牛巴贝斯虫巢式PCR检测方法的建立
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摘要
为建立一种适用于基层兽医实验室特异、敏感、快速检测牛巴贝斯虫感染的方法,以牛巴贝斯虫球状体蛋白4(SBP4)基因为靶基因设计引物,建立检测牛巴贝斯虫的巢式PCR方法。结果表明,该方法可特异性地检测牛巴贝斯虫感染,与14种动物梨形虫无交叉反应。该方法能够检测到1 copy的SBP4基因,其敏感性分别是对应的内、外引物普通PCR的10倍和1 000倍。应用该巢式PCR方法,对田间随机采集的136份野外样品进行检测,检出阳性样品9份,显著高于AbouLaila等报道的方法(5份)。这些数据表明,建立的巢式PCR方法可用于基层兽医实验室敏感、准确地检测牛巴贝斯虫感染。
To establish a specific,sensitive and rapid detection method for Babesia bovis that is easy-to-use in practice in local veterinary laboratories,a nested PCR was established using the primers designed targeting SBP4 gene of spherical body protein(SBP) of B.bovis.As a result,this method was specific for B.bovis and no cross-reaction was observed with 14 related piroplasm samples.The analytic sensitivity of the method showed to detect as few as 1 copy of SBP4 gene.When compared with the conventional PCR with both outer primers and inner primers of the nested PCR,the sensitivity increased 1 000 times and 10 times,respectively.Using the developed method,136 field samples were detected,and it gave much higher positives(9 samples) than the method of Abou Laila M et al.(5 samples).In conclusion,the established nested PCR can be implied in practice in local veterinary laboratories due to its satisfactory sensitivity and specificity for the detection of B.bovis.
To establish a specific,sensitive and rapid detection method for Babesia bovis that is easy-to-use in practice in local veterinary laboratories,a nested PCR was established using the primers designed targeting SBP4 gene of spherical body protein(SBP) of B.bovis.As a result,this method was specific for B.bovis and no cross-reaction was observed with 14 related piroplasm samples.The analytic sensitivity of the method showed to detect as few as 1 copy of SBP4 gene.When compared with the conventional PCR with both outer primers and inner primers of the nested PCR,the sensitivity increased 1 000 times and 10 times,respectively.Using the developed method,136 field samples were detected,and it gave much higher positives(9 samples) than the method of Abou Laila M et al.(5 samples).In conclusion,the established nested PCR can be implied in practice in local veterinary laboratories due to its satisfactory sensitivity and specificity for the detection of B.bovis.
引文
[1] LEWIS R L.Blood transfusion in a case of bovine piroplasmosis[J].Veterinary Record,1946,58(48):568.
[2] YIN H,LUW S,LUO J X.Babesiosis in China[J].Tropical Animal Health and Production,1997,29(4):11S-15S.
[3] LUO J X,YIN H,GUAN G Q,et al.Description of a new Babesia sp.infective for cattle in China[J].Parasitology Research,2002,88(13):13-15.
[4]刘钟灵,赵俊龙,马丽华,等.寄生于中国水牛Bubalus bubalis的巴贝斯虫一新种(梨形虫目:巴贝斯科)[J].畜牧兽医学报,1997,28(1):84-89.LIU Zhong-ling,ZHAO Jun-long,MA Li-hua,et al.Babesia orientalis sp.nov.parasitized in buffalo Bubalus bubalis in China(Piroplasmida:Babesiidae)[J].Acta Veterinaria et Zootechnica Sinica,1997,28(1):84-89.(in Chinese)
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[6] MORZARIA S,KATENDE J,KAIRO A,et al.New methods for the diagnosis of Babesia bigemina infection[J].Memórias do Instituto Oswaldo Cruz,1992,87(Suppl 3):201-205.
[7] DUH D,JELOVSEK M,AVSIC-ZUPANC T.Evaluation of an indirect fluorescence immunoassay for the detection of serum antibodies against Babesia divergens in humans[J].Parasitology,2007,134(2):179-185.
[8] GUAN G Q,CHAUVIN A,ROGNIAUX H,et al.Merozoite proteins from Babesia sp.BQ1(Lintan)as potential antigens for serodiagnosis by ELISA[J]. Parasitology,2010,137(6):927-938.
[9] FAHRIMAL Y,GOFF W L,JASMER D P.Detection of Babesia bovis carrier cattle by using polymerase chain reaction amplification of parasite DNA[J].Journal of Clinical Microbiology,1992,30(6):1374-1379.
[10] LIU J L,YIH H,LIU G Y,et al.Discrimination of Babesia major and Babesia ovata based on ITS1-5.8S-ITS2 region sequences of rRNA gene[J].Parasitology Research,2008,102(4):709-713.
[11] MTSHALI M S,MTSHALI P S.Molecular diagnosis and phylogenetic analysis of Babesia bigemina and Babesia bovis hemoparasites from cattle in South Africa[J].BMC Veterinary Research,2013,9:154.
[12] ABOULAILA M,YOKOYAMA N,IGARASHI I.Development and evaluation of two nested PCR assays for the detection of Babesia bovis from cattle blood[J].Veterinary Parasitology,2010,172(1-2):65-70.
[13] LIU A H,GUAN G Q,DU P F,et al.Loop-mediated isothermal amplification(LAMP)method based on two species-specific primer sets for the rapid identification of Chinese Babesia bovis and B.bigemina[J].Parasitology International,2012,61(4):658-663.
[14] BULING A, CRIADO-FORNELIO A, ASENZO G, et al.A quantitative PCR assay for the detection and quantification of Babesia bovis and B.bigemina[J].VeterinaryParasitology,2007,147(1-2):16-25.
[15] QUROLLO B A,ARCHER N R,SCHREEG M E,et al.Improved molecular detection of Babesia infections in animals using a novel quantitative real-time PCR diagnostic assay targeting mitochondrial DNA[J].Parasites&Vectors,2017,10(1):128.
[16]刘启生,王振宝,王真,等.牛巴贝斯虫实时荧光PCR检测方法的建立[J].中国预防兽医学报,2013,35(1):58-61.LIU Qi-sheng,WANG Zhen-bao,WANG Zhen,et al.Development of real-time PCR assay for detection of Babesia bovis[J].Chinese Journal of Preventive Veterinary Medicine,2013,35(1):58-61.(in Chinese)
[17] GOO Y K,JIA H,ABOGE G O,et al.Babesia gibsoni:serodiagnosis of infection in dogs by an enzyme-linked immunosorbent assay with recombinant BgTRAP[J].Experimental Parasitology,2008,118(4):555-560.
[18] TERKAWI M A,HUYEN N X,WIBOWO P E,et al.Spherical body protein 4 is a new serological antigen for global detection of Babesia bovis infection in cattle[J].Clinical and Vaccine Immunology,2011,18(2):337-342.
[19]武鑫,张宇霞,史晗,等.致病性沙门菌SYBR GreenⅠ实时荧光定量PCR检测方法的建立[J].中国兽医科学,2015,45(3):270-274.WU Xin,ZHANG Yu-xia,SHI Han,et al.Development of SYBR GreenⅠreal-time fluorescent quantitative PCR for detection of pathogenic Salmonella strains[J].Chinese Veterinary Science,2015,45(3):270-274.(in Chinese)
[20] MCLAUGHLIN G L,EDLIND T D,IHLER G M.Detection of Babesia bovis using DNA hybridization[J].The Journal of Protozoology,1986,33(1):125-128.
[21] DE VRIES E,CORTON C,HARRIS B,et al.Expressed sequence tag(EST)analysis of the erythrocytic stages of Babesia bovis[J].Veterinary Parasitology,2006,138(1-2):61-74.
[22] TERKAWI M A,SEUSEU F J,EKO-WIBOWO P,et al.Secretion of a new spherical body protein of Babesia bovis into the cytoplasm of infected erythrocytes[J].Molecular and Biochemical Parasitology,2011,178(1-2):40-45.
[2] YIN H,LUW S,LUO J X.Babesiosis in China[J].Tropical Animal Health and Production,1997,29(4):11S-15S.
[3] LUO J X,YIN H,GUAN G Q,et al.Description of a new Babesia sp.infective for cattle in China[J].Parasitology Research,2002,88(13):13-15.
[4]刘钟灵,赵俊龙,马丽华,等.寄生于中国水牛Bubalus bubalis的巴贝斯虫一新种(梨形虫目:巴贝斯科)[J].畜牧兽医学报,1997,28(1):84-89.LIU Zhong-ling,ZHAO Jun-long,MA Li-hua,et al.Babesia orientalis sp.nov.parasitized in buffalo Bubalus bubalis in China(Piroplasmida:Babesiidae)[J].Acta Veterinaria et Zootechnica Sinica,1997,28(1):84-89.(in Chinese)
[5] KIM C M,BLANCO L B,ALHASSAN A,et al.Development of a rapid immunochromatographic test for simultaneous serodiagnosis of bovine babesioses caused by Babesia bovis and Babesia bigemina[J].American Journal of Tropical Medicine and Hygiene,2008,78(1):117-121.
[6] MORZARIA S,KATENDE J,KAIRO A,et al.New methods for the diagnosis of Babesia bigemina infection[J].Memórias do Instituto Oswaldo Cruz,1992,87(Suppl 3):201-205.
[7] DUH D,JELOVSEK M,AVSIC-ZUPANC T.Evaluation of an indirect fluorescence immunoassay for the detection of serum antibodies against Babesia divergens in humans[J].Parasitology,2007,134(2):179-185.
[8] GUAN G Q,CHAUVIN A,ROGNIAUX H,et al.Merozoite proteins from Babesia sp.BQ1(Lintan)as potential antigens for serodiagnosis by ELISA[J]. Parasitology,2010,137(6):927-938.
[9] FAHRIMAL Y,GOFF W L,JASMER D P.Detection of Babesia bovis carrier cattle by using polymerase chain reaction amplification of parasite DNA[J].Journal of Clinical Microbiology,1992,30(6):1374-1379.
[10] LIU J L,YIH H,LIU G Y,et al.Discrimination of Babesia major and Babesia ovata based on ITS1-5.8S-ITS2 region sequences of rRNA gene[J].Parasitology Research,2008,102(4):709-713.
[11] MTSHALI M S,MTSHALI P S.Molecular diagnosis and phylogenetic analysis of Babesia bigemina and Babesia bovis hemoparasites from cattle in South Africa[J].BMC Veterinary Research,2013,9:154.
[12] ABOULAILA M,YOKOYAMA N,IGARASHI I.Development and evaluation of two nested PCR assays for the detection of Babesia bovis from cattle blood[J].Veterinary Parasitology,2010,172(1-2):65-70.
[13] LIU A H,GUAN G Q,DU P F,et al.Loop-mediated isothermal amplification(LAMP)method based on two species-specific primer sets for the rapid identification of Chinese Babesia bovis and B.bigemina[J].Parasitology International,2012,61(4):658-663.
[14] BULING A, CRIADO-FORNELIO A, ASENZO G, et al.A quantitative PCR assay for the detection and quantification of Babesia bovis and B.bigemina[J].VeterinaryParasitology,2007,147(1-2):16-25.
[15] QUROLLO B A,ARCHER N R,SCHREEG M E,et al.Improved molecular detection of Babesia infections in animals using a novel quantitative real-time PCR diagnostic assay targeting mitochondrial DNA[J].Parasites&Vectors,2017,10(1):128.
[16]刘启生,王振宝,王真,等.牛巴贝斯虫实时荧光PCR检测方法的建立[J].中国预防兽医学报,2013,35(1):58-61.LIU Qi-sheng,WANG Zhen-bao,WANG Zhen,et al.Development of real-time PCR assay for detection of Babesia bovis[J].Chinese Journal of Preventive Veterinary Medicine,2013,35(1):58-61.(in Chinese)
[17] GOO Y K,JIA H,ABOGE G O,et al.Babesia gibsoni:serodiagnosis of infection in dogs by an enzyme-linked immunosorbent assay with recombinant BgTRAP[J].Experimental Parasitology,2008,118(4):555-560.
[18] TERKAWI M A,HUYEN N X,WIBOWO P E,et al.Spherical body protein 4 is a new serological antigen for global detection of Babesia bovis infection in cattle[J].Clinical and Vaccine Immunology,2011,18(2):337-342.
[19]武鑫,张宇霞,史晗,等.致病性沙门菌SYBR GreenⅠ实时荧光定量PCR检测方法的建立[J].中国兽医科学,2015,45(3):270-274.WU Xin,ZHANG Yu-xia,SHI Han,et al.Development of SYBR GreenⅠreal-time fluorescent quantitative PCR for detection of pathogenic Salmonella strains[J].Chinese Veterinary Science,2015,45(3):270-274.(in Chinese)
[20] MCLAUGHLIN G L,EDLIND T D,IHLER G M.Detection of Babesia bovis using DNA hybridization[J].The Journal of Protozoology,1986,33(1):125-128.
[21] DE VRIES E,CORTON C,HARRIS B,et al.Expressed sequence tag(EST)analysis of the erythrocytic stages of Babesia bovis[J].Veterinary Parasitology,2006,138(1-2):61-74.
[22] TERKAWI M A,SEUSEU F J,EKO-WIBOWO P,et al.Secretion of a new spherical body protein of Babesia bovis into the cytoplasm of infected erythrocytes[J].Molecular and Biochemical Parasitology,2011,178(1-2):40-45.