸的iiPCR方法。结果显示该方法仅特异性检测出IBRV,而对牛病毒性腹泻-黏膜病病毒、牛副流感3型病毒、牛呼吸道合胞体病毒、牛腺病毒3型、牛冠状病毒、牛支原体、多杀性巴氏杆菌和溶血性曼氏杆菌等病原则不能检出。该方法的检测下限为2.4拷贝/μL质粒标准品,并具有良好的重复性。对临床样品的检测结果与OIE推荐的荧光定量PCR方法的检测结果一致;该方法配合PetNAD核酸萃取试剂盒,从核酸提取到报告检测结果仅需1 h,具有操作简便、快速、可现地化的特点。对9个省29份商品化冻精检测结果显示,IBRV阳性率为24%(7/29),表明我国流通的商品化冻精携带IBRV情况普遍。本研究为IBRV的现地检测提供了一个可靠的方法。
The purpose of this study was to develop an insulated isothermal PCR(iiPCR) assay for detecting the infectious bovine rhinotracheitis virus(IBRV). After selection of a pair of primers and a fluorescent Taq Man probe according to previous report and optimization the reaction conditions, an ii PCR assay was successfully developed and validated for the first time. The results showed that the assay only specificly reacted with IBRV and did not cross-react with any of the bovine viral diarrhoea virus, bovine parainfluenza virus type 3, bovine respiratory syncytial virus, bovine adenovirus type 3, bovine coronavirus,Mycoplasma bovis, Pasteurella multocida and Mannheimia haemolytica, indicating that the assay is highly specific. Meanwhile, the ii PCR assay is highly sensitive and reproducible, the detection limit of the ii PCR assay for IBRV was estimated to be 2.4 copies/μL.The detection ratio for clinical samples was the same as that of a real-time quantitative PCR recommended by OIE. Notably,combined with the PetNAD nucleic acid extraction kit, it takes only 1 hour from nucleic acid extraction to report test result, with the characteristics of easy operation, fast and on-site. Furthermore, 29 commercial frozen semen samples collected from 9 provinces were detected as 24% IBRV positive, indicating that IBRV in commercial frozen semen was common in China. In conclusion, this study provides a reliable method for on-site detecting of IBRV.
The purpose of this study was to develop an insulated isothermal PCR(iiPCR) assay for detecting the infectious bovine rhinotracheitis virus(IBRV). After selection of a pair of primers and a fluorescent Taq Man probe according to previous report and optimization the reaction conditions, an ii PCR assay was successfully developed and validated for the first time. The results showed that the assay only specificly reacted with IBRV and did not cross-react with any of the bovine viral diarrhoea virus, bovine parainfluenza virus type 3, bovine respiratory syncytial virus, bovine adenovirus type 3, bovine coronavirus,Mycoplasma bovis, Pasteurella multocida and Mannheimia haemolytica, indicating that the assay is highly specific. Meanwhile, the ii PCR assay is highly sensitive and reproducible, the detection limit of the ii PCR assay for IBRV was estimated to be 2.4 copies/μL.The detection ratio for clinical samples was the same as that of a real-time quantitative PCR recommended by OIE. Notably,combined with the PetNAD nucleic acid extraction kit, it takes only 1 hour from nucleic acid extraction to report test result, with the characteristics of easy operation, fast and on-site. Furthermore, 29 commercial frozen semen samples collected from 9 provinces were detected as 24% IBRV positive, indicating that IBRV in commercial frozen semen was common in China. In conclusion, this study provides a reliable method for on-site detecting of IBRV.
引文
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[11]Tsai Yun-Long,Lin Yu-Chan,Chou Pin-Hsing,et al.Detection of white spot syndrome virus by polymerase chain reaction performed under insulated isothermal conditions[J].J Virol Methods,2012,181(1):134-137.
[12]Ambagala A,Pahari S,Fisher M,et al.A rapid field-deployable reverse transcription-insulated isothermal polymerase chain reaction assay for sensitive and specific detection of Bluetongue virus[J].Transbound Emerg Dis,2017,64(2):476-486.
[13]Ambagala A,Fisher M,Goolia M,et al.Field deployable reverse transcription insulated isothermal PCR(RT iiPCR)assay for rapid and sensitive detection of Foot and mouth disease virus[J].Transbound Emerg Dis,2017,64(5):1610-1623.
[14]张颖慧,王牧川,张斌,等.检测牛传染性鼻气管炎病毒和牛支原体的双重PCR方法的建立及应用[J].中国兽医科学,2018,48(7):824-829.
[15]Raaperi K,Orro T,Viltrop A.Epidemiology and control of bovine herpesvirus 1 infection in Europe[J].Vet J,2014,201(3):249-256.
[16]Pawar S S,Meshram C D,Singh N K,et al.Development of a SYBR Green I based duplex real-time PCR for detection of bovine herpesvirus-1 in semen[J].J Virol Methods,2014,208:6-10.
[17]Krishnan M,Ugaz V M,Burns M A.PCR in a Rayleigh-Bénard convection cell[J].Science,2002,298(5594):793.
[18]季新成,牛国辉,员丽娟,等.牛精液中IBRV荧光PCR检测方法的建立及精液带毒与血清抗体的关系[J].西北农林科技大学学报(自然科学版),2010,38(5):1-7.
[2]Jones C,Chowdhury S.Bovine herpesvirus type 1(BHV-1)is an important cofactor in the bovine respiratory disease complex[J].Vet Clin North Am Food Anim Pract,2010,26(2):303-321.
[3]陈茹,毕英佐,曹永长,等.牛传染性鼻气管炎病毒LUXTM新型荧光PCR检测方法的建立[J].中国预防兽医学报,2007,29(4):303-307.
[4]何小丽,李凡飞,张凯,等.国内外牛传染性鼻气管炎的流行现状及防控措施的研究进展[J].现代畜牧兽医,2018,(6):53-57.
[5]Biswas S,Bandyopadhyay S,Dimri U,et al.Bovine herpesvirus-1(BHV-1)-a re-emerging concern in livestock:a revisit to its biology,epidemiology,diagnosis,and prophylaxis[J].Vet Q,2013,33(2):68-81.
[6]Wang J,O'Keefe J,Orr D,et al.Validation of a real-time PCRassay for the detection of bovine herpesvirus 1 in bovine semen[J].J Virol Methods,2007,144(1):103-108.
[7]徐晓琴,冷雪,李真光,等.牛传染性鼻气管炎诊断方法研究进展[J].动物医学进展,2010,31(1):81-86.
[8]OIE.OIE Terrestrial Manual[M].Paris:OIE,2010.
[9]Tsai Y L,Wang H T,Chang H G,et al.Development of TaqMan probe-based insulated isothermal PCR(iiPCR)for sensitive and specific on-site pathogen detection[J].PLo S One,2012,7(9):e45278.
[10]李凡,岳华,邱莞思,等.基于恒温隔绝式荧光PCR技术现场检测牛轮状病毒方法的建立及应用[J].畜牧兽医学报,2017,48(6):1092-1098.
[11]Tsai Yun-Long,Lin Yu-Chan,Chou Pin-Hsing,et al.Detection of white spot syndrome virus by polymerase chain reaction performed under insulated isothermal conditions[J].J Virol Methods,2012,181(1):134-137.
[12]Ambagala A,Pahari S,Fisher M,et al.A rapid field-deployable reverse transcription-insulated isothermal polymerase chain reaction assay for sensitive and specific detection of Bluetongue virus[J].Transbound Emerg Dis,2017,64(2):476-486.
[13]Ambagala A,Fisher M,Goolia M,et al.Field deployable reverse transcription insulated isothermal PCR(RT iiPCR)assay for rapid and sensitive detection of Foot and mouth disease virus[J].Transbound Emerg Dis,2017,64(5):1610-1623.
[14]张颖慧,王牧川,张斌,等.检测牛传染性鼻气管炎病毒和牛支原体的双重PCR方法的建立及应用[J].中国兽医科学,2018,48(7):824-829.
[15]Raaperi K,Orro T,Viltrop A.Epidemiology and control of bovine herpesvirus 1 infection in Europe[J].Vet J,2014,201(3):249-256.
[16]Pawar S S,Meshram C D,Singh N K,et al.Development of a SYBR Green I based duplex real-time PCR for detection of bovine herpesvirus-1 in semen[J].J Virol Methods,2014,208:6-10.
[17]Krishnan M,Ugaz V M,Burns M A.PCR in a Rayleigh-Bénard convection cell[J].Science,2002,298(5594):793.
[18]季新成,牛国辉,员丽娟,等.牛精液中IBRV荧光PCR检测方法的建立及精液带毒与血清抗体的关系[J].西北农林科技大学学报(自然科学版),2010,38(5):1-7.