巨片形吸虫病快速诊断胶体金免疫层析试条的研制与评价
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摘要
目的建立快速、简便诊断巨片形吸虫病的胶体金免疫层析试条方法。方法提取巨片形吸虫总RNA,通过RT-PCR获得FgCL1-YN基因片段,并克隆入pET28a(+)表达载体,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达得到重组蛋白(r FgCL1-YN);将r FgCL1-YN和羊抗鼠IgG抗体分别包被于硝酸纤维素膜的适宜位置作为检测带和质控带,以胶体金标记抗人IgG4抗体的鼠单抗作为检测试剂,制备检测特异性IgG4型抗体的免疫层析试条。用该试条检测巨片形吸虫病患者(30份)、日本血吸虫病患者(15份)以及健康人(32份)血清,评价其诊断价值,同时用ELISA法进行平行检测作为对照。结果试条检测法的敏感性为100%(30/30),特异性为97.9%(46/47),总体符合率为98.7%(76/77)。ELISA法检测的敏感性、特异性和总体符合率分别100%(30/30)、100%(47/47)和100%(77/77)。试条法与ELISA法的敏感性、特异性和总体符合率,经四格表确切概率法检验,P值分别为1、0.5和0.5,全部大于0.05。显示,试条法与ELISA法检测巨片形吸虫病患者血清的结果高度一致。另外,试条法的检测时间为10min,血清用量低于10μL,且具较高的敏感度。结论以r FgCL1-YN建立的快速诊断胶体金免疫层析试条,检测巨片形吸虫病具有较高的诊断价值。
To establish and evaluate a colloid gold-immunochromatographic assay(GICA)dynamic flow strip for the diagnosis of human fascioliasis,total RNA was prepared from Fascioliasis gigantica(F.gigantica)collected from Dali,Yunnan Province,China.FgCL1-YN gene was obtained by reverse transcription-polymerase chain reaction(RT-PCR).The PCR product was sequenced and cloned into pET28a(+)vector.The recombinant plasmid was expressed and induced by isopropyl-β-Dthiogalactopyranoside(IPTG)to obtain recombinant protein,r FgCL1-YN.The mouse anti-human IgG4 monoclonal antibodies was conjugated with colloid gold as detecting reagent;the r FgCL1-YN and goat anti-mouse IgG antibody were immobilized on nitrocellulose in proper position as test line and control line,separately.The prepared immunochromatographic strip was evaluated using serum samples from fascioliasis gigantica patients(30cases),schistosomiasis japonica patients(15cases)and healthy donors(32cases).Sensitivity detected by the GICA strip test was 100%(30/30).One serum sample from a healthy control was an invalid result with the GICA.Reference sera from schistosomiasis were all negatives for anti- F.giganticaIgG4 antibodies using the GICA.The sensitivity,specificity and total diagnostic coincidence rate of r FgCL1-YNbased the enzyme-linked immunosorbent assay(ELISA)was 100%(30/30),100%(47/47)and 100%(77/77)respectively.Comparison between the GICA and the ELISA was made by exact probabilities in 2×2table analysis.The Pvalue of the total diagnostic coincidence rate,sensitivity and specificity was 1,0.5and 0.5,respectively(P>0.05).High degree of agreement was observed between the GICA and ELISA.The detection time of GICA was 10 min,and the serum concentration was less than 10μL with highly sensitive.The developed immunochromatographic strip test using recombinant FgCL1-YN antigen as coated antigen is a sensitive,simple and rapid assay for diagnosing human fascioliasis.
To establish and evaluate a colloid gold-immunochromatographic assay(GICA)dynamic flow strip for the diagnosis of human fascioliasis,total RNA was prepared from Fascioliasis gigantica(F.gigantica)collected from Dali,Yunnan Province,China.FgCL1-YN gene was obtained by reverse transcription-polymerase chain reaction(RT-PCR).The PCR product was sequenced and cloned into pET28a(+)vector.The recombinant plasmid was expressed and induced by isopropyl-β-Dthiogalactopyranoside(IPTG)to obtain recombinant protein,r FgCL1-YN.The mouse anti-human IgG4 monoclonal antibodies was conjugated with colloid gold as detecting reagent;the r FgCL1-YN and goat anti-mouse IgG antibody were immobilized on nitrocellulose in proper position as test line and control line,separately.The prepared immunochromatographic strip was evaluated using serum samples from fascioliasis gigantica patients(30cases),schistosomiasis japonica patients(15cases)and healthy donors(32cases).Sensitivity detected by the GICA strip test was 100%(30/30).One serum sample from a healthy control was an invalid result with the GICA.Reference sera from schistosomiasis were all negatives for anti- F.giganticaIgG4 antibodies using the GICA.The sensitivity,specificity and total diagnostic coincidence rate of r FgCL1-YNbased the enzyme-linked immunosorbent assay(ELISA)was 100%(30/30),100%(47/47)and 100%(77/77)respectively.Comparison between the GICA and the ELISA was made by exact probabilities in 2×2table analysis.The Pvalue of the total diagnostic coincidence rate,sensitivity and specificity was 1,0.5and 0.5,respectively(P>0.05).High degree of agreement was observed between the GICA and ELISA.The detection time of GICA was 10 min,and the serum concentration was less than 10μL with highly sensitive.The developed immunochromatographic strip test using recombinant FgCL1-YN antigen as coated antigen is a sensitive,simple and rapid assay for diagnosing human fascioliasis.
引文
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[3]Mas-Coma S,Bargues MD,Valero MA,et al.Fascioliasis and other plant-borne trematode zoonoses[J].Int J Parasitol,2005,35:1255-1278.DOI:10.1016/j.ijpara.2005.07.010
[4]Fang W,Li TM,Li KR,et al.Experimental infection of Galba pervia,Radix swinhoei and Physa acuta with Fasciola hepatica in Dali,Yunnan[J].Chin J Parasitol Parasit Dis,2014,32(4):285-288.(in Chinese)方文,李天美,李科荣,等.大理地区肝片形吸虫感染中间宿主实验研究[J].中国寄生虫学与寄生虫病杂志,2014,32(4):285-288.
[5]Chen MX,Ai L,Xu XN,et al.Twenty six cases of human Fasciola gigantica infection in Dali,Yunnan province[J].Chin J Endemiol,2012,31(6):595-598.(in Chinese)陈木新,艾琳,许学年,等.云南省大理州巨片形吸虫群体感染26例分析[J].中国地方病学杂志,2012,31(6):595-598.
[6]Chen JX,Chen MX,Ai L,et al.An outbreak of human fascioliasis gigantic in southwest China[J].PLoS One,2013,8(8):e71520.DOI:10.1371/journal.pone.0071520
[7]Zhang GL,Su HY,Zhou J,et al.Clinical analysis of 11cases of fascioliasis[J].Infect Dis Info,2012,25(4):242-246.(in Chinese)张国丽,苏慧勇,周俊,等.片形吸虫病11例临床分析[J].传染病信息,2012,25(4):242-246.
[8]Yang RJ,Liu YC,Yang XM,et al.Analysis of 18human cases infected with Fasciola gigantica[J].Int J Med Parasit Dis,2012,39(40):246-249.(in Chinese)杨汝军,刘彦才,杨锡铭,等.人体巨片形吸虫病18例分析[J].国际医学寄生虫病杂志,2012,39(40):246-249.
[9]Wang YQ,Zhou Y,Cheng N,et al.Cloning,expression and immunodiagnostic evaluation of the Fasciola gigantica thioredoxin peroxidase[J].Chin J Parasitol Parasit Dis,2015,33(2):81-85.(in Chinese)王月琪,周岩,程娜,等.巨片形吸虫硫氧还蛋白过氧化物酶克隆、表达和免疫学诊断价值评价[J].中国寄生虫学与寄生虫病杂志,2015,33(2):81-85.
[10]Zhou J,Gu W,Jiao JM,et al.First outbreak of Fasciola giganticainfection in Yunnan province:An clinical analysis of10cases[J].World Chin J Dis,2012,20(21):1978-1981.(in Chinese)邹静,顾伟,焦建明,等.云南省首次爆发流行巨片形吸虫感染10例[J].世界华人消化杂志,2012,20(21):1978-1981.
[11]Duthaler U,Rinaldi L,Maurelli MP,et al.Fasciola hepatica:comparison of the sedimentation and FLOTAC techniques for the detection and quantification of faecal egg counts in rats[J].Exp Parasitol,2010,126:161-166.DOI:10.1016/j.exppara.2010.04.020
[12]Hillyer GV.Immunodiagnosis of human and animal fasciolosis[M].In:Dalton JP,ed.Fasciolosis.Wallingford:CABI Publishing,1999:435-447.
[13]Espino AM,Diaz A,Perez A,et al.Dynamics of antigenemia and coproantigens during human Fasciola hepatica outbreak[J].J Clin Microbiol,1998,l36:2723-2726.
[14]Carnevale S,Rodriguez MI,Guarnera EA,et al.Immunodiagnosis of fasciolosis using recombinant procathepsin L cystein proteinase[J].Diagn Microbiol Infect Dis,2001,41:43-49.DOI:10.1016/S0732-8893(01)00288-7
[15]Carnevale S,Rodriguez MI,Santillan G,et al.Immunodiagnosis of human fasciolosis by enzyme-linked immunosorbent assay(ELISA)and a micro-ELISA[J].Clin Diagn Lab Immunol2001,8:174-177.DOI:10.1128/CDLI.8.1.174-177.2001
[16]Intapan PM,Tantrawatpan C,Maleewong W,et al.Potent epitopes derived from Fasciola gigantica cathepsin L1in peptide-based immunoassay for the serodiagnosis of human fascioliasis[J].Diagn Microbiol Infect Dis,2005,53:125-129.DOI:10.1016/j.diagmicrobio.2005.05.010
[17]Mezo M,Gonzalez-Warleta M,Ubeira FM,et al.Optimized serodiagnosis of sheep fascioliasis by Fast-D protein liquid chromatography fractionation of Fasciola hepatica excretory-secretory antigens[J].J Parasitol,2003,89:843-849.
[18]Wongkham C,Tantrawatpan C,Intapan PM,et al.Evaluation of immunoglobulin G subclass antibodies against recombinant Fasciola gigantica cathepsin L1in an enzyme-linked immunosorbent assay for serodiagnosis of human fasciolosis[J].Clin Diagn Lab Immunol,2005,12:1152-1156.DOI:10.1128/CDLI.12.10.1152-1156.2005
[19]O’Neill SM,Parkinson M,Strauss W,et al.Immunodiagnosis of Fasciola hepaticainfection(fascioliasis)in a human population in the Bolivian Altiplano using purified cathepsin L cysteine proteinase[J].Am J Trop Med Hyg,1998,58:417-423.
[20]Raina OK,Yadav SC,Sriveny D,et al.Immuno-diagnosis of bubaline fasciolosis with Fasciola gigantica cathepsin-l and recombinant cathepsin l 1-d proteases[J].Acta Trop,2006,98(2):145-151.DOI:10.1016/j.actatropica.2006.03.004
[21]Frens G.Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions[J].Nat Phys Sci,1973,241(105):20-22.
[22]Hayat MA.Colloidal Gold,Volume 1:Principles,Methods and Applications(Colloidal Gold:Principles,Methods and Application)[M].San Diego:Academic Press,1989:1-39.
[23]Hillyer GV,Sanchez Z,Deleon D,et al.Immunodiagnosis of bovine fascioliasis by enzyme-linked immunosorbent-assay and immunoprecipitation methods[J].J Parasitol,1985,71:449-454.DOI:10.2307/3281536
[24]O’Neil SM,Parkinson M,Dowd AJ,et al.Short report immunodiagnosis of human Fascio liasis using recombinant Fasciola hepatica cathepsin L1cysteine proteinase[J].Am J Trop Med Hyg,1999,60(5):749-751.
[25]Yadav SC,Saini M,Raina OK,et al.Fasciola gigantic cathepsin L cysteine proteinase in the detection of early experimental fasciolosis in ruminants[J].Paraitol Res,2005,97:527-534.DOI:10.1007/s00436-005-1466-8
[26]Chaisiri W,Chairat T,Pewpan MI,et al.Evaluation of immunoglobulin G subclass antibodies against recombinant Fasciola gigantica cathepsin L1in an enzyme-linked immunoso rbent assay for serodiagnosis of human fasciolosis[J].Clin Diagn Lab Immunol,2005,12(10):1152-1156.DOI:10.1128/CDLI.12.10.1152-1156.2005
[27]Victoria MS,Laura M,Mar1aJP,et al.Development and evaluation of a new lateral flow immunoassay for serodiagnosis of human fciolosis[J].PLoS Negl Trop Dis,2011,5(11):e1376.DOI:10.1371/journal.pntd.0001376
[2]Mas-Coma S,Valero MA,Bargues MD,et al.Fasciola lymnaeids and human fascioliasis,with a global overview on disease transmission,epidemiology,evolutionary genetics,molecular epidemiology and control[J].J Adv Parasitol,2009,l69:41-146.DOI:10.1016/S0065-308X(09)69002-3
[3]Mas-Coma S,Bargues MD,Valero MA,et al.Fascioliasis and other plant-borne trematode zoonoses[J].Int J Parasitol,2005,35:1255-1278.DOI:10.1016/j.ijpara.2005.07.010
[4]Fang W,Li TM,Li KR,et al.Experimental infection of Galba pervia,Radix swinhoei and Physa acuta with Fasciola hepatica in Dali,Yunnan[J].Chin J Parasitol Parasit Dis,2014,32(4):285-288.(in Chinese)方文,李天美,李科荣,等.大理地区肝片形吸虫感染中间宿主实验研究[J].中国寄生虫学与寄生虫病杂志,2014,32(4):285-288.
[5]Chen MX,Ai L,Xu XN,et al.Twenty six cases of human Fasciola gigantica infection in Dali,Yunnan province[J].Chin J Endemiol,2012,31(6):595-598.(in Chinese)陈木新,艾琳,许学年,等.云南省大理州巨片形吸虫群体感染26例分析[J].中国地方病学杂志,2012,31(6):595-598.
[6]Chen JX,Chen MX,Ai L,et al.An outbreak of human fascioliasis gigantic in southwest China[J].PLoS One,2013,8(8):e71520.DOI:10.1371/journal.pone.0071520
[7]Zhang GL,Su HY,Zhou J,et al.Clinical analysis of 11cases of fascioliasis[J].Infect Dis Info,2012,25(4):242-246.(in Chinese)张国丽,苏慧勇,周俊,等.片形吸虫病11例临床分析[J].传染病信息,2012,25(4):242-246.
[8]Yang RJ,Liu YC,Yang XM,et al.Analysis of 18human cases infected with Fasciola gigantica[J].Int J Med Parasit Dis,2012,39(40):246-249.(in Chinese)杨汝军,刘彦才,杨锡铭,等.人体巨片形吸虫病18例分析[J].国际医学寄生虫病杂志,2012,39(40):246-249.
[9]Wang YQ,Zhou Y,Cheng N,et al.Cloning,expression and immunodiagnostic evaluation of the Fasciola gigantica thioredoxin peroxidase[J].Chin J Parasitol Parasit Dis,2015,33(2):81-85.(in Chinese)王月琪,周岩,程娜,等.巨片形吸虫硫氧还蛋白过氧化物酶克隆、表达和免疫学诊断价值评价[J].中国寄生虫学与寄生虫病杂志,2015,33(2):81-85.
[10]Zhou J,Gu W,Jiao JM,et al.First outbreak of Fasciola giganticainfection in Yunnan province:An clinical analysis of10cases[J].World Chin J Dis,2012,20(21):1978-1981.(in Chinese)邹静,顾伟,焦建明,等.云南省首次爆发流行巨片形吸虫感染10例[J].世界华人消化杂志,2012,20(21):1978-1981.
[11]Duthaler U,Rinaldi L,Maurelli MP,et al.Fasciola hepatica:comparison of the sedimentation and FLOTAC techniques for the detection and quantification of faecal egg counts in rats[J].Exp Parasitol,2010,126:161-166.DOI:10.1016/j.exppara.2010.04.020
[12]Hillyer GV.Immunodiagnosis of human and animal fasciolosis[M].In:Dalton JP,ed.Fasciolosis.Wallingford:CABI Publishing,1999:435-447.
[13]Espino AM,Diaz A,Perez A,et al.Dynamics of antigenemia and coproantigens during human Fasciola hepatica outbreak[J].J Clin Microbiol,1998,l36:2723-2726.
[14]Carnevale S,Rodriguez MI,Guarnera EA,et al.Immunodiagnosis of fasciolosis using recombinant procathepsin L cystein proteinase[J].Diagn Microbiol Infect Dis,2001,41:43-49.DOI:10.1016/S0732-8893(01)00288-7
[15]Carnevale S,Rodriguez MI,Santillan G,et al.Immunodiagnosis of human fasciolosis by enzyme-linked immunosorbent assay(ELISA)and a micro-ELISA[J].Clin Diagn Lab Immunol2001,8:174-177.DOI:10.1128/CDLI.8.1.174-177.2001
[16]Intapan PM,Tantrawatpan C,Maleewong W,et al.Potent epitopes derived from Fasciola gigantica cathepsin L1in peptide-based immunoassay for the serodiagnosis of human fascioliasis[J].Diagn Microbiol Infect Dis,2005,53:125-129.DOI:10.1016/j.diagmicrobio.2005.05.010
[17]Mezo M,Gonzalez-Warleta M,Ubeira FM,et al.Optimized serodiagnosis of sheep fascioliasis by Fast-D protein liquid chromatography fractionation of Fasciola hepatica excretory-secretory antigens[J].J Parasitol,2003,89:843-849.
[18]Wongkham C,Tantrawatpan C,Intapan PM,et al.Evaluation of immunoglobulin G subclass antibodies against recombinant Fasciola gigantica cathepsin L1in an enzyme-linked immunosorbent assay for serodiagnosis of human fasciolosis[J].Clin Diagn Lab Immunol,2005,12:1152-1156.DOI:10.1128/CDLI.12.10.1152-1156.2005
[19]O’Neill SM,Parkinson M,Strauss W,et al.Immunodiagnosis of Fasciola hepaticainfection(fascioliasis)in a human population in the Bolivian Altiplano using purified cathepsin L cysteine proteinase[J].Am J Trop Med Hyg,1998,58:417-423.
[20]Raina OK,Yadav SC,Sriveny D,et al.Immuno-diagnosis of bubaline fasciolosis with Fasciola gigantica cathepsin-l and recombinant cathepsin l 1-d proteases[J].Acta Trop,2006,98(2):145-151.DOI:10.1016/j.actatropica.2006.03.004
[21]Frens G.Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions[J].Nat Phys Sci,1973,241(105):20-22.
[22]Hayat MA.Colloidal Gold,Volume 1:Principles,Methods and Applications(Colloidal Gold:Principles,Methods and Application)[M].San Diego:Academic Press,1989:1-39.
[23]Hillyer GV,Sanchez Z,Deleon D,et al.Immunodiagnosis of bovine fascioliasis by enzyme-linked immunosorbent-assay and immunoprecipitation methods[J].J Parasitol,1985,71:449-454.DOI:10.2307/3281536
[24]O’Neil SM,Parkinson M,Dowd AJ,et al.Short report immunodiagnosis of human Fascio liasis using recombinant Fasciola hepatica cathepsin L1cysteine proteinase[J].Am J Trop Med Hyg,1999,60(5):749-751.
[25]Yadav SC,Saini M,Raina OK,et al.Fasciola gigantic cathepsin L cysteine proteinase in the detection of early experimental fasciolosis in ruminants[J].Paraitol Res,2005,97:527-534.DOI:10.1007/s00436-005-1466-8
[26]Chaisiri W,Chairat T,Pewpan MI,et al.Evaluation of immunoglobulin G subclass antibodies against recombinant Fasciola gigantica cathepsin L1in an enzyme-linked immunoso rbent assay for serodiagnosis of human fasciolosis[J].Clin Diagn Lab Immunol,2005,12(10):1152-1156.DOI:10.1128/CDLI.12.10.1152-1156.2005
[27]Victoria MS,Laura M,Mar1aJP,et al.Development and evaluation of a new lateral flow immunoassay for serodiagnosis of human fciolosis[J].PLoS Negl Trop Dis,2011,5(11):e1376.DOI:10.1371/journal.pntd.0001376