鸡传染性喉气管炎病毒可视化LAMP快速诊断方法的建立和应用
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摘要
试验旨在建立简易、快速、高效的鸡传染性喉气管炎病毒(infectious laryngotracheitis virus,ILTV)检测和诊断方法。根据GenBank上公布的ILTV TK基因序列,设计检测ILTV的特异性环介导的等温扩增(loop media-ted isothermal amplification,LAMP)技术反应引物,通过对LAMP反应体系和反应条件的优化,以及特异性、敏感性和临床样品的检测,建立了ILTV LAMP检测方法。结果显示,以内引物ILT9-FIP和ILT9-BIP、外引物ILT9-F3和ILT9-B3、环引物ILT9-LB和ILT9-LF为LAMP反应引物,反应温度为66℃时,所建立的LAMP检测方法反应效率最高;所建立的LAMP检测方法能够特异性地检测ILTV(匈牙利株和王岗株),不与新城疫病毒(NDV,B株)、鸡传染性支气管炎病毒(IBV,H52株和H120株)、大肠杆菌、鸡副嗜血杆菌、巴氏杆菌等发生交叉反应,且能够检测到的病毒最低浓度达到0.06pg/μL,其灵敏度是普通PCR方法的100倍;采用建立LAMP方法对50个临床样本进行检测,阳性率为14%,且与PCR检测结果的符合率达96%。本研究建立了特异性强、灵敏度高、操作简单的LAMP检测方法,适用于临床上ILTV的快速检测和诊断。
This study was aimed to establish a simple,rapid and efficient method for detection and diagnosis of infectious laryngotracheitis virus(ILTV).The specific perimers were designed for the loop mediated isothermal amplification(LAMP)reaction based on the conservative region of ILTV TKgene in GenBank.The LAMP detection method for ILTV was established through the optimization of the LAMP reaction system and reaction conditions,as well as the specificity,sensitivity and detection of clinical samples.The results showed that the established LAMP reaction exhibited the highest amplification efficiency under the parameters of internal primers(ILT9 FIPand ILT9 BIP),outer primers(ILT9-F3 and ILT9-B3),ring primers(ILT9-LB and ILT9-LF)and reaction temperature(66 ℃).The established LAMP detection method could specifically detect ILTV(Hungarian and Wanggang strains),there was no cross-react with Newcastle disease virus(NDV,B strain),avian infectious bronchitis virus(IBV,H52 and H120 strains),Escherichia coli,Haemophilus parasuis,Pasteurella,etc.Meanwhile,the sensitivity of the established LAMP detection method was 0.06 pg/L,and its sensitivity was 100 times of the conventional PCR method.50 clinical samples were tested by establishing LAMP method,the positive rate was 14%,and the coincidence rate was 96% with PCR method.This study established a LAMP detection method with high specificity,high sensitivity and simple operation,which was suitable for rapid detection and diagnosis of ILTV in clinical application.
This study was aimed to establish a simple,rapid and efficient method for detection and diagnosis of infectious laryngotracheitis virus(ILTV).The specific perimers were designed for the loop mediated isothermal amplification(LAMP)reaction based on the conservative region of ILTV TKgene in GenBank.The LAMP detection method for ILTV was established through the optimization of the LAMP reaction system and reaction conditions,as well as the specificity,sensitivity and detection of clinical samples.The results showed that the established LAMP reaction exhibited the highest amplification efficiency under the parameters of internal primers(ILT9 FIPand ILT9 BIP),outer primers(ILT9-F3 and ILT9-B3),ring primers(ILT9-LB and ILT9-LF)and reaction temperature(66 ℃).The established LAMP detection method could specifically detect ILTV(Hungarian and Wanggang strains),there was no cross-react with Newcastle disease virus(NDV,B strain),avian infectious bronchitis virus(IBV,H52 and H120 strains),Escherichia coli,Haemophilus parasuis,Pasteurella,etc.Meanwhile,the sensitivity of the established LAMP detection method was 0.06 pg/L,and its sensitivity was 100 times of the conventional PCR method.50 clinical samples were tested by establishing LAMP method,the positive rate was 14%,and the coincidence rate was 96% with PCR method.This study established a LAMP detection method with high specificity,high sensitivity and simple operation,which was suitable for rapid detection and diagnosis of ILTV in clinical application.
引文
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[7]LIU W,ZOU D,LI Y,et al.Sensitive and rapid detection of the new Delhi metallo-beta-lactamase gene by loop-mediated isothermal amplification[J].Journal of Clinical Microbiology,2012,50(5):1580-1585.
[8]许青荣,周祖涛,毕丁仁,等.蛋鸡传染性喉气管炎病毒的分离、PCR检测及TK基因序列分析[J].黑龙江畜牧兽医,2017,24:122-123.XU Q R,ZHOU Z T,BI D R,et al.Isolation,PCR detection and TK gene sequence analysis of infectious laryngotracheitis virus in laying hens[J].Heilongjiang Animal Science and Veterinary Medicine,2017,24:122-123.(in Chinese)
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[10]田莉莉,李林.鸡传染性喉气管炎病毒套式PCR检测方法的建立及应用[J].中国家禽,2011,33(17):57-59.TIAN L L,LI L.Establishment and application of nested PCR for detection of infectious laryngotracheitis virus[J].China Poultry,2011,33(17):57-59.(in Chinese)
[11]张小荣,张林,刘怡,等.一种检测传染性喉气管炎病毒的夹心ELISA方法的建立[J].中国家禽,2017,39(9):16-19.ZHANG X R,ZHANG L,LIU Y,et al.Establishment of sandwich ELISA method for detection of infectious laryngotracheitis virus[J].China Poultry,2017,39(9):16-19.(in Chinese)
[12]NOTOMI T,OKAYAMA H,MASUBUCHI H.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Research,2000,28(12):E63.
[13]KANEKO H,KAWANA T,FUKUSHIMA E.Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances[J].Journal of Biochemical and Biophysical Methods,2007,70(3):499-501.
[14]CALVERT A E,BIGGERSTAFF B J,TANNER N A,et al.Rapid colorimetric detection of Zika virus from serum and urine specimens by reverse transcription loop-mediated isothermal amplification(RT-LAMP)[J].PLoS One,2017,12(9):e0185340.
[15]OLONINIYI O K,KUROSAKI Y,MIYANOTO H,et al.Rapid detection of all known ebolavirus species by reverse transcription-loop-mediated isothermal amplification(RT-LAMP)[J].Journal of Virological Methods,2017,246:8-14.
[16]HOOS J,PETERS R M,TABATABAI J.Reversetranscription loop-mediated isothermal amplification for rapid detection of respiratory syncytial virus directly from nasopharyngeal swabs[J].Journal of Virological Methods,2017,242:53-57.
[17]谢志勤,谢芝勋,邓显文.鸡传染性喉气管炎病毒LAMP检测方法的建立[J].畜牧与兽医,2012,44(11):52-55.XIE Z Q,XIE Z X,DENG X W.Establishment of LAMP for detection of avian infectious laryngotracheitis virus[J].Animal Husbandry and Veterinary Medicine,2012,44(11):52-55.(in Chinese)
[18]孔令辰,侯佳蕾,蒋文泓,等.应用环介导逆转录等温扩增技术快速检测新城疫病毒[J].华南农业大学学报,2008,29(4):75-78.KONG L C,HOU J L,JIANG W H,et al.Reverse transcriptase loop-mediated isothermal amplification technique for rapid detection of Newcastle disease virus[J].Journal of South China Agricultural University,2008,29(4):75-78.(in Chinese)
[19]KEELER C L,KINGSLEY D H,BURTON C R.Identification of the thymidinekinase gene of infectious laryngotracheitis virus[J].Avian Disease,1991,35(4):920-929.
[20]李家伟,郭利,杨勇,等.牛病毒性腹泻病毒LAMP检测方法的建立与应用[J].中国畜牧兽医,2015,42(12):3111-3118.LI J W,GUO L,YANG Y,et al.Establishment and application of LAMP detection method of bovine viral diarrhea virus[J].China Animal Husbandry&Veterinary Medicine,2015,42(12):3111-3118.(in Chinese)
[21]张日腾,张乔亚,姜辰龙.猪繁殖与呼吸综合征病毒RT-LAMP可视化检测方法的建立与应用[J].中国兽医科学,2018,48(1):7-12.ZHANG R T,ZHANG Q Y,JIANG C L.Establishment and applications of RT-LAMP for the visual detection of porcine reproductive and respiratory syndrome virus[J].Chinese Veterinary Science,2018,48(1):7-12.(in Chinese)
[22]汤小真,陈露薇,卢荣彬,等.基于钙黄绿素显色的可视化LAMP检测猪流行性腹泻病毒的研究[J].中国畜牧兽医,2015,42(2):331-336.TANG X Z,CHEN L W,LU R B,et al.Detection of porcine epidemic diarrhea virus by calcein-based visual loop-mediated isothermal amplification(LAMP)assay[J].China Animal Husbandry&Veterinary Medicine,2015,42(2):331-336.(in Chinese)
[2]MCGEOCH D J,DOLAN A,RALPH A C.Toward a comprehensive phylogeny for mammalian and avian herpesviruses[J].Journal of Virology,2000,74(22):10401-10406.
[3]HUMBERD J,GARCIA M,RIBLET S M,et al.Detection of infectious laryngotracheitis virus in formalin-fixed,paraffin-embedded tissues by nested polymerase chain reaction[J].Avian Diseases,2002,46(1):64.
[4]CREELAN J L,CALVERT V M,GRAHAM D A,et al.Rapid detection and characterization from field cases of infectious laryngotracheitis virus by Realtime polymerase chain reaction and restriction fragment length polymorphism[J].Avian Pathology,2006,35(2):173-179.
[5]YORK J J,FAHEY K J.Diagnosis of infectious laryngotracheitis using a monoclonal antibody ELISA[J].Avian Pathology,1998,17(1):173-182.
[6]刘文俊,黄晕茂,阳佑天,等.鹅新城疫病毒RT-LAMP可视化检测方法的建立[J].中国畜牧兽医,2018,45(1):22-31.LIU W J,HUANG Y M,YANG Y T,et al.Establishment of RT-LAMP virual detection method for goose Newcastle disease virus[J].China Aninal Husbandry&Veterinary Medicine,2018,45(1):22-31.(in Chinese)
[7]LIU W,ZOU D,LI Y,et al.Sensitive and rapid detection of the new Delhi metallo-beta-lactamase gene by loop-mediated isothermal amplification[J].Journal of Clinical Microbiology,2012,50(5):1580-1585.
[8]许青荣,周祖涛,毕丁仁,等.蛋鸡传染性喉气管炎病毒的分离、PCR检测及TK基因序列分析[J].黑龙江畜牧兽医,2017,24:122-123.XU Q R,ZHOU Z T,BI D R,et al.Isolation,PCR detection and TK gene sequence analysis of infectious laryngotracheitis virus in laying hens[J].Heilongjiang Animal Science and Veterinary Medicine,2017,24:122-123.(in Chinese)
[9]赵妍,孔聪聪,张晓敏,等.鸡传染性喉气管炎病毒TaqMan Real-time PCR检测方法的建立[J].中国预防兽医学报,2012,34(8):642-646.ZHAO Y,KONG C C,ZHANG X M,et al.Development of a TaqMan Real-time PCR for detection of infectious laryngotracheitis virus[J].Chinese Journal of Preventive Veterinary Medicine,2012,34(8):642-646.(in Chinese)
[10]田莉莉,李林.鸡传染性喉气管炎病毒套式PCR检测方法的建立及应用[J].中国家禽,2011,33(17):57-59.TIAN L L,LI L.Establishment and application of nested PCR for detection of infectious laryngotracheitis virus[J].China Poultry,2011,33(17):57-59.(in Chinese)
[11]张小荣,张林,刘怡,等.一种检测传染性喉气管炎病毒的夹心ELISA方法的建立[J].中国家禽,2017,39(9):16-19.ZHANG X R,ZHANG L,LIU Y,et al.Establishment of sandwich ELISA method for detection of infectious laryngotracheitis virus[J].China Poultry,2017,39(9):16-19.(in Chinese)
[12]NOTOMI T,OKAYAMA H,MASUBUCHI H.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Research,2000,28(12):E63.
[13]KANEKO H,KAWANA T,FUKUSHIMA E.Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances[J].Journal of Biochemical and Biophysical Methods,2007,70(3):499-501.
[14]CALVERT A E,BIGGERSTAFF B J,TANNER N A,et al.Rapid colorimetric detection of Zika virus from serum and urine specimens by reverse transcription loop-mediated isothermal amplification(RT-LAMP)[J].PLoS One,2017,12(9):e0185340.
[15]OLONINIYI O K,KUROSAKI Y,MIYANOTO H,et al.Rapid detection of all known ebolavirus species by reverse transcription-loop-mediated isothermal amplification(RT-LAMP)[J].Journal of Virological Methods,2017,246:8-14.
[16]HOOS J,PETERS R M,TABATABAI J.Reversetranscription loop-mediated isothermal amplification for rapid detection of respiratory syncytial virus directly from nasopharyngeal swabs[J].Journal of Virological Methods,2017,242:53-57.
[17]谢志勤,谢芝勋,邓显文.鸡传染性喉气管炎病毒LAMP检测方法的建立[J].畜牧与兽医,2012,44(11):52-55.XIE Z Q,XIE Z X,DENG X W.Establishment of LAMP for detection of avian infectious laryngotracheitis virus[J].Animal Husbandry and Veterinary Medicine,2012,44(11):52-55.(in Chinese)
[18]孔令辰,侯佳蕾,蒋文泓,等.应用环介导逆转录等温扩增技术快速检测新城疫病毒[J].华南农业大学学报,2008,29(4):75-78.KONG L C,HOU J L,JIANG W H,et al.Reverse transcriptase loop-mediated isothermal amplification technique for rapid detection of Newcastle disease virus[J].Journal of South China Agricultural University,2008,29(4):75-78.(in Chinese)
[19]KEELER C L,KINGSLEY D H,BURTON C R.Identification of the thymidinekinase gene of infectious laryngotracheitis virus[J].Avian Disease,1991,35(4):920-929.
[20]李家伟,郭利,杨勇,等.牛病毒性腹泻病毒LAMP检测方法的建立与应用[J].中国畜牧兽医,2015,42(12):3111-3118.LI J W,GUO L,YANG Y,et al.Establishment and application of LAMP detection method of bovine viral diarrhea virus[J].China Animal Husbandry&Veterinary Medicine,2015,42(12):3111-3118.(in Chinese)
[21]张日腾,张乔亚,姜辰龙.猪繁殖与呼吸综合征病毒RT-LAMP可视化检测方法的建立与应用[J].中国兽医科学,2018,48(1):7-12.ZHANG R T,ZHANG Q Y,JIANG C L.Establishment and applications of RT-LAMP for the visual detection of porcine reproductive and respiratory syndrome virus[J].Chinese Veterinary Science,2018,48(1):7-12.(in Chinese)
[22]汤小真,陈露薇,卢荣彬,等.基于钙黄绿素显色的可视化LAMP检测猪流行性腹泻病毒的研究[J].中国畜牧兽医,2015,42(2):331-336.TANG X Z,CHEN L W,LU R B,et al.Detection of porcine epidemic diarrhea virus by calcein-based visual loop-mediated isothermal amplification(LAMP)assay[J].China Animal Husbandry&Veterinary Medicine,2015,42(2):331-336.(in Chinese)