5种常见犬腹泻病毒基因芯片检测方法的建立
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摘要
为建立一种简便、快速、高效的可同时区分犬腺病毒1型(canine adenovirus type 1,CAV-1)、犬腺病毒2型(canine adenovirus type 2,CAV-2)、犬冠状病毒(canine coronavirus,CCV)、犬瘟热病毒(canine distemper virus,CDV)、犬细小病毒(canine parvovirus,CPV) 5种常见犬腹泻病毒的基因芯片诊断方法,本研究以CAV-1、CAV-2、CCV、CDV、CPV 5种犬腹泻病毒为靶病毒,根据NCBI上收录的病毒基因序列在其保守区域内设计引物,在此基础上根据变异区域设计针对每种病毒的探针2～3条,优化检测体系的各反应条件,确定该检测方法的特异性与敏感性,建立可同时区分5种病毒的基因芯片检测方法。结果显示,建立的基因芯片检测方法可同时检测以上述5种犬腹泻病毒,其中PCR的退火温度为55℃、延伸时间为1 min 15 s;探针与PCR产物的杂交温度40℃、杂交时间2.5 h时,该方法对CAV-1、CAV-2、CCV、CDV和CPV 5种病毒标准品的检测限分别为0.2 fg/μL、2 fg/μL、2 fg/μL、20 pg/μL和0.02 fg/μL,具有较高的灵敏性;同时对犬副流感病毒进行特异性试验,发现无阳性信号出现,具有较强的特异性;对14份临床腹泻样品检测结果显示,基因芯片方法对CAV-1、CAV-2、CCV、CDV和CPV 5种病毒的阳性检出率分别为28.57%、50.00%、64.28%、14.28%和85.71%,并且基因芯片检测方法的敏感性较PCR要高10～100倍。以上结果表明,本研究建立的基因芯片检测方法具有特异、敏感等特点,对临床中犬类混合感染病毒检测具有一定的诊断意义。
This study was aimed to establish a simple,rapid and efficient method for the diagnosis of canine adenovirus type 1(CAV-1),canine adenovirus type 2(CAV-2),canine coronavirus(CCV),canine distemper virus(CDV) and canine parvovirus(CPV).5 canine diarrhea viruses CAV-1,CAV-2,CCV,CDV and CPV were used as target viruses.The primers were designed in the conserved region according to the viral gene sequences included in NCBI.Then 2 to 3 probes for each virus were designed according to the variation region.After optimizing the reaction conditions of the detection system and detecting the specificity and sensitivity of the detection method,the gene chip detection method which could simultaneously distinguish 5 viruses was established.The results showed that the chip was able to simultaneously detect the above five kinds of canine diarrhea viruses.The optimized reaction system in this study was that the annealing temperature of PCR was 55 ℃,the extension time was 1 min 15 s,the hybridization temperature was 40 ℃,and the hybridization time was 2.5 h.The detection limit of 5 virus standard were as follows:CAV-1 0.2 fg/μL,CAV-2 2 fg/μL,CCV 2 fg/μL,CDV 20 pg/μL and CPV 0.02 fg/μL.The specificity results showed that no positive signals were found for the CPIV.The detection of 14 clinical diarrhea samples showed that the positive detection rates of CAV-1,CAV-2,CCV,CDV and CPV by the gene chip method were 28.57%,50.00%,64.28%,14.28% and 85.71%,respectively,and the sensitivity of the microarray assay was 10 to 100 times higher than that of PCR.These results indicated that the developed method in this study was specific and sensitive,and had diagnostic meaning for the clinical mixed infection of canine viruses.
This study was aimed to establish a simple,rapid and efficient method for the diagnosis of canine adenovirus type 1(CAV-1),canine adenovirus type 2(CAV-2),canine coronavirus(CCV),canine distemper virus(CDV) and canine parvovirus(CPV).5 canine diarrhea viruses CAV-1,CAV-2,CCV,CDV and CPV were used as target viruses.The primers were designed in the conserved region according to the viral gene sequences included in NCBI.Then 2 to 3 probes for each virus were designed according to the variation region.After optimizing the reaction conditions of the detection system and detecting the specificity and sensitivity of the detection method,the gene chip detection method which could simultaneously distinguish 5 viruses was established.The results showed that the chip was able to simultaneously detect the above five kinds of canine diarrhea viruses.The optimized reaction system in this study was that the annealing temperature of PCR was 55 ℃,the extension time was 1 min 15 s,the hybridization temperature was 40 ℃,and the hybridization time was 2.5 h.The detection limit of 5 virus standard were as follows:CAV-1 0.2 fg/μL,CAV-2 2 fg/μL,CCV 2 fg/μL,CDV 20 pg/μL and CPV 0.02 fg/μL.The specificity results showed that no positive signals were found for the CPIV.The detection of 14 clinical diarrhea samples showed that the positive detection rates of CAV-1,CAV-2,CCV,CDV and CPV by the gene chip method were 28.57%,50.00%,64.28%,14.28% and 85.71%,respectively,and the sensitivity of the microarray assay was 10 to 100 times higher than that of PCR.These results indicated that the developed method in this study was specific and sensitive,and had diagnostic meaning for the clinical mixed infection of canine viruses.
引文
[1] HU R L,HUANG G,QIU W,et al.Detection and differentiation of CAV-1 and CAV-2 by polymerase chain reaction[J].Veterinary Research Communications,2001,25(1):77-84.
[2] 张洋,袁子国,姜秋杰,等.犬2型腺病毒的分离鉴定及初步特性分析[J].军事医学科学院院刊,2008,32(6):541-544.ZHANG Y,YUAN Z G,JIANG Q J,et al.Isolation,identification and preliminary characteristic analysis of a CAV2[J].Bulletin of the Academy of Military Medical Sciences,2008,32(6):541-544.(in Chinese)
[3] NTAFIS V,MARI V,DECARO N,et al.Canine coronavirus,Greece.Molecular analysis and genetic diversity characterization[J].Infection Genetics and Evolution,2013,16:129-136.
[4] COSTA E M,DE CASTRO T X,BOTTINO FDE O,et al.Molecular characterization of canine coronavirus strains circulating in Brazil[J].Veterinary Microbiology,2014,168(1):8-15.
[5] DWCARO N,CORDONNIER N,DEMETER Z,et al.European surveillance for pantropic canine coronavirus[J].Journal of Clinical Microbiology,2013,51(1):83-88.
[6] DI MARTION B,DI FELICE E,CECI C,et al.Canine kobuviruses in diarrhoeic dogs in Italy[J].Veterinary Microbiology,2013,166(1-2):246-249.
[7] PINTO L D,BARROS I N,BUDASZEWSKI R F,et al.Characterization of pantropic canine coronavirus from Brazil[J].Veterinary Journal,2014,202(3):659-662.
[8] TERADA Y,MATSUI N,NOGUCHI K,et al.Emergence of pathogenic coronaviruses in cats by homologous recombination between feline and canine coronaviruses[J].PLoS One,2014,9(9):e106534.
[9] GAO F S,HU G X,XIA X Z,et al.Isolation and identification of a canine coronavirus strain from giant pandas (Ailuropoda melanoleuca)[J].Journal of Veterinary Science,2009,10(3):261-263.
[10] 张昕,黄坚,张萍,等.成都地区宠物犬感染犬瘟热病毒和犬呼吸道冠状病毒的分子流行病学调查[J].中国畜牧兽医,2018,45(2):486-492.ZHANG X,HUANG J,ZHANG P,et al.Molecular epidemiologic investigation on infections of canine distemper virus and canine respiratory coronavirus in pet dogs in Chengdu area[J].China Animal Husbandry & Veterinary Medicine,2018,45(2):486-492.(in Chinese)
[11] NGUYEN D V,SUZUKI J,MINAMI S,et al.Isolation and phylogenetic analysis of canine distemper virus among domestic dogs in Vietnam[J].Journal of Veterinary Medical Science,2017,79(1):123-127.
[12] LOOTS A K,MITCHELL E,DALTON D L,et al.Advances in canine distemper virus pathogenesis research:A wildlife perspective[J].Journal of General Virology,2017,98(3):311-321.
[13] MELI M L,SIMMLER P,CATTORI V,et al.Importance of canine distemper virus (CDV) infection in free-ranging Iberian lynxes (Lynx pardinus)[J].Veterinary Microbiology,2010,146(1-2):132-137.
[14] 赵屹钦,曾梦颖,郭娟,等.犬细小病毒昆明流行株的分离鉴定与VP2基因序列分析[J].中国畜牧兽医,2019,46(1):206-214.ZHAO Y Q,ZENG M Y,GUO J,et al.Isolation and identification of Kunming popular strain of canine parvovirus and sequence analysis of VP2 gene[J].China Animal Husbandry & Veterinary Medicine,2019,46(1):206-214.(in Chinese)
[15] ZHANG R,YANG S,ZHANG W,et al.Phylogenetic analysis of the VP2 gene of canine parvovirus circulating in China[J].Virus Genes,2010,40(3):397-402.
[16] 庄向婷,白军,张振仓.新型分子诊断技术在动物疫病检测中的应用与发展[J].畜牧与饲料科学,2018,39(6):108-112.ZHUANG X T,BAI J,ZHANG Z C.Application and development of new molecular diagnostic techniques in animal epidemics detection[J].Animal Husbandry and Feed Science,2018,39(6):108-112.(in Chinese)
[17] 俞向前,艾旻,张忠海,等.犬细小病毒与犬冠状病毒双重PCR检测方法的建立[J].上海畜牧兽医通讯,2017,4:10-12.YU X Q,AI M,ZHANG Z H,et al.Establishment of double PCR detection method for canine parvovirus and canine coronavirus[J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2017,4:10-12.(in Chinese)
[18] BRAY E A.Classification of genes differentially expressed during water-deficit stress in Arabidopsis thaliana:An analysis using microarray and differential expression data[J].Annals of Botany,2002,89:803-811.
[19] BIDARD F,IMBEAUD S,REYMOND N,et al.A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina[J].BMC Research Notes,2010,3:171.
[20] CHOU C C,CHEN C H,LEE T T,et al.Optimization of probe length and the number of probes per gene for optimal microarray analysis of gene expression[J].Nucleic Acids Research,2004,32(12):e99.
[21] DOH E J,OH S E.Multiplex PCR method to discriminate Artemisia iwayomogi from other Artemisia plants[J].Methods in Molecular Biology,2012,862:149-160.
[22] 杨国淋,马锐,赵玉佳,等.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[J].农业生物技术学报,2018,26(3):530-539.YANG G L,MA R,ZHAO Y J,et al.The optimization and preliminary application of visual microarray for detecting four respiratory disease of chicken (Callus gallus)[J].Journal of Agricultural Biotechnology,2018,26(3):530-539.(in Chinese)
[23] 苏霞,朱瑞豪,陈小玲,等.鸡传染性贫血病毒、网状内皮增生症病毒与禽白血病病毒基因芯片检测方法的建立[J].华北农学报,2015,30(6):91-96.SU X,ZHU R H,CHEN X L,et al.The establishment of gene chip for chicken infectious anemia virus,reticuloeotheliosis virus and avian leucosis virus[J].Acta Agriculturae Boreali-Sinica,2015,30(6):91-96.
[24] 张悦,徐福洲,陈小玲,等.禽白血病病毒B、E和J亚群基因芯片检测方法的建立[J].中国动物检疫,2012,29(6):37-42.ZHANG Y,XU F Z,CHEN X L,et al.Development of a gene chip assay for the detection of avian leucosis virus subgroups B,E and J[J].China Animal Health Inspection,2012,29(6):37-42.(in Chinese)
[25] 赵雪丽,刘毅,班付国,等.犬瘟热病毒、犬细小病毒二重TaqMan MGB探针实时荧光定量PCR检测方法的建立与应用[J].中国畜牧兽医,2018,45(8):2086-2094.ZHAO X L,LIU Y,BAN F G,et al.Establishment and application of a double TaqMan MGB Real-time PCR assay for detection of canine distemper virus and canine parvovirus[J].China Animal Husbandry & Veterinary Medicine,2018,45(8):2086-2094.(in Chinese)
[26] 苏霞,周宏专,常彦嫣,等.犬瘟热病毒、犬细小病毒和犬冠状病毒三重PCR检测方法的建立[J].畜牧与兽医,2018,50(7):103-107.SU X,ZHOU H Z,CHANG Y Y,et al.Establishment of a multiplex PCR for simultaneous detection of CDV,CPV and CCV[J].Animal Husbandry & Veterinary Medicine,2018,50(7):103-107.(in Chinese)
[27] 刘大飞,姜一曈,戚亭,等.同时检测犬瘟热病毒、犬细小病毒、Ⅰ型和Ⅱ型犬腺病毒多重PCR方法的建立[J].中国预防兽医学报,2012,34(11):911-914.LIU D F,JIANG Y T,QI T,et al.Establishment of the multiplex PCR to the detection of CDV,CPV,CAV-Ⅰ and CAV-Ⅱ[J].Chinese Journal of Preventive Veterinary Medicine,2012,34(11):911-914.(in Chinese)
[2] 张洋,袁子国,姜秋杰,等.犬2型腺病毒的分离鉴定及初步特性分析[J].军事医学科学院院刊,2008,32(6):541-544.ZHANG Y,YUAN Z G,JIANG Q J,et al.Isolation,identification and preliminary characteristic analysis of a CAV2[J].Bulletin of the Academy of Military Medical Sciences,2008,32(6):541-544.(in Chinese)
[3] NTAFIS V,MARI V,DECARO N,et al.Canine coronavirus,Greece.Molecular analysis and genetic diversity characterization[J].Infection Genetics and Evolution,2013,16:129-136.
[4] COSTA E M,DE CASTRO T X,BOTTINO FDE O,et al.Molecular characterization of canine coronavirus strains circulating in Brazil[J].Veterinary Microbiology,2014,168(1):8-15.
[5] DWCARO N,CORDONNIER N,DEMETER Z,et al.European surveillance for pantropic canine coronavirus[J].Journal of Clinical Microbiology,2013,51(1):83-88.
[6] DI MARTION B,DI FELICE E,CECI C,et al.Canine kobuviruses in diarrhoeic dogs in Italy[J].Veterinary Microbiology,2013,166(1-2):246-249.
[7] PINTO L D,BARROS I N,BUDASZEWSKI R F,et al.Characterization of pantropic canine coronavirus from Brazil[J].Veterinary Journal,2014,202(3):659-662.
[8] TERADA Y,MATSUI N,NOGUCHI K,et al.Emergence of pathogenic coronaviruses in cats by homologous recombination between feline and canine coronaviruses[J].PLoS One,2014,9(9):e106534.
[9] GAO F S,HU G X,XIA X Z,et al.Isolation and identification of a canine coronavirus strain from giant pandas (Ailuropoda melanoleuca)[J].Journal of Veterinary Science,2009,10(3):261-263.
[10] 张昕,黄坚,张萍,等.成都地区宠物犬感染犬瘟热病毒和犬呼吸道冠状病毒的分子流行病学调查[J].中国畜牧兽医,2018,45(2):486-492.ZHANG X,HUANG J,ZHANG P,et al.Molecular epidemiologic investigation on infections of canine distemper virus and canine respiratory coronavirus in pet dogs in Chengdu area[J].China Animal Husbandry & Veterinary Medicine,2018,45(2):486-492.(in Chinese)
[11] NGUYEN D V,SUZUKI J,MINAMI S,et al.Isolation and phylogenetic analysis of canine distemper virus among domestic dogs in Vietnam[J].Journal of Veterinary Medical Science,2017,79(1):123-127.
[12] LOOTS A K,MITCHELL E,DALTON D L,et al.Advances in canine distemper virus pathogenesis research:A wildlife perspective[J].Journal of General Virology,2017,98(3):311-321.
[13] MELI M L,SIMMLER P,CATTORI V,et al.Importance of canine distemper virus (CDV) infection in free-ranging Iberian lynxes (Lynx pardinus)[J].Veterinary Microbiology,2010,146(1-2):132-137.
[14] 赵屹钦,曾梦颖,郭娟,等.犬细小病毒昆明流行株的分离鉴定与VP2基因序列分析[J].中国畜牧兽医,2019,46(1):206-214.ZHAO Y Q,ZENG M Y,GUO J,et al.Isolation and identification of Kunming popular strain of canine parvovirus and sequence analysis of VP2 gene[J].China Animal Husbandry & Veterinary Medicine,2019,46(1):206-214.(in Chinese)
[15] ZHANG R,YANG S,ZHANG W,et al.Phylogenetic analysis of the VP2 gene of canine parvovirus circulating in China[J].Virus Genes,2010,40(3):397-402.
[16] 庄向婷,白军,张振仓.新型分子诊断技术在动物疫病检测中的应用与发展[J].畜牧与饲料科学,2018,39(6):108-112.ZHUANG X T,BAI J,ZHANG Z C.Application and development of new molecular diagnostic techniques in animal epidemics detection[J].Animal Husbandry and Feed Science,2018,39(6):108-112.(in Chinese)
[17] 俞向前,艾旻,张忠海,等.犬细小病毒与犬冠状病毒双重PCR检测方法的建立[J].上海畜牧兽医通讯,2017,4:10-12.YU X Q,AI M,ZHANG Z H,et al.Establishment of double PCR detection method for canine parvovirus and canine coronavirus[J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2017,4:10-12.(in Chinese)
[18] BRAY E A.Classification of genes differentially expressed during water-deficit stress in Arabidopsis thaliana:An analysis using microarray and differential expression data[J].Annals of Botany,2002,89:803-811.
[19] BIDARD F,IMBEAUD S,REYMOND N,et al.A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina[J].BMC Research Notes,2010,3:171.
[20] CHOU C C,CHEN C H,LEE T T,et al.Optimization of probe length and the number of probes per gene for optimal microarray analysis of gene expression[J].Nucleic Acids Research,2004,32(12):e99.
[21] DOH E J,OH S E.Multiplex PCR method to discriminate Artemisia iwayomogi from other Artemisia plants[J].Methods in Molecular Biology,2012,862:149-160.
[22] 杨国淋,马锐,赵玉佳,等.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[J].农业生物技术学报,2018,26(3):530-539.YANG G L,MA R,ZHAO Y J,et al.The optimization and preliminary application of visual microarray for detecting four respiratory disease of chicken (Callus gallus)[J].Journal of Agricultural Biotechnology,2018,26(3):530-539.(in Chinese)
[23] 苏霞,朱瑞豪,陈小玲,等.鸡传染性贫血病毒、网状内皮增生症病毒与禽白血病病毒基因芯片检测方法的建立[J].华北农学报,2015,30(6):91-96.SU X,ZHU R H,CHEN X L,et al.The establishment of gene chip for chicken infectious anemia virus,reticuloeotheliosis virus and avian leucosis virus[J].Acta Agriculturae Boreali-Sinica,2015,30(6):91-96.
[24] 张悦,徐福洲,陈小玲,等.禽白血病病毒B、E和J亚群基因芯片检测方法的建立[J].中国动物检疫,2012,29(6):37-42.ZHANG Y,XU F Z,CHEN X L,et al.Development of a gene chip assay for the detection of avian leucosis virus subgroups B,E and J[J].China Animal Health Inspection,2012,29(6):37-42.(in Chinese)
[25] 赵雪丽,刘毅,班付国,等.犬瘟热病毒、犬细小病毒二重TaqMan MGB探针实时荧光定量PCR检测方法的建立与应用[J].中国畜牧兽医,2018,45(8):2086-2094.ZHAO X L,LIU Y,BAN F G,et al.Establishment and application of a double TaqMan MGB Real-time PCR assay for detection of canine distemper virus and canine parvovirus[J].China Animal Husbandry & Veterinary Medicine,2018,45(8):2086-2094.(in Chinese)
[26] 苏霞,周宏专,常彦嫣,等.犬瘟热病毒、犬细小病毒和犬冠状病毒三重PCR检测方法的建立[J].畜牧与兽医,2018,50(7):103-107.SU X,ZHOU H Z,CHANG Y Y,et al.Establishment of a multiplex PCR for simultaneous detection of CDV,CPV and CCV[J].Animal Husbandry & Veterinary Medicine,2018,50(7):103-107.(in Chinese)
[27] 刘大飞,姜一曈,戚亭,等.同时检测犬瘟热病毒、犬细小病毒、Ⅰ型和Ⅱ型犬腺病毒多重PCR方法的建立[J].中国预防兽医学报,2012,34(11):911-914.LIU D F,JIANG Y T,QI T,et al.Establishment of the multiplex PCR to the detection of CDV,CPV,CAV-Ⅰ and CAV-Ⅱ[J].Chinese Journal of Preventive Veterinary Medicine,2012,34(11):911-914.(in Chinese)
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