同时检测禽类六种病毒的金标银染可视化基因芯片的构建及初步应用
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摘要
本研究旨在利用金标银染显色技术,构建同时检测禽白血病病毒(ALV)、鸡马立克病病毒(MDV)、传染性法氏囊病毒(IBDV)、新城疫病毒(NDV)、禽流感病毒(AIV)、鸡传染性喉气管炎病毒(ILTV)的可视化基因芯片。利用T-A法分别克隆得到ALV-env、MDV-meq、IBDV-vp_2基因,并利用本实验室已保存的AIV-np、NDV-f、ILTV-tk基因序列,设计寡核苷酸探针,制备ALV-MDV-IBDV-AIV-NDV-ILTV基因芯片阵列。引入生物素标记的引物进行PCR扩增,并与芯片进行杂交银染显色,肉眼观察检测结果,并对芯片的特异性、敏感性、稳定性进行评价,以及临床初步验证。成功克隆ALV-env、MDV-meq及IBDV-vp_2共3个靶基因,测序鉴定其正确性。发现寡核苷酸探针最优使用浓度为50μmol·L~(-1),40℃条件下杂交2 h,Nanogold-Streptavidin链霉亲和素溶液的质量浓度为4μg·mL~(-1),银染5 min,可见明显的检测信号。特异性试验显示,ALV、MDV、IBDV、NDV、AIV、ILTV之间无交叉反应,且以鸡传染性支气管炎病毒为模板制备的PCR扩增标记不与ALV-MDV-IBDV-AIV-NDV-ILTV芯片杂交。芯片检测的灵敏度为1 pg·μL~(-1)。芯片在常温条件下保存60 d,在4℃条件下保存75 d仍可进行有效检测。临床病料检测结果与PCR检测结果一致。本研究成功构建同时检测ALV、MDV、IBDV、NDV、AIV和ILTV的金标银染可视化基因芯片,并确定其最佳反应条件,为临床标准化应用奠定了基础。
The aim of the present study was to develop a visual chip for simultaneously detecting avian leukosis virus( ALV),chicken Marek's disease virus( MDV),infectious bursal disease virus( IBDV),Newcastle disease virus( NDV),avian influenza virus( AIV),and infectious laryngotracheitis virus( ILTV) using gold label silver stain method. The gene sequences of ALV-env、MDV-meq and IBDV-vp_2 were cloned using T-A method. These sequences,also the sequences of AIV-np,NDV-f,ILTV-tk stored by our laboratory,were used for probes design,following which the simultaneously detecting ALV-MDV-IBDV-AIV-NDV-ILTV chip was developed. Following the PCR amplification using a pair of biotin-labeled primers,the chip was used for naked-eyes observation after hybridization and silver stain. The chip was then validated by specificity and sensitivity identification,stability evaluation,and clinical verification.The sequences of ALV-env,MDV-meq,IBDV-vp_2 were cloned successfully. The hybridization,at 40 ℃for 2 h,of 50 μmol·L~(-1) oligonucleotide probes with the biotin-labeled PCR products resulted in visible silver stain signal. No cross reaction was observed among ALV,MDV,IBDV,NDV,AIV and ILTV by specificity test. The specificity of the chip was confirmed by its negative hybridization with the PCR products from infectious bronchitis virus. The minimum-does of the plasmid as PCR template was 1 pg·μL~(-1) by sensitivity test. The chip can be stored at room temperature for 60 d,or at 4 ℃ for 75 d according to stability test. The results of chip test consistent with that of PCR/RT-PCR method. The present study successfully developed a visual chip for simultaneously detecting ALV,MDV,IBDV,NDV,AIV and ILTV with an optimization reaction condition,which will facilitate the clinical application of the chip in the future.
The aim of the present study was to develop a visual chip for simultaneously detecting avian leukosis virus( ALV),chicken Marek's disease virus( MDV),infectious bursal disease virus( IBDV),Newcastle disease virus( NDV),avian influenza virus( AIV),and infectious laryngotracheitis virus( ILTV) using gold label silver stain method. The gene sequences of ALV-env、MDV-meq and IBDV-vp_2 were cloned using T-A method. These sequences,also the sequences of AIV-np,NDV-f,ILTV-tk stored by our laboratory,were used for probes design,following which the simultaneously detecting ALV-MDV-IBDV-AIV-NDV-ILTV chip was developed. Following the PCR amplification using a pair of biotin-labeled primers,the chip was used for naked-eyes observation after hybridization and silver stain. The chip was then validated by specificity and sensitivity identification,stability evaluation,and clinical verification.The sequences of ALV-env,MDV-meq,IBDV-vp_2 were cloned successfully. The hybridization,at 40 ℃for 2 h,of 50 μmol·L~(-1) oligonucleotide probes with the biotin-labeled PCR products resulted in visible silver stain signal. No cross reaction was observed among ALV,MDV,IBDV,NDV,AIV and ILTV by specificity test. The specificity of the chip was confirmed by its negative hybridization with the PCR products from infectious bronchitis virus. The minimum-does of the plasmid as PCR template was 1 pg·μL~(-1) by sensitivity test. The chip can be stored at room temperature for 60 d,or at 4 ℃ for 75 d according to stability test. The results of chip test consistent with that of PCR/RT-PCR method. The present study successfully developed a visual chip for simultaneously detecting ALV,MDV,IBDV,NDV,AIV and ILTV with an optimization reaction condition,which will facilitate the clinical application of the chip in the future.
引文
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[13]XUE L,FEI J J,SONG Y,et al. Visual DNA microarray for detection of epidermal growth factor receptor(EGFR)gene mutations[J]. Scand J Clin Lab Inv,2014,74(8):693-699.
[14]HE Y Q,ZHANG X B,ZHANG S Q,et al. Visual detection of single-base mismatches in DNA using hairpin oligonucleotide with double-target DNA binding sequences and gold nanoparticles[J]. Biosens Bioelectro,2012,34(1):37-43.
[15]曹三杰.鸡新城疫、鸡传染性支气管炎基因芯片的构建及其检测技术研究[D].成都:四川农业大学,2004.CAO S J. A study on constructing of NDV-IBV microarray and its detection method[D]. Chengdu:Sichuan Agricultural University,2004.(in Chinese)
[16]DAVIDSON I,SKODA I. The impact of feathers use on the detection and study of DNA viral pathogens in commercial poultry[J]. World’s Poult Sci J,2005,61(3):407-417.
[17]BROWN A C,BAIGENT S J,SMITH L P,et al. Interaction of MEQ protein and C-terminal-binding protein is critical for induction of lymphomas by Marek’s disease virus[J]. Proc Natl Acad Sci U S A,2006,103(6):1687-1692.
[18]张文通,魏凤,苗立中,等.传染性法氏囊病病毒RT-PCR检测及VP2基因序列分析[J].动物医学进展,2014,35(5):8-11.ZHANG W T,WEI F,MIAO L Z,et al. RT-PCR detection of infectious bursal disease virus and sequence analysis of its VP2 gene[J]. Progress in Veterinary Medicine,2014,35(5):8-11.(in Chinese)
[19]GANAR K,DAS M,SINHA S,et al. Newcastle disease virus:Current status and our understanding[J]. Virus Res,2014,184:71-81.
[20]彭海燕,迟莹,李燕,等.高致病性禽流感病毒H5N1亚型核蛋白NP真核表达载体的构建、表达与鉴定[J].江苏预防医学,2014,25(5):6-9.PENG H Y,CHI Y,LI Y,et al. Construction of eukaryotic expression vector of avian influenza virus H5N1nuclear protein(NP)and expression,confirmation of recombinant NP protein in mammalian eukaryotic cells[J]. Jiangsu Journal of Preventive Medicine,2014,25(5):6-9.(in Chinese)
[21]WANG L G,MA J,XUE C Y,et al. Dynamic distribution and tissue tropism of Infectious laryngotracheitis virus in experimentally infected chickens[J]. Arch Virol,2013,158(3):659-666.
[22]WEI Q,LIU S Z,HUANG J F,et al. Comparison of hybridization behavior between double and single strand of targets and the application of asymmetric PCR targets in c DNA microarray[J]. J Biochem Mol Biol,2004,37(4):439-444.
[23]RASHID S,NAEEM K,AHMED Z,et al. Multiplex polymerase chain reaction for the detection and differentiation of avian influenza viruses and other poultry respiratory pathogens[J]. Poult Sci, 2009, 88(12):2526-2531.
[24]XIE Z X,LUO S S,XIE L J,et al. Simultaneous typing of nine avian respiratory pathogens using a novel Ge XP analyzer-based multiplex PCR assay[J]. J Virol Methods,2014,207:188-195.
[25]常晓霞.猪伪狂犬病毒、猪细小病毒和猪圆环病毒2型联合共检可视化基因芯片检测技术研究[D].成都:四川农业大学,2015.CHANG X X. Detection technology research of simultaneous detection PRV-PPV-PCV2 visual microarray[D].Chengdu:Sichuan Agricultural University,2015.(in Chinese)
[2]WANG Y F,ZHANG Y Q,JIANG X D. The application of nanoparticles in biochips[J]. Recent Pat Biotechnol,2008,2(1):55-59.
[3]UYEKI T M,COX N J. Global concerns regarding novel influenza A(H7N9)virus infections[J]. New Engl J Med,2013,368(20):1862-1864.
[4]LAM T T Y,WANG J,SHEN Y Y,et al. The genesis and source of the H7N9 Influenza viruses causing human infections in China[J]. Nature,2013,502(7470):241-244.
[5]IVSHINA A V,VODEIKO G M,KUZNETSOV V A,et al. Mapping of genomic segments of influenza B virus strains by an oligonucleotide microarray method[J]. J Clin Microbiol,2004,42(12):5793-5801.
[6]LEE D H,KIM J H,YUK S S,et al. Rapid hemagglutinin subtyping of novel avian-origin Influenza A(H7N9)virus using a diagnostic microarray[J]. Biochip J,2014,8(1):55-59.
[7]SULTANKULOVA K T, KOZHABERGENOV N S,STROCHKOV V M,et al. New oligonucleotide microarray for rapid diagnosis of avian viral diseases[J]. Virol J,2017,14:69.
[8]WANG L C,HUANG D A,PU C E,et al. Avian oncogenic virus differential diagnosis in chickens using oligonucleotide microarray[J]. J Virol Methods,2014,210:45-50.
[9]WANG L C,HUANG D A,CHENG M C,et al. H5 Avian influenza virus pathotyping using oligonucleotide microarray[J]. J Virol Methods,2015,220:39-42.
[10]WANG X Q,DANG E L,GAO J Z,et al. Development of a gold nanoparticle-based oligonucleotide microarray for simultaneous detection of seven swine viruses[J]. J Virol Methods,2013,191(1):9-15.
[11]ZOU N L,ZANG Y C,MAO H J,et al. A visual highsensitivity microarray for detection of Hepatitis B virus DNA and its single nucleotide polymorphisms[J]. Chin J Anal Chem,2012,40(4):569-573.
[12]CHEN S Y,DENG T,WANG T Z,et al. Visualization of high-throughput and label-free antibody-polypeptide binding for drug screening based on microarrays and surface plasmon resonance imaging[J]. J Biomed Opt,2012,17(1):015005.
[13]XUE L,FEI J J,SONG Y,et al. Visual DNA microarray for detection of epidermal growth factor receptor(EGFR)gene mutations[J]. Scand J Clin Lab Inv,2014,74(8):693-699.
[14]HE Y Q,ZHANG X B,ZHANG S Q,et al. Visual detection of single-base mismatches in DNA using hairpin oligonucleotide with double-target DNA binding sequences and gold nanoparticles[J]. Biosens Bioelectro,2012,34(1):37-43.
[15]曹三杰.鸡新城疫、鸡传染性支气管炎基因芯片的构建及其检测技术研究[D].成都:四川农业大学,2004.CAO S J. A study on constructing of NDV-IBV microarray and its detection method[D]. Chengdu:Sichuan Agricultural University,2004.(in Chinese)
[16]DAVIDSON I,SKODA I. The impact of feathers use on the detection and study of DNA viral pathogens in commercial poultry[J]. World’s Poult Sci J,2005,61(3):407-417.
[17]BROWN A C,BAIGENT S J,SMITH L P,et al. Interaction of MEQ protein and C-terminal-binding protein is critical for induction of lymphomas by Marek’s disease virus[J]. Proc Natl Acad Sci U S A,2006,103(6):1687-1692.
[18]张文通,魏凤,苗立中,等.传染性法氏囊病病毒RT-PCR检测及VP2基因序列分析[J].动物医学进展,2014,35(5):8-11.ZHANG W T,WEI F,MIAO L Z,et al. RT-PCR detection of infectious bursal disease virus and sequence analysis of its VP2 gene[J]. Progress in Veterinary Medicine,2014,35(5):8-11.(in Chinese)
[19]GANAR K,DAS M,SINHA S,et al. Newcastle disease virus:Current status and our understanding[J]. Virus Res,2014,184:71-81.
[20]彭海燕,迟莹,李燕,等.高致病性禽流感病毒H5N1亚型核蛋白NP真核表达载体的构建、表达与鉴定[J].江苏预防医学,2014,25(5):6-9.PENG H Y,CHI Y,LI Y,et al. Construction of eukaryotic expression vector of avian influenza virus H5N1nuclear protein(NP)and expression,confirmation of recombinant NP protein in mammalian eukaryotic cells[J]. Jiangsu Journal of Preventive Medicine,2014,25(5):6-9.(in Chinese)
[21]WANG L G,MA J,XUE C Y,et al. Dynamic distribution and tissue tropism of Infectious laryngotracheitis virus in experimentally infected chickens[J]. Arch Virol,2013,158(3):659-666.
[22]WEI Q,LIU S Z,HUANG J F,et al. Comparison of hybridization behavior between double and single strand of targets and the application of asymmetric PCR targets in c DNA microarray[J]. J Biochem Mol Biol,2004,37(4):439-444.
[23]RASHID S,NAEEM K,AHMED Z,et al. Multiplex polymerase chain reaction for the detection and differentiation of avian influenza viruses and other poultry respiratory pathogens[J]. Poult Sci, 2009, 88(12):2526-2531.
[24]XIE Z X,LUO S S,XIE L J,et al. Simultaneous typing of nine avian respiratory pathogens using a novel Ge XP analyzer-based multiplex PCR assay[J]. J Virol Methods,2014,207:188-195.
[25]常晓霞.猪伪狂犬病毒、猪细小病毒和猪圆环病毒2型联合共检可视化基因芯片检测技术研究[D].成都:四川农业大学,2015.CHANG X X. Detection technology research of simultaneous detection PRV-PPV-PCV2 visual microarray[D].Chengdu:Sichuan Agricultural University,2015.(in Chinese)