鸡柔嫩艾美耳球虫和巨型艾美耳球虫双重PCR检测方法的建立
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摘要
为了建立一种能够快速实现对鸡柔嫩艾美耳球虫(Eimeria tenella)和巨型艾美耳球虫(E.maxima)同时进行检测的双重PCR方法,基于2种艾美耳球虫各自的ITS-1基因筛选2对特异性引物,对制备的阳性DNA样品进行PCR扩增,凝胶电泳检测结果显示,分别在约450bp和150bp处出现目的条带。对PCR反应中的退火温度、MgCl2浓度、DNA模板量等进行优化。结果表明,当退火温度为59℃、MgCl2浓度为2.5mmol/L、DNA模板量为2μL时,双重PCR扩增结果最佳。特异性检测结果显示,所建立的双重PCR对环形泰勒虫、羊巴贝斯虫、隐孢子虫和蓝氏贾第虫的扩增结果均呈阴性,表明建立的方法具有较高的特异性。成功建立了柔嫩艾美耳球虫和巨型艾美耳球虫双重PCR检测方法,为临床中鸡球虫病的快速诊断提供了一种可靠的检测方法。
In order to establish a fast duplex PCR method to simultaneously detect Eimeria tenella and E.maximaof chickens,two pairs of specific primers of the ITS-1 gene from two Eimeriaspecies were respectively screened.Two target bands(450 bp and 150 bp)were observed in agarose gel electrophoresis after PCR amplification of positive DNA samples.The duplex PCR system was then optimized with annealing temperature,MgCl2 concentration and the amount of DNA template.The optimal amplification was achieved with conditions of annealing temperature at 59℃,MgCl2 concentration at 2.5 mmol/L,and the amount of DNA template at 2μL.Specificity test showed no amplification of duplex PCR to Theileria annulata,Babesia ovis,Cryptosporidium and Giardia lamblia,indicating high specificity of duplex PCR system.These results indicated that a specific and efficient duplex PCR method was successfully established for detecting E.tenella and E.maxima,providing a reliable method for quickly diagnose clinical chicken coccidiosis.
In order to establish a fast duplex PCR method to simultaneously detect Eimeria tenella and E.maximaof chickens,two pairs of specific primers of the ITS-1 gene from two Eimeriaspecies were respectively screened.Two target bands(450 bp and 150 bp)were observed in agarose gel electrophoresis after PCR amplification of positive DNA samples.The duplex PCR system was then optimized with annealing temperature,MgCl2 concentration and the amount of DNA template.The optimal amplification was achieved with conditions of annealing temperature at 59℃,MgCl2 concentration at 2.5 mmol/L,and the amount of DNA template at 2μL.Specificity test showed no amplification of duplex PCR to Theileria annulata,Babesia ovis,Cryptosporidium and Giardia lamblia,indicating high specificity of duplex PCR system.These results indicated that a specific and efficient duplex PCR method was successfully established for detecting E.tenella and E.maxima,providing a reliable method for quickly diagnose clinical chicken coccidiosis.
引文
[1]索勋,李国清.鸡球虫病学[M].北京:中国农业大学出版社,1998.
[2] Guo A,Cai J,Gong W,et al.Transcriptome analysis in chicken cecal epithelia upon infection by Eimeria tenella in vivo[J].PLoS One,2013,8(5):e64236.
[3]张龙现,宁长申,赵金凤,等.鸡球虫病流行病学概况[J].国外畜牧学:猪与禽,1998(2):36-41.
[4]李武尧.育成期蛋鸡暴发小肠球虫病的诊断与治疗[J].养禽与禽病防治,2012(5):21-22.
[5]陆亚琦,刘瑞丽,郝晓宇,等.柔嫩艾美耳球虫细胞培养液对鸡胚盲肠上皮细胞活性的影响[J].畜牧与兽医,2016,48(1):84-86.
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[7]徐奇武.肉鸡慢性球虫病的防治[J].农技服务,2012,29(6):735.
[8]杨晓伟,蒙晓雷,何志霞.鸡球虫病与综合防治[J].畜牧与饲料科学,2009,30(10):80-82.
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[10] Cha J O,Zhao J,Yang M S,et al.Oocyst-shedding patterns of three Eimeriaspecies in chickens and chedding pattern variation depending on the storage period of Eimeria tenella oocysts[J].J Parasitol,2018,104(1):18-22.
[11]王鑫,韩红玉,黄兵,等.特异性引物PCR鉴定鸡球虫种类与卵囊纯度的研究[J].寄生虫与医学昆虫学报,2010,17(4):203-207.
[12]刘群,蒋金书.不同种、株鸡球虫DNA多态性的比较研究[J].畜牧兽医学报,2000,31(4):360-365.
[13]丛培君.鸡堆型艾美尔球虫乳酸脱氢酶增强型核酸疫苗构建和免疫保护研究[D].黑龙江哈尔滨:东北农业大学,2014.
[14] Williams R B.Quantification of the crowding effect during infections with the seven Eimeriaspecies of the domesticated fowl:its importance for experimental designs and the production of oocyst stocks[J].Int J Parasitol,2001,31(10):1056-1069.
[15] Carvalho F S,Wenceslau A A,Teixeira M,et al.Diagnosis of Eimeria species using traditional and molecular methods in field studies[J].Vet Parasitol,2011,176(2-3):95-100.
[16]侯瑞生,王丽,张勤,等.多重PCR技术及其在病原体检测中的应用[J].中华全科医学,2011,9(8):1288-1290.
[17]邓雯,马彦博,刘玉梅.鸡球虫对机体的致病作用研究进展[J].家禽生态,2004(2):59-62.
[18]辛玲,许金俊,陶建平.多重PCR检测3种鸡球虫方法的建立[J].中国兽医杂志,2008(3):6-8.
[19]李巍,韩彩霞,李微,等.基于ITS1序列的鸡艾美耳球虫种的套式PCR鉴别方法[J].中国兽医科学,2013,43(3):289-293.
[20]杨霞,卢中华,张红英,等.PCR技术在畜禽疾病诊断中的应用[J].河南农业大学学报,2001(S1):77-78.
[21]李文超,陶建平,沈永林,等.PCR及相关技术在鸡球虫种株鉴别方面的应用[J].动物医学进展,2006(9):18-22.
[22] Gadelhaq S M,Arafa W M,Aboelhadid S M.Molecular characterization of Eimeria species naturally infecting egyptian baldi chickens[J].Iran J Parasitol,2015,10(1):87-95.
[23] You M J.Detection of four important Eimeriaspecies by multiplex PCR in a single assay[J].Parasitol Int,2014,63(3):527-532.
[24]薛方民.鸡艾美耳球虫不同种株ITS-1、ITS-2序列的克隆和分析比较[D].山东济南:山东师范大学,2007.
[25]陈明洁,方倜,柯涛,等.多重PCR-一种高效快速的分子生物学技术[J].武汉理工大报,2005(10):37-40.
[26]刘高鹏.多重PCR检测和鉴定五种猪腹泻病毒研究[D].浙江杭州:浙江理工大学,2017.
[2] Guo A,Cai J,Gong W,et al.Transcriptome analysis in chicken cecal epithelia upon infection by Eimeria tenella in vivo[J].PLoS One,2013,8(5):e64236.
[3]张龙现,宁长申,赵金凤,等.鸡球虫病流行病学概况[J].国外畜牧学:猪与禽,1998(2):36-41.
[4]李武尧.育成期蛋鸡暴发小肠球虫病的诊断与治疗[J].养禽与禽病防治,2012(5):21-22.
[5]陆亚琦,刘瑞丽,郝晓宇,等.柔嫩艾美耳球虫细胞培养液对鸡胚盲肠上皮细胞活性的影响[J].畜牧与兽医,2016,48(1):84-86.
[6]李国清.兽医寄生虫学(中英双语)[M].2版.北京:中国农业大学出版社,2015.
[7]徐奇武.肉鸡慢性球虫病的防治[J].农技服务,2012,29(6):735.
[8]杨晓伟,蒙晓雷,何志霞.鸡球虫病与综合防治[J].畜牧与饲料科学,2009,30(10):80-82.
[9] Allen P C,Fetterer R H.Recent advances in biology and immunobiology of Eimeriaspecies and in diagnosis and control of infection with these coccidian parasites of poultry[J].Clin Microbiol Rev,2002,15(1):58-65.
[10] Cha J O,Zhao J,Yang M S,et al.Oocyst-shedding patterns of three Eimeriaspecies in chickens and chedding pattern variation depending on the storage period of Eimeria tenella oocysts[J].J Parasitol,2018,104(1):18-22.
[11]王鑫,韩红玉,黄兵,等.特异性引物PCR鉴定鸡球虫种类与卵囊纯度的研究[J].寄生虫与医学昆虫学报,2010,17(4):203-207.
[12]刘群,蒋金书.不同种、株鸡球虫DNA多态性的比较研究[J].畜牧兽医学报,2000,31(4):360-365.
[13]丛培君.鸡堆型艾美尔球虫乳酸脱氢酶增强型核酸疫苗构建和免疫保护研究[D].黑龙江哈尔滨:东北农业大学,2014.
[14] Williams R B.Quantification of the crowding effect during infections with the seven Eimeriaspecies of the domesticated fowl:its importance for experimental designs and the production of oocyst stocks[J].Int J Parasitol,2001,31(10):1056-1069.
[15] Carvalho F S,Wenceslau A A,Teixeira M,et al.Diagnosis of Eimeria species using traditional and molecular methods in field studies[J].Vet Parasitol,2011,176(2-3):95-100.
[16]侯瑞生,王丽,张勤,等.多重PCR技术及其在病原体检测中的应用[J].中华全科医学,2011,9(8):1288-1290.
[17]邓雯,马彦博,刘玉梅.鸡球虫对机体的致病作用研究进展[J].家禽生态,2004(2):59-62.
[18]辛玲,许金俊,陶建平.多重PCR检测3种鸡球虫方法的建立[J].中国兽医杂志,2008(3):6-8.
[19]李巍,韩彩霞,李微,等.基于ITS1序列的鸡艾美耳球虫种的套式PCR鉴别方法[J].中国兽医科学,2013,43(3):289-293.
[20]杨霞,卢中华,张红英,等.PCR技术在畜禽疾病诊断中的应用[J].河南农业大学学报,2001(S1):77-78.
[21]李文超,陶建平,沈永林,等.PCR及相关技术在鸡球虫种株鉴别方面的应用[J].动物医学进展,2006(9):18-22.
[22] Gadelhaq S M,Arafa W M,Aboelhadid S M.Molecular characterization of Eimeria species naturally infecting egyptian baldi chickens[J].Iran J Parasitol,2015,10(1):87-95.
[23] You M J.Detection of four important Eimeriaspecies by multiplex PCR in a single assay[J].Parasitol Int,2014,63(3):527-532.
[24]薛方民.鸡艾美耳球虫不同种株ITS-1、ITS-2序列的克隆和分析比较[D].山东济南:山东师范大学,2007.
[25]陈明洁,方倜,柯涛,等.多重PCR-一种高效快速的分子生物学技术[J].武汉理工大报,2005(10):37-40.
[26]刘高鹏.多重PCR检测和鉴定五种猪腹泻病毒研究[D].浙江杭州:浙江理工大学,2017.