巢式PCR在弓形虫检测和基因研究中的应用进展
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摘要
弓形虫是一种专性细胞内寄生的寄生虫,人感染后多呈隐性感染,临床检测及诊断困难。巢式PCR是利用2套引物,对靶细胞基因2次识别并进行2轮扩增的方法,它可从极少量的模板中获得较高浓度和纯度的靶片段,在弓形虫的检测和基因研究中具有敏感性高、特异性强、稳定性好的优点。本文对巢式PCR在弓形虫中的应用进行了综述。
Toxoplasma gondii is an obligate intracellular parasite that is difficult to detect and diagnose clinically in people who are infected.Nested PCR uses two sets of primers to amplify genes of target cells twice to identify them,resulting in a higher concentration and purity of target fragments from a tiny amount of template.When detecting T.gondii,nested PCR also has advantages such as sensitivity,specificity,and consistency.This paper reviews advances in the use of nested PCR to detect and genetically study T.gondii.
Toxoplasma gondii is an obligate intracellular parasite that is difficult to detect and diagnose clinically in people who are infected.Nested PCR uses two sets of primers to amplify genes of target cells twice to identify them,resulting in a higher concentration and purity of target fragments from a tiny amount of template.When detecting T.gondii,nested PCR also has advantages such as sensitivity,specificity,and consistency.This paper reviews advances in the use of nested PCR to detect and genetically study T.gondii.
引文
[1]刘长生.异常妊娠史孕妇弓形虫感染情况调查[J].中国病原生物学杂志,2011,6(9):718,660.
[2]赵娜.768名孕妇弓形虫特异性IgM的检测[J].中国城乡企业卫生,2008,6(6):82.
[3]Li S,Cui L,Zhao J,et al.Seroprevalence of Toxoplasma gondiiinfection in female sterility patients in China[J].J Parasitol,2011,97(3):529-30.
[4]岳林,王丽芬,陈敏淑.弓形虫感染与男性不育的相关性研究[J].陕西医学杂志,2006,35(9):1138-44.
[5]Terpsidis KI,Papazahariadou MG,Taitzoglou IA,et al.Toxo-plasma gondii:Reproductive parameters in experimentally infec-ted male rats[J].Exp Parasitol,2009,121(3):238-41.
[6]Qi R,Su XP,Gao XL,et al.Toxoplasmainfection in males withsterility in Shenyang,China[J].Nat J Androl,2005,11(7):503-4.
[7]Su C,Zhang X,Dubey JP.Genotyping of Toxoplasma gondii bymultilocus PCR-RFLP markers:a high resolution and simplemethod for identification of parasites[J].Int J Parasitol,2006,36(7):841-8.
[8]Brandao GP,Ferreira AM,Melo MN,et al.Characterization ofToxoplasma gondii from domestic animals from Minas Gerais,Brazil[J].Parasite,2006,13(2):143-9.
[9]Howe DK,Honore S,Derouin F,et al.Determination of geno-types of Toxoplasma gondii strains isolated from patients withtoxoplasmosis[J].J Clin Microbiol,1997,35(6):1411-4.
[10]Khan A,Su C,German M,et al.Genotyping of Toxoplasmagondii strains from immunocompromised patients reveals highprevalence of type I strains[J].J Clin Microbiol,2005,43(12):5881-7.
[11]傅斌,刘克义,韩广东,等.巢式PCR法检测弓形虫的应用研究[J].中国热带医学,2005,5(5):929-31.
[12]Miller MA,Grigg ME,Kreuder C,et al.An unusual genotypeof Toxoplasma gondii is common in California sea otters(Enhydralutris nereis)and is a cause of mortality[J].Int J Parasitol,2004,34(3):275-84.
[13]甘绍伯.病原生物学与感染性疾病[J].中国热带医学,2006,6(12):2103-14.
[14]Aspinall TV,Marlee D,Hyde JE,et al.Prevalence of Toxo-plasma gondii in commercial meat products as monitored by poly-merase chain reaction-food for thought?[J].Int J Parasitol,2002,32(9):1193-9.
[15]王权,王巧全,潘淑娟,等.动物源性食品中弓形Nested PCR-RFLP分子检测技术的建立[J].中国兽医寄生虫病,2007,15(6):1-7.
[16]任常娥.河南省部分地区猪弓形虫分离株基因型研究和弓形虫RH株的细胞培养[D].河南农业大学,2009.10-6.
[17]Fuentes I,Rodriguez M,Domingo CJ,et al.Urine sample usedfor cogenital toxoplamosis diagnosis by PCR[J].J Clin Microbi-ol,1996,34(10):2368-71.
[18]Alfonso Y,Fraga J,Jimenez N,et al.Detection of Toxoplasmagondii in cerebrospinal fluid from AIDS patients by nested PCRand rapid identification of type I allele at B1gene by RFLP analy-sis[J].Exp Parasitol,2009,122(3):203-7.
[19]Horiuchi K,Yabe I,Tajima Y,et al.Case of Toxoplasma en-cephalopathy with specific MRI findings,diagnosed by IgG avidityindex and nested PCR[J].Rinsho Shinkeigaku,2010,50(4):252-6.
[20]Mahalakshmi B,Therese KL,Madhavan HN,et al.Diagnosticvalue of specific local antibody production and nucleic acid amplifi-cation technique-nested polymerase chain reaction(nPCR)in clini-cally suspected ocular toxoplasmosis[J].Ocul Immunol Inflamm,2006,14(2):105-12.
[21]董莉,何莉,周智辉,等.低体质量新生儿和新生儿肺炎常见病原体筛查[J].实用儿科临床杂志,2008,23(10):739-41.
[22]程宁,周智辉,何莉,等.巢式PCR筛查早产儿TORCH病原感染的研究[J].中国病原生物学杂志,2010,5(10):729-31.
[23]Lee JY,Lee SE,Lee EG,et al.Nested PCR-based detection ofToxoplasma gondii in German shepherd dogs and stray cats inSouth Korea[J].Res Vet Sci,2008,85(1):125-7.
[24]李文姝,陆惠民,闵太善,等.弓形虫棒状体蛋白ROP2基因的克隆与表达[J].中国人兽共患病杂志,2005,21(10):883-6.
[25]王素华.弓形虫半巢式PCR检测方法的建立[J].检验检疫科学,2008,18(1):6-8.
[26]孔得翔,王素华,曲道峰,等.弓形虫半巢式PCR检测方法的建立[J].中国兽医学报,2009,29(1):52-62.
[27]黄留玉主编.PCR最新技术原理、方法及应用[M].第1版.北京:化学工业出版社,2005.260.
[28]Pujol-Rique M,Derouin F,García-Quintanilla A,et al.Designof a one-tube hemi-nested PCR for detection of Toxoplasma gon-dii and comparison of three DNA purification methods[J].J MedMicrobiol,1999,48(9):857-62.
[29]Hurtado A,Aduriz G,Moreno B,et al.Single tube nested PCRfor the detection of Toxoplasma gondii in fetal tissues from natu-rally aborted ewes[J].Vet Parasitol,2001,102(1-2):17-27.
[30]郑秀红,贾立军,邢莹,等.猪附红细胞体单管巢式PCR诊断方法的建立及应用[J].中国兽医学报,2011,31(2):205-6.
[31]糜祖煌,秦玲.弓形虫B1基因套式PCR检测技术研究[J].中国人兽共患病杂志,1999,15(2):63-4.
[32]黄璋琼,李春花,罗志武,等.弓形虫(Toxoplasma gondii)的PCR检测方法在笼养猕猴群中的应用[J].中国比较医学杂志,2008,18(6):21-4.
[33]陈俏梅,张俐,何国声.检测实验动物弓形虫感染的两种PCR方法的建立和比较[J].中国兽医寄生虫病,2003,11(2):6-8.
[34]Calderaro A,Piccolo G,Gorrini C,et al.Comparison betweentwo real-time PCR assays and a nested-PCR for the detection ofToxoplasma gondii[J].Acta Biomed,2006,77(2):75-80.
[35]Putignani L,Mancinelli L,Del Chierico F,et al.Investigation ofToxoplasma gondii presence in farmed shellfish by nested-PCRand real-time PCR fluorescent amplicon generation assay(FLAG)[J].Exp Parasitol,2011,127(2):409-17.
[36]王胜昌,谢建云,邵伟娟,等.弓形虫PCR检测方法的建立及初步应用[J].上海实验动物科学,2003,23(1):15-34.
[2]赵娜.768名孕妇弓形虫特异性IgM的检测[J].中国城乡企业卫生,2008,6(6):82.
[3]Li S,Cui L,Zhao J,et al.Seroprevalence of Toxoplasma gondiiinfection in female sterility patients in China[J].J Parasitol,2011,97(3):529-30.
[4]岳林,王丽芬,陈敏淑.弓形虫感染与男性不育的相关性研究[J].陕西医学杂志,2006,35(9):1138-44.
[5]Terpsidis KI,Papazahariadou MG,Taitzoglou IA,et al.Toxo-plasma gondii:Reproductive parameters in experimentally infec-ted male rats[J].Exp Parasitol,2009,121(3):238-41.
[6]Qi R,Su XP,Gao XL,et al.Toxoplasmainfection in males withsterility in Shenyang,China[J].Nat J Androl,2005,11(7):503-4.
[7]Su C,Zhang X,Dubey JP.Genotyping of Toxoplasma gondii bymultilocus PCR-RFLP markers:a high resolution and simplemethod for identification of parasites[J].Int J Parasitol,2006,36(7):841-8.
[8]Brandao GP,Ferreira AM,Melo MN,et al.Characterization ofToxoplasma gondii from domestic animals from Minas Gerais,Brazil[J].Parasite,2006,13(2):143-9.
[9]Howe DK,Honore S,Derouin F,et al.Determination of geno-types of Toxoplasma gondii strains isolated from patients withtoxoplasmosis[J].J Clin Microbiol,1997,35(6):1411-4.
[10]Khan A,Su C,German M,et al.Genotyping of Toxoplasmagondii strains from immunocompromised patients reveals highprevalence of type I strains[J].J Clin Microbiol,2005,43(12):5881-7.
[11]傅斌,刘克义,韩广东,等.巢式PCR法检测弓形虫的应用研究[J].中国热带医学,2005,5(5):929-31.
[12]Miller MA,Grigg ME,Kreuder C,et al.An unusual genotypeof Toxoplasma gondii is common in California sea otters(Enhydralutris nereis)and is a cause of mortality[J].Int J Parasitol,2004,34(3):275-84.
[13]甘绍伯.病原生物学与感染性疾病[J].中国热带医学,2006,6(12):2103-14.
[14]Aspinall TV,Marlee D,Hyde JE,et al.Prevalence of Toxo-plasma gondii in commercial meat products as monitored by poly-merase chain reaction-food for thought?[J].Int J Parasitol,2002,32(9):1193-9.
[15]王权,王巧全,潘淑娟,等.动物源性食品中弓形Nested PCR-RFLP分子检测技术的建立[J].中国兽医寄生虫病,2007,15(6):1-7.
[16]任常娥.河南省部分地区猪弓形虫分离株基因型研究和弓形虫RH株的细胞培养[D].河南农业大学,2009.10-6.
[17]Fuentes I,Rodriguez M,Domingo CJ,et al.Urine sample usedfor cogenital toxoplamosis diagnosis by PCR[J].J Clin Microbi-ol,1996,34(10):2368-71.
[18]Alfonso Y,Fraga J,Jimenez N,et al.Detection of Toxoplasmagondii in cerebrospinal fluid from AIDS patients by nested PCRand rapid identification of type I allele at B1gene by RFLP analy-sis[J].Exp Parasitol,2009,122(3):203-7.
[19]Horiuchi K,Yabe I,Tajima Y,et al.Case of Toxoplasma en-cephalopathy with specific MRI findings,diagnosed by IgG avidityindex and nested PCR[J].Rinsho Shinkeigaku,2010,50(4):252-6.
[20]Mahalakshmi B,Therese KL,Madhavan HN,et al.Diagnosticvalue of specific local antibody production and nucleic acid amplifi-cation technique-nested polymerase chain reaction(nPCR)in clini-cally suspected ocular toxoplasmosis[J].Ocul Immunol Inflamm,2006,14(2):105-12.
[21]董莉,何莉,周智辉,等.低体质量新生儿和新生儿肺炎常见病原体筛查[J].实用儿科临床杂志,2008,23(10):739-41.
[22]程宁,周智辉,何莉,等.巢式PCR筛查早产儿TORCH病原感染的研究[J].中国病原生物学杂志,2010,5(10):729-31.
[23]Lee JY,Lee SE,Lee EG,et al.Nested PCR-based detection ofToxoplasma gondii in German shepherd dogs and stray cats inSouth Korea[J].Res Vet Sci,2008,85(1):125-7.
[24]李文姝,陆惠民,闵太善,等.弓形虫棒状体蛋白ROP2基因的克隆与表达[J].中国人兽共患病杂志,2005,21(10):883-6.
[25]王素华.弓形虫半巢式PCR检测方法的建立[J].检验检疫科学,2008,18(1):6-8.
[26]孔得翔,王素华,曲道峰,等.弓形虫半巢式PCR检测方法的建立[J].中国兽医学报,2009,29(1):52-62.
[27]黄留玉主编.PCR最新技术原理、方法及应用[M].第1版.北京:化学工业出版社,2005.260.
[28]Pujol-Rique M,Derouin F,García-Quintanilla A,et al.Designof a one-tube hemi-nested PCR for detection of Toxoplasma gon-dii and comparison of three DNA purification methods[J].J MedMicrobiol,1999,48(9):857-62.
[29]Hurtado A,Aduriz G,Moreno B,et al.Single tube nested PCRfor the detection of Toxoplasma gondii in fetal tissues from natu-rally aborted ewes[J].Vet Parasitol,2001,102(1-2):17-27.
[30]郑秀红,贾立军,邢莹,等.猪附红细胞体单管巢式PCR诊断方法的建立及应用[J].中国兽医学报,2011,31(2):205-6.
[31]糜祖煌,秦玲.弓形虫B1基因套式PCR检测技术研究[J].中国人兽共患病杂志,1999,15(2):63-4.
[32]黄璋琼,李春花,罗志武,等.弓形虫(Toxoplasma gondii)的PCR检测方法在笼养猕猴群中的应用[J].中国比较医学杂志,2008,18(6):21-4.
[33]陈俏梅,张俐,何国声.检测实验动物弓形虫感染的两种PCR方法的建立和比较[J].中国兽医寄生虫病,2003,11(2):6-8.
[34]Calderaro A,Piccolo G,Gorrini C,et al.Comparison betweentwo real-time PCR assays and a nested-PCR for the detection ofToxoplasma gondii[J].Acta Biomed,2006,77(2):75-80.
[35]Putignani L,Mancinelli L,Del Chierico F,et al.Investigation ofToxoplasma gondii presence in farmed shellfish by nested-PCRand real-time PCR fluorescent amplicon generation assay(FLAG)[J].Exp Parasitol,2011,127(2):409-17.
[36]王胜昌,谢建云,邵伟娟,等.弓形虫PCR检测方法的建立及初步应用[J].上海实验动物科学,2003,23(1):15-34.