上转发光免疫层析技术快速检测猪抗旋毛虫IgG抗体方法的建立
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摘要
基于上转发光免疫层析(UPT-LF)技术,旨在建立UPT-LF快速检测猪抗旋毛虫IgG抗体的方法。采用间接法制备UPT-LF试纸条,通过共价偶联使二抗山羊抗猪IgG和上转发光纳米颗粒(UCP)结合,并加入样品垫和被检血清结合,以旋毛虫肌幼虫丝氨酸蛋白酶抑制因子基因(WM5)表达的蛋白WM5为猪抗旋毛虫IgG特异性结合抗原,WM5(2 g/L)与兔抗山羊IgG(0.5 g/L)喷点于分析膜上作为UPT-LF试纸条检测带(T)与质控带(C),该试纸条命名为Tsp-UPT-LF。Tsp-UPT-LF通过对95份阴性血清检测,确定Tsp-UPT-LF的Cutoff值为0.099。Tsp-UPT-LF和ELISA分别对273份旋毛虫感染猪血清样本进行检测,总符合率为92.31%,一致性系数Kappa(K)值为0.837,表明两种方法具有高度一致性。Tsp-UPT-LF检测旋毛虫、猪囊虫、华支睾吸虫感染的猪血清Tsp-UPT-LF呈现对旋毛虫良好的特异性。Tsp-UPT-LF检测感染50 000条旋毛虫肌幼虫28 d猪血清为阳性,T/C值0.109,122 d阳性最强,T/C值为0.207,至425 d时阳性值有所下降,抗旋毛虫IgG阳性率为71.4%。本研究建立了基于UPT-LF技术快速检测猪抗旋毛虫IgG抗体的方法,为猪旋毛虫即时感染检测、确保食品安全提供了一种可参考的检测方法。
The study is to develop an up-converting phosphor-lateral flow(UPT-LF) assay for rapid detection of anti-Trichinella IgG antibodies on site.The UPT-LF strip was prepared by indirect method.The second antibodies of the goat anti-pig IgG was covalent coupled with up-converting phosphor(UCP) particles and then bound with serum samples in sample pad.The protein WM5 expressed by the serine protease inhibitor gene(WM5) of Trichinella spiralis(T.spiralis)larvae was used as binding antigen in the strip.WM5(2 g/L) and rabbit anti-goat IgG(0.5 g/L) were fixed on the test line(T) and control line(C) of the nitrocellulose membrane,respectively.The test strip was named as Tsp-UPT-LF.The cutoff value of Tsp-UPT-LF was defined as 0.099 by detection of 95 negative serum samples.Tsp-UPT-LF also showed excellent specificity in detection of pig serum infected with T.spiralis,Cysticercus or Clonorchis sinensis.273 pig serum samples were detected by Tsp-UPT-LF and ELISA,respectively.The total consistent rate of TspUPT-LF and ELISA was 92.31%,while Kappa value is 0.837,with the excellent consistency.TspUPT-LF positive result was 0.109 T/C for pig serum infected with 50 000 T.spiralis on the day28,while the max detection value was 0.207 T/C on the day 122.T/C descended on the day 425.The rate of positive results was 71.4%for anti-TrichinellaIgG of pig serum infected with 50 000 T.spiralis.The established UPT-LF assay for rapid detection of anti-Trichinella IgG antibody in swine,provides a reference method for the point-of-care testing(POCT)of T.spiralis and food safety.
The study is to develop an up-converting phosphor-lateral flow(UPT-LF) assay for rapid detection of anti-Trichinella IgG antibodies on site.The UPT-LF strip was prepared by indirect method.The second antibodies of the goat anti-pig IgG was covalent coupled with up-converting phosphor(UCP) particles and then bound with serum samples in sample pad.The protein WM5 expressed by the serine protease inhibitor gene(WM5) of Trichinella spiralis(T.spiralis)larvae was used as binding antigen in the strip.WM5(2 g/L) and rabbit anti-goat IgG(0.5 g/L) were fixed on the test line(T) and control line(C) of the nitrocellulose membrane,respectively.The test strip was named as Tsp-UPT-LF.The cutoff value of Tsp-UPT-LF was defined as 0.099 by detection of 95 negative serum samples.Tsp-UPT-LF also showed excellent specificity in detection of pig serum infected with T.spiralis,Cysticercus or Clonorchis sinensis.273 pig serum samples were detected by Tsp-UPT-LF and ELISA,respectively.The total consistent rate of TspUPT-LF and ELISA was 92.31%,while Kappa value is 0.837,with the excellent consistency.TspUPT-LF positive result was 0.109 T/C for pig serum infected with 50 000 T.spiralis on the day28,while the max detection value was 0.207 T/C on the day 122.T/C descended on the day 425.The rate of positive results was 71.4%for anti-TrichinellaIgG of pig serum infected with 50 000 T.spiralis.The established UPT-LF assay for rapid detection of anti-Trichinella IgG antibody in swine,provides a reference method for the point-of-care testing(POCT)of T.spiralis and food safety.
引文
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[16] CORSTJENS P L,HOEKSTRA P T,CLAUDIA J,et al.Utilizing the ultrasensitive Schistosoma up-converting phosphor lateral flow circulating anodic antigen (UCP-LF CAA) assay for sample pooling-strategies[J].Infect Dis Poverty,2017,6(1):155.
[17] CORSTJENS P L,VAN LIESHOUT,ZUIDERWIJK M,et al.Up-converting phosphor technology-based lateral flow assay for detection of Schistosoma circulating anodic antigen in serum[J].J Clin Microbiol,2008,46(1):171-176.
[18] WU X P,FU B Q,WANG X L,et al.Identification of antigenic genes in Trichinella spiralis by immunoscreening of cDNA libraries[J].Vet Parasitol,2009,159(3/4):272-275.
[19] 夏慧卿.旋毛虫肌幼虫WM5抗原基因的克隆、表达及鉴定[D].吉林长春:吉林大学,2007.
[20] WILHELM S,HIRSCH T,PATTERSON W M,et al.Multicolor upconversion nanoparticles for protein conjugation[J].Theranostics,2013,3(4):239-248.
[21] OUELLETTE A L,LI J J,COOPER D E,et al.Evolving point-of-care diagnostics using up-converting phosphor bioanalytical systems[J].Anal Chem,2009,81(9):3216-3221.
[22] 吴秀萍.旋毛虫不同发育时期抗原基因的筛选与鉴定[D].吉林长春:吉林大学,2009.
[23] 徐建余,程道新.旋毛虫感染期幼虫抗原的特异性分析[J].中国寄生虫学与寄生虫病杂志,1998,16(2):149-152.
[24] 赵光辉,张龙现,王选年,等.旋毛虫排泄分泌抗原的成分及应用价值[J].中国病原生物学杂志,2006(5):395-398.
[2] MURRELL K D.Zoonotic foodborne parasites and their surveillance[J].Rev Sci Tech,2013,32(2):559-569.
[3] Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO).Multicriteria-based ranking for risk management of food-borne parasites.microbiological risk assessment series.FAO/WHO,2014,23:302.
[4] 王安琪,丁静,王楠,等.旋毛虫成虫DNaseⅡ-T3223-7基因的克隆及其表达特性鉴定[J].中国兽医学报,2017,37(1):87-91.
[5] BAI X,HU X,LIU X,et al.Current research of trichinellosis in China[J].Front Microbiol,2017( 8):1472.
[6] MAYER-SCHOLl A,POZIO E,GAYDA J,et al.Magnetic stirrer method for the detection of trichinella larvae in muscle samples[J].Jove-J Vis Exp,2017(3):121.
[7] 翟铖铖,陈家旭,陈韶红,等.旋毛虫病诊断方法研究综述[J].中国动物检疫,2017,34(6):68-73.
[8] GOTTSTEIN B E,POZIO K.Epidemiology,diagnosis,treatment,and control of trichinellosis[J].Clin Microbiol Rev,2009,22(1):127-145.
[9] GAMBLE H R,POZIO E,BRUSCHI F,et al.International commission on trichinellosis:recommendations on the use of serological tests for the detection of trichinella infection in animals and man[J].Parasite,2004,11(1):3-13.
[10] GONDEK M,BIE.Use of ELISA and Western blot for serological detection of antibodies to ES antigens of Trichinella spiralis muscle larvae in sera of swine experimentally infected with Trichinella spiralis[J].Vet Immunol Immunopathol,2018,203:13-20.
[11] GóMEZ-MORALES M A,LUDOVISI A,AMATI M,et al.Indirect versus direct detection methods of Trichinella spp.infection in wild boar ( Sus scrofa )[J].Parasite Vector,2014,7(1):171.
[12] ZHANG P P,LIU X,WANG C,et al.Evaluation of up-converting phosphor technology-based lateral flow strips for rapid detection of Bacillus anthracis Spore,Brucella spp and Yersinia pestis[J].PloS One,2014,9(8):e105305.
[13] HUA F,ZHANG P P,ZHANG F,et al.Development and evaluation of an up-converting phosphor technology-based lateral flow assay for rapid detection of Francisella tularensis[J].Sci Rep,2015(5):17178.
[14] LI L,ZHOU L,YU Y,et al.Development of up-converting phosphor technology-based lateral-flow assay for rapidly quantitative detection of hepatitis B surface antibody[J].Diagn Microbiol Infect Dis,2009,63(2):165-172.
[15] CORSTJENS P L,DE DOOD C J,KORNELIS D,et al.Tools for diagnosis,monitoring and screening of Schistosoma infections utilizing lateral-flow based assays and upconverting phosphor labels[J].Parasitology,2014,141(14):1841-1855.
[16] CORSTJENS P L,HOEKSTRA P T,CLAUDIA J,et al.Utilizing the ultrasensitive Schistosoma up-converting phosphor lateral flow circulating anodic antigen (UCP-LF CAA) assay for sample pooling-strategies[J].Infect Dis Poverty,2017,6(1):155.
[17] CORSTJENS P L,VAN LIESHOUT,ZUIDERWIJK M,et al.Up-converting phosphor technology-based lateral flow assay for detection of Schistosoma circulating anodic antigen in serum[J].J Clin Microbiol,2008,46(1):171-176.
[18] WU X P,FU B Q,WANG X L,et al.Identification of antigenic genes in Trichinella spiralis by immunoscreening of cDNA libraries[J].Vet Parasitol,2009,159(3/4):272-275.
[19] 夏慧卿.旋毛虫肌幼虫WM5抗原基因的克隆、表达及鉴定[D].吉林长春:吉林大学,2007.
[20] WILHELM S,HIRSCH T,PATTERSON W M,et al.Multicolor upconversion nanoparticles for protein conjugation[J].Theranostics,2013,3(4):239-248.
[21] OUELLETTE A L,LI J J,COOPER D E,et al.Evolving point-of-care diagnostics using up-converting phosphor bioanalytical systems[J].Anal Chem,2009,81(9):3216-3221.
[22] 吴秀萍.旋毛虫不同发育时期抗原基因的筛选与鉴定[D].吉林长春:吉林大学,2009.
[23] 徐建余,程道新.旋毛虫感染期幼虫抗原的特异性分析[J].中国寄生虫学与寄生虫病杂志,1998,16(2):149-152.
[24] 赵光辉,张龙现,王选年,等.旋毛虫排泄分泌抗原的成分及应用价值[J].中国病原生物学杂志,2006(5):395-398.