检测四种鸡呼吸道疫病病毒可视化芯片技术的优化及初步应用
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摘要
可视化芯片技术是在传统芯片技术的基础上发展起来的一项新的疾病诊断和基因分析技术,对于临床疾病检测和诊断具有重要意义。本研究针对禽流感病毒(Avian influenza virus,AIV)的核蛋白(nucleprotein,NP)基因设计一对特异性引物,以H9亚型禽流感病毒分离株c DNA为模板,经PCR扩增、连接、转化和核酸序列鉴定后,得到含有AIV-NP基因的重组质粒。同时复苏本实验室保存的3株分别含有新城疫病毒(Newcastle disease virus,NDV)的融合(fusion,F)蛋白基因、鸡传染性喉气管炎病毒(Infectious laryngotracheitis virus,ILTV)胸苷激酶(haemaggluttinin-neuraminidase,TK)基因和鸡传染性支气管炎病毒(Infectious bronchitis virus,IBV)核衣壳(nucleocapsid,N)蛋白基因的重组菌。以上述4种病原的靶基因核酸序列片段的正义链为模板,设计寡核苷酸探针,喷样到尼龙膜上制备成芯片,利用不对称PCR技术扩增生物素标记的靶基因,与芯片进行杂交后,检测结果直接用肉眼就可以判定。本研究对芯片制备流程和检测过程中主要条件进行优化。结果表明当寡核苷酸探针喷样浓度为25μmol/L、芯片杂交反应的时间为1 h、杂交温度为50℃、Streptavidin HRP Conjugate(1.0 mg/m L)稀释2 000倍、二氨基联苯胺(diaminobenzidine,DAB)显色时间为5 min时,芯片检测技术的结果最佳。用该方法与PCR/RT-PCR技术同时对临床采集的96份疑似病料进行检测,两种方法的检测结果一致。本研究构建的检测4种禽呼吸道疾病病毒可视化基因芯片技术具有高通量、快速、准确等优点,为鸡病的临床诊断提供了新的技术。
Visual microarray technology based on the traditional microarray technology, has a great significance for the detection of clinical disease detection and diagnosis. In this study, the specific primers were designed, according to the conservative gene nucleprotein(NP) of Avian influenza virus(AIV), c DNA was obtained from RT-PCR. The recombinant plasmid of AIV-NP was successfully constructed. Meanwhile,fusion(F) gene of Newcastle disease virus(NDV), nucleocapsid(N) gene of Infectious bronchitis virus(IBV)and thymidine kinase(TK) gene of Infectious laryngotracheitis virus(ILTV) were successfully recovered from the conserved bacteria. The oligonucleotide probes were designed on the basis of target gene sequences. The visual microarray was prepared and hybridized to the PCR products labeled biotin. The parameters of the visual microarray were optimized. The results showed that 25 μ mol/L of oligonucleotide probes, 1 h of hybridization time, 50 ℃ of hybridization temperature, 2 000 times diluted of 1.0 mg/m L Streptavidin HRP Conjugate and 5 min of diaminobenzidine(DAB) staining time, were the optimal condition for microarray detection. The 96 clinical samples were simultaneously detected by the visual microarray and PCR/RT-PCR,the results showed that the detection rate of two methods was consensus. The visual microarray of AIV-NDVIBV-ILTV developed in this study is a fast, accurately and high-throughput detection methods, which could provide a new technology for chicken disease clinical diagnosis.
Visual microarray technology based on the traditional microarray technology, has a great significance for the detection of clinical disease detection and diagnosis. In this study, the specific primers were designed, according to the conservative gene nucleprotein(NP) of Avian influenza virus(AIV), c DNA was obtained from RT-PCR. The recombinant plasmid of AIV-NP was successfully constructed. Meanwhile,fusion(F) gene of Newcastle disease virus(NDV), nucleocapsid(N) gene of Infectious bronchitis virus(IBV)and thymidine kinase(TK) gene of Infectious laryngotracheitis virus(ILTV) were successfully recovered from the conserved bacteria. The oligonucleotide probes were designed on the basis of target gene sequences. The visual microarray was prepared and hybridized to the PCR products labeled biotin. The parameters of the visual microarray were optimized. The results showed that 25 μ mol/L of oligonucleotide probes, 1 h of hybridization time, 50 ℃ of hybridization temperature, 2 000 times diluted of 1.0 mg/m L Streptavidin HRP Conjugate and 5 min of diaminobenzidine(DAB) staining time, were the optimal condition for microarray detection. The 96 clinical samples were simultaneously detected by the visual microarray and PCR/RT-PCR,the results showed that the detection rate of two methods was consensus. The visual microarray of AIV-NDVIBV-ILTV developed in this study is a fast, accurately and high-throughput detection methods, which could provide a new technology for chicken disease clinical diagnosis.
引文
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马锐,黄小波,杨国淋,等.2016.猪病毒性腹泻病金标银染可视化芯片技术实验条件优化研究及应用[J].农业生物技术学报,24(8):1233-1242.(Ma R,Huang X BYang G L et al.2016.The optimization and application of swine diarrhea virus visual microarray based on gold labeled silver staining[J].Journal of Agricultural Biotechnology.,24(8):1233-1242.)
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杨国淋,赵松,尹人杰,等.2016.结合不对称PCR技术构建鸡NDV-IBV-ILTV联合检测基因芯片[J].农业生物技术学报,24(1):142-150.(Yang G L,Zhao S,Yin R J,et al2016.Construction of gene chip for detecting NDV-IBV-ILTV of chicken(Gallus gallus)with asymmetric PCR[J]Journal of Agricultural Biotechnology,24(1):142-150.)
张仙,常晓霞,赵松,等.2015.可视化芯片技术研究进展[J]动物医学进展,23(4):100-103.(Zhang X,Chang X XZhao S,et al.2015.Research progress of visual microarray technology[J].Progress in Veterinary Medicine,23(4):100-103.
赵玉佳,文心田,马锐,等,2017.可视化芯片显色技术在病原学检测中的研究进展[J].中国预防兽医学报,39(2)159-162.(Zhao Y J,Wen X T,Ma R,et al.2017.Research progress of visualization microarray in pathogen detection[J].Chinese Journal of Preventive Veterinary Medicine,39(2):159-162.)
Aktas G,Skouridou V,Masip L.2015.Novel signal amplification approach for HRP-based colorimetric genosensors using DNA binding protein tags[J].Biosensors and Bioelectronics,74(8),:1005-1010.
Ali A M A,Mansour S M G,Mohamed M H A,et al.2014Molecular characterization of thymidine kinase and glycoprotein G genes from a possible vaccine induced infectious laryngotracheitis outbreak in Egypt[J].Pakistan Veterinary Journal,34(3):2074-7764.
Ferrari M D L,Resende M R,Sakai K,et al.2013.Visual Analysis of DNA microarray data for accurate molecular identification of non-albicans candida isolates from patients with candidemia episodes[J].Journal of Clinical Microbiology,51(11):3826-3829.
Ding L,Rui C,Zhan S J,et al.2010.Detection of hepatitis Bvirus genotypes A to D by the fluorescence polarization assay based on asymmetric PCR[J].Journal of Virological Methods,168(2):31-37.
Gilbert M,Pfeiffer D U.2012.Risk factor modelling of the spatio-temporal patterns of Highly pathogenic avian influenza(HPAIV)H5N1:A review[J].Spatial and spatio-temporal epidemiology,3(3):173-183.
Kirkpatrick NC,Mahmoudian A,ORourke D,et al.2009.Differentiation of infectious laryngotracheitis virus isolates by restriction fragment length polymorphic analysis of polymerase chain reaction products amplified from multiple genes[J].Avian Diseases,50(1):28-34.
Lee C W,Saif Y M.2009.Avian influenza virus[J].Comparative immunology,microbiology and infectious diseases,32(4):301-310.
Li Y,Xiong T,Wu H,et al.2016.Visual DNA microarray coupled with multiplex-PCR for the rapid detection of twelve genetically modified maize[J].Bio Chip Journal,10(1):42-47.
Sterrenburg E,Turk R,Boer J M,et al.2002.A common reference for c DNA microarray hybridizations[J].Nucleic Acids Research,30(21):2-6.
Tao Q,Wang X,Bao H,et al.2009.Detection and differentiation of four poultry diseases using asymmetric reverse transcription polymerase chain reaction in combination with oligonucleotide microarrays[J].Journal of Veterinary Diagnostic Investigation,21(5):623-632.
Tong L,Wu J,Li J,et al.2013.Hybridization chain reaction engineered DNA nanopolylinker for amplified electrochemical sensing of biomarkers[J].Analyst,138(17):4870-4876.
Wang X,Dang E,Gao J,et al.2013.Development of a gold nanoparticle-based oligonucleotide microarray for simultaneous detection of seven swine viruses[J].Journal of Virological Methods,191(1):9-15.
Wang Z X,Gao S M,Zheng Q W,et al.2015.Epidemiology and evolutionary characteristics of Avian infectious bronchitis virus in China.[J].Kafkasüniversitesi Veteriner Fakültesi Dergisi,22(2):179-186.
黄书林,吴彤,蒋菲,等.2016.H5亚型禽流感病毒环介导等温扩增检测方法的建立[J].农业生物技术学报24(9):1384-1391.(Huang S L,Wu T,Jiang F,et al.2016.Detection of H5 Avian influenza virus by loop-mediated isothermal amplification[J].Journal of Agricultural Biotechnology,24(9):1384-1391.)
黄溢泓,韦正吉,李志源,等.2010.五种禽呼吸道病病原多重PCR检测方法的建立和应用[J].中国兽医科学,40(2):164-168.(Huang Y H,Wei Z J,Li Z Y et al.2010.Establishment and application of a multiplex PCR assay for simultaneous detection of AIV,NDV,IBV,ILTV and MG[J].Chinese Veterinary Science,40(2):164-168.)
孔强,曾伟,赵焕云,等.2016.某规模场产蛋量下降鸡群新城疫病毒分离与鉴定[J].畜牧与兽医48(5):100-104(Kon G Q,Zeng W,Zhao H Y,et al.2016.Isolation and identification of Newcastle disease virus isolated from chicken flocks in a scale field[J].Animal Husbandry&Veterinary Medicine,48(5):100-104.)
马锐,黄小波,杨国淋,等.2016.猪病毒性腹泻病金标银染可视化芯片技术实验条件优化研究及应用[J].农业生物技术学报,24(8):1233-1242.(Ma R,Huang X BYang G L et al.2016.The optimization and application of swine diarrhea virus visual microarray based on gold labeled silver staining[J].Journal of Agricultural Biotechnology.,24(8):1233-1242.)
王善辉,谢金文,王文秀,等.2014.NDV江苏分离株F基因的克隆与序列分析[J].中国动物检疫,31(3):64-67(Wang S H,Xie J W,Wang W X,et al.2014.Cloning and sequence analysis of fusion gene of NDV Jiangsu Isolates[J].China Animal Health Inspection,31(3):64-67.)
杨国淋,赵松,尹人杰,等.2016.结合不对称PCR技术构建鸡NDV-IBV-ILTV联合检测基因芯片[J].农业生物技术学报,24(1):142-150.(Yang G L,Zhao S,Yin R J,et al2016.Construction of gene chip for detecting NDV-IBV-ILTV of chicken(Gallus gallus)with asymmetric PCR[J]Journal of Agricultural Biotechnology,24(1):142-150.)
张仙,常晓霞,赵松,等.2015.可视化芯片技术研究进展[J]动物医学进展,23(4):100-103.(Zhang X,Chang X XZhao S,et al.2015.Research progress of visual microarray technology[J].Progress in Veterinary Medicine,23(4):100-103.
赵玉佳,文心田,马锐,等,2017.可视化芯片显色技术在病原学检测中的研究进展[J].中国预防兽医学报,39(2)159-162.(Zhao Y J,Wen X T,Ma R,et al.2017.Research progress of visualization microarray in pathogen detection[J].Chinese Journal of Preventive Veterinary Medicine,39(2):159-162.)
Aktas G,Skouridou V,Masip L.2015.Novel signal amplification approach for HRP-based colorimetric genosensors using DNA binding protein tags[J].Biosensors and Bioelectronics,74(8),:1005-1010.
Ali A M A,Mansour S M G,Mohamed M H A,et al.2014Molecular characterization of thymidine kinase and glycoprotein G genes from a possible vaccine induced infectious laryngotracheitis outbreak in Egypt[J].Pakistan Veterinary Journal,34(3):2074-7764.
Ferrari M D L,Resende M R,Sakai K,et al.2013.Visual Analysis of DNA microarray data for accurate molecular identification of non-albicans candida isolates from patients with candidemia episodes[J].Journal of Clinical Microbiology,51(11):3826-3829.
Ding L,Rui C,Zhan S J,et al.2010.Detection of hepatitis Bvirus genotypes A to D by the fluorescence polarization assay based on asymmetric PCR[J].Journal of Virological Methods,168(2):31-37.
Gilbert M,Pfeiffer D U.2012.Risk factor modelling of the spatio-temporal patterns of Highly pathogenic avian influenza(HPAIV)H5N1:A review[J].Spatial and spatio-temporal epidemiology,3(3):173-183.
Kirkpatrick NC,Mahmoudian A,ORourke D,et al.2009.Differentiation of infectious laryngotracheitis virus isolates by restriction fragment length polymorphic analysis of polymerase chain reaction products amplified from multiple genes[J].Avian Diseases,50(1):28-34.
Lee C W,Saif Y M.2009.Avian influenza virus[J].Comparative immunology,microbiology and infectious diseases,32(4):301-310.
Li Y,Xiong T,Wu H,et al.2016.Visual DNA microarray coupled with multiplex-PCR for the rapid detection of twelve genetically modified maize[J].Bio Chip Journal,10(1):42-47.
Sterrenburg E,Turk R,Boer J M,et al.2002.A common reference for c DNA microarray hybridizations[J].Nucleic Acids Research,30(21):2-6.
Tao Q,Wang X,Bao H,et al.2009.Detection and differentiation of four poultry diseases using asymmetric reverse transcription polymerase chain reaction in combination with oligonucleotide microarrays[J].Journal of Veterinary Diagnostic Investigation,21(5):623-632.
Tong L,Wu J,Li J,et al.2013.Hybridization chain reaction engineered DNA nanopolylinker for amplified electrochemical sensing of biomarkers[J].Analyst,138(17):4870-4876.
Wang X,Dang E,Gao J,et al.2013.Development of a gold nanoparticle-based oligonucleotide microarray for simultaneous detection of seven swine viruses[J].Journal of Virological Methods,191(1):9-15.
Wang Z X,Gao S M,Zheng Q W,et al.2015.Epidemiology and evolutionary characteristics of Avian infectious bronchitis virus in China.[J].Kafkasüniversitesi Veteriner Fakültesi Dergisi,22(2):179-186.