PCR-防污染核酸快速诊断HPV及分型试剂的研究和开发
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摘要
目的:建立一种基于PCR扩增结合封闭式核酸快速检测装置(内含核酸检测试纸条)新的基因分型方法,对四种常见人乳头瘤病毒(HPV)进行快速诊断与分型,以区分生殖道感染中最常见的低危性HPV6,11和高危性HPV16,18基因型,为临床常见HPV诊断和分型提供一种快速、准确、无需昂贵仪器、;廉价的诊断试剂。
方法与结果: 137例临床HPV16-18和HPV6-11阳性的标本通过DNA提取,PCR扩增,封闭式核酸快速检测装置的检测与分型,将其产物部分测序和与广州达安基因公司荧光定量PCR结果做比较,其符合率达到97%以上。
结论:结果表明该方法能够对四种最常见人乳头瘤病毒(HPV)进行快速诊断与分型,该方法简单、快速、准确、经济,不需要高级的检测设备,对宫颈棉拭子样本和瘤体样本均适用。该方法适合于各级医院尤其是中小型医院对人乳头瘤病毒(HPV)进行快速诊断与分型,具有重要的临床推广价值。
Objective: Develops a novel genotyping method for detecting the four types of human papillimovirus (HPV6, HPV11, HPV16, HPV18) which based on the polymerase chain reaction (PCR) and cross-contamination-proof (XCP) nucleic acid detection device.
Methods and Results: After the sample preparation and PCR amplification, the XCP nucleic acid detection device was used for HPV detection and genotyping .Compared the results of 137 clinical HPV positive samples with real time PCR, the coordination rate was beyond 97 percent.
Conclusion: The results showed that the new genotyping method can detect HPV genotypes and is simpler, faster, more accurate, cheaper, no expensive-equipment needed and cross-contamination-proof comparing with other method. It could be used in the small hospital and the clinics in resource-limited area and have an important promote value.
方法与结果: 137例临床HPV16-18和HPV6-11阳性的标本通过DNA提取,PCR扩增,封闭式核酸快速检测装置的检测与分型,将其产物部分测序和与广州达安基因公司荧光定量PCR结果做比较,其符合率达到97%以上。
结论:结果表明该方法能够对四种最常见人乳头瘤病毒(HPV)进行快速诊断与分型,该方法简单、快速、准确、经济,不需要高级的检测设备,对宫颈棉拭子样本和瘤体样本均适用。该方法适合于各级医院尤其是中小型医院对人乳头瘤病毒(HPV)进行快速诊断与分型,具有重要的临床推广价值。
Objective: Develops a novel genotyping method for detecting the four types of human papillimovirus (HPV6, HPV11, HPV16, HPV18) which based on the polymerase chain reaction (PCR) and cross-contamination-proof (XCP) nucleic acid detection device.
Methods and Results: After the sample preparation and PCR amplification, the XCP nucleic acid detection device was used for HPV detection and genotyping .Compared the results of 137 clinical HPV positive samples with real time PCR, the coordination rate was beyond 97 percent.
Conclusion: The results showed that the new genotyping method can detect HPV genotypes and is simpler, faster, more accurate, cheaper, no expensive-equipment needed and cross-contamination-proof comparing with other method. It could be used in the small hospital and the clinics in resource-limited area and have an important promote value.
引文
[1]徐耀先,周晓峰,刘立德.分子病毒学[M].湖北科技出版社2000,第1版:356一366
[2]许延贵,王敬云,张惠珍等.HPV的分子生物学特征及其致病机理.国外医学病毒学分册[J] 2004,11(1):1-5
[3]LaiminLA.Regulation of transcriptinon and replication by human papillomavirusin McCanceDJ(ed.).Human Tumor Biurses[M],ASM press,Washington,1998,201-203
[4]Boner Winifred, Morgan I M.Novel celluar interacting partnersn of the human papillomavirous 16 transcription/replication factor E2.Virol [J], 2002, 90 (6):113-118
[5]Nead M A,Mc Cance D J.Activitier of the Transforming proteits of human papillomavirus.In Mc Cance D(2ed).Human tumor biruses[M],ASM press,washington,1998,2:25-31
[6]Lechner M S,Laimins L A.Inhibition of p53 DNA binding by human papillmavurus E6 proteins.J virol[J],1984,68:4262-73
[7] APt D,Chong T,Liu Y.Nuclear factor 1 and epithelial cell-specific transcription of human papillomavirus type 16.J Virol[J] ,1993,67:4455-4463
[8] Um SJ.Abrogation of IRF-1 respense by high-risk HPV E7 Protein in vivo.Cancer Letters[J],2000,179:205-212
[9] Harald zur Hausen.Papillomaviruses Causing Cancer:Evasion from Host- Cell Control in Early Events in Carcinogenesis Journal of the National Cancer[J].Insititute,2000,92(9):690-698
[10]Fausch S C, Da Silva D M,Rudolf M P,etal.Human Papillomavirus virus-like Particles do not activaet Langerhans cells: a possible immune escape mechanism uesd by human papillomaviruses.J Immunol,2002,169(6):3242-3249
[11]Frazer I H.The role of the immune system in anogenital human papillomavirous.Aust J Dermatol [J], 1998, 39 (9):55-57
[12]Parkin DM, Bray F, Ferlay J, ct al.Estimating the world cancer burden: global cancer 2000.Int J cancer [M], 2001, 94-153
[13]World Health Organization.Cytological screening in the control of cervical cancer:Technical guidelines [J].Geneva: World Health Organization, 1988
[14]Anttila A,Pukkala E,Sodeman B,et al.Effect of organized screening on cervical cancer incidence and moratality in Finland,1963~1995:Recent increase in cervical cancer incidence.Int J Cancer[J],1999,83-89
[15]Lombard I,Vincent-salomon A,Validire P,et al.Human papillomavirus genotype as a major determinant of the course of cervical cancer.J Clin Oncol[J],1998,16:216
[16]Mclachlin CM.Human papillomavirus in cervical neoplasia: Role, risk, and implications.Clin Lab Med [J], 2000, 20(2):257
[17]Phelps W C, Barnes J A, Lobe D C.Molecular tagets for human papillomaviruses: prospects for antiviral therapy.Antiviral Chemistry & Chemo therapy [J], 1998, 9: 359-377
[18]Williams H C, Pottier A, Strachan D.The descriptive epidemiology of warts in British school children.Br J Dermato [J], 1993, 128:504-511
[19]Larsson P A,Liden S.Prevalance of skin diseases among adolescents 12-16 years of age.Acta Derm Venerol[J],1980,60:415-423
[20]Phelps W C, Barnes J A, Lobe D C.Molecular tagets for human papillomaviruses: prospects for antiviral therapy. Antiviral Chemistry & Chemotherapy [J], 1998, 9: 359-377
[21]Bauknecht T,Randelzhofer B,Schmitt B,et al.Response to IL-6 of HPV-18 cervical carcinoma cell lines Virology[J],1999,258:344-354
[22]Kirwan J M, Herrington C S.Human papillomavirus and cervical cancer: where are you now? Br J Obstet Gynaecol [J], 2001, 108:1204-13
[23]Cuschieri K S,Cubie H A,Whitley M W,et al.Multiple high risk HPV infections are common in cervical neoplasia and young women in a cervical screening population.J.Clin.Pathol[J],2004,57:68-72
[24]Peyton C L,Gravitt P E,Hunt W C,et al.Determinants of genital human papillomavirus detection in a US population.J Infect Dis[J],2001,183:1554-1564
[25]Molano M,Posso H,Weiderpass E,et al.Prevalence and determinants of HPV infection among Columbion women with normal cytology.Br J Cancer,2002,87:324-333
[26]Lidia Rosi Medeiros,Anaelena Braganca,de Moraes Ethur,et al.Roselaine Ruviaro Zanini 1 Otavio Berwanger, Mary Clarisse Bozzetti,Luciane Calil Mylius Cad.Saude Publica,Rio de Janeiro,2005,21(4):1006-1015
[27]Robet E, Bristow F J.Montz Human Papillomavirous: Moleclar biology and screening applications in cervical neoplasia-a primer for primary care physician primcare update ob/gyns [J], 1998, 5(5):238-246
[28]金奇,医学分子病毒学[M].北京:科学出版社,2002,500-502
[29]Syrjanen K, Syrjanen S.Papaillomavirus infections in human pathology. Chichester [J]: Wiley, 2000, 58:118-121
[30]Matsukura T.Sugase M.Relationships between 80 human papillomavirus genotypes and different gredes of cervical intraepithelial neoplasia: association and causality.Viology [J], 2001, 283:139-147
[31]Chan S Y,Delius H,Halpern AL,et al.Analysis of genomic sequences of 95 papillomavirus types:uniting typing,phylogeny,and taxonomy.J Virol[J],1995,69(5)3074-3083
[32]Hesselink A T, van-den-Brule AJ, Brink AA, ET al.Comparison of hybrid capture 2 with in situ hybridization for the detection of high-risk human papillomavirus in liquid-based cervical samples.Cancer [M], 2004, 102(1):11-18
[33]Pretet JL,Dalstein V,Monnier-Benoit S,et al.High risk HPV load estimated by hybrid captureⅡcorrelates with HPV16 load measured by real-time PCR in cervical smears of HPV16-infected women.J Clin Virol[J],2004,31(2):140-147
[34]Castle PE, Schiffman M, Wheeler CM.Hybrid capture 2 viral load and the 2-year cumulative risk of cervical intraepithelial neoplasia grade 3 or caner.Am J Obstet Gynecol[J], 2004, 191(5):1590-1597
[35]Castle PE, Wheeler CM, Solomon D, ET al.Interlaboratory reliability of hybrid capture 2.Am J Clin Pathol [J], 2004, 122(2):238-245
[36]Castle PE,Schiffman M,Burk RD,et al.Restricted cross-reactivity of hybrid capture2 with nononcogenic human papinomavirus types.Cancer Epidemiol Biomarkers Prev[J],2002,11(11):1394-1399
[37]Castle PE,Lorincz AT,Scott DR,et al.Comparison between prototype hybridcapture 3 and hybrid capture 2 and human papillonavirus DNA assays for detection of high-grade cervical intraepithelial neoplasia and cancer.J Clin Microbiol[J],2003,41(9):4022-4030
[38]Gravitt PE,Peyton CL,Alessi TQ,et al.Improved amplification of genital human papillomaviruses.J Clin Microbiol[J],2000,38(1):357-361
[39]Jacpbs MV,Snijders PJ,van den Brule AJ,et al.A general primer GP5+/GP6(+)-mediated PCR-enzyme immunoassay method for rapid detection of 14 highrisk and 6 low-risk human papillomavirus genotypes in cervical scrapings.J Clin Microbiol[J],1997,35(3):791-795
[40]Kleter B,van Doorn LJ,ter Schegget J,et al.A novel short-fragment PCR assay for highly sensitive broad-spectrum detection of anogenital human papillomaviruses.Am J Pathol[J],1998,153(6):1731-1739
[41]Moberg M,Gustavsson I,Gyllensten U.Real-time PCR-based system for simultaneous quantification of human papillomavirus type associated with high risk of cervical cancer.J Clin Microbiol,2003,41(7):3221-3228
[42]Cubie HA,Seagar AL,Mc Googan E,et al.Rapid real-time PCR to distinguish between human papillomavirus types 16 and 18.J Clin Pathol Mol Pathol[J],2001,54(1):24-29
[43]Hart KW,Williams OM,Thelwell N,et al.Novel method for detection,typing,and quantification of human papillomavirus in clinical samples.J Clin Microbiol[J],2001,39(9):3204-3212
[44]Lamarcq L,Deeds J,Ginzinger D,et al.Quantitation of human papillomavirus transcripts by real time quantitative reverse transcription-polymerase chain reaction in samples collected for cervical cancer screening,J Mol Diagn[J],2002,4(2):97-102
[45]Wang JF,Lu DW,Wang Y,et al.Quantitation of human papillomavirus 16 E6 and E7 DNA in residual material from ThinPrep papanicolaou tests using realtime polymerase chain reaction analysis.Cancer[J],2002,94(8):2199-2210
[46]Kraus I,Molden T,Erno LE,et al.Human papillomavirus oncogenic expression in the dysplastic portio;an investigation of biopsies from 190 cervical cones.Br J Cancer[J],2004,90(7):1407-1413
[47]Hwang TS,Jeong JK,Park M,et al.Detection and typing of HPV genotypes in various cervical lesions by HPV oligonucleotide microarray.Gynecol Oncol[J],2003,90(1):51-56
[48]Kim KH,Yoon MS,Na YJ,et al.Development and evaluation of a highly sensitive human papillomavirus genotyping DNA chip.Gynecol Oncol[J],2006,100(1):38-43
[49]Wallace J,Woda BA,Pihan G.Facile,comprehensive,high-throughput genotyping of human genital papillomaviruses using spectrally addressable liquid bead microarrays. J Mol Diagn [J], 2005, 7(1):72-80
[50]Valkirs GE and Barton R.Immuno concentrationa new format for solid -phace immunoassays. Clin Chem [J], 1985; 31:1427– 1431
[51]Spielberg F, Kabeya CM, Ryder RW, ET al.Field testing and comparative evaluation of rapid visually read screening assays for antibody to human immuneodeficiency virus.Lancet [J], 1989, 1:580-584
[52]Huang Q, Lan X, Tong T, ET al.Dotimmuneogold filtration assay as a screening test for syphilis.J Clin Microbiol [J], 1996, 34(8):201l-2013
[53]刘瑛.快速斑点免疫结合试验的研究进展[J].微生物学与免疫学进展1998,26 (4):84-86
[54]Genaro O, Melo MA, Costa RT da, ET al.Evaluation of the Immunochromatography assay for the diagnosis of canine visceral leishmaniasis.Aprospective study on experimentally infected dogs with leishmania chagasi.Memorias do Instituto Oswaldo Cruz [J], 1996, 91:192-193
[55]Kwok S, Higuchi R.Avoiding false positives with PCR.Nature.1989 May 18; 339(6221):237-8. Erratum in: Nature [J], 1989 Jun 8; 339(6224):490
[2]许延贵,王敬云,张惠珍等.HPV的分子生物学特征及其致病机理.国外医学病毒学分册[J] 2004,11(1):1-5
[3]LaiminLA.Regulation of transcriptinon and replication by human papillomavirusin McCanceDJ(ed.).Human Tumor Biurses[M],ASM press,Washington,1998,201-203
[4]Boner Winifred, Morgan I M.Novel celluar interacting partnersn of the human papillomavirous 16 transcription/replication factor E2.Virol [J], 2002, 90 (6):113-118
[5]Nead M A,Mc Cance D J.Activitier of the Transforming proteits of human papillomavirus.In Mc Cance D(2ed).Human tumor biruses[M],ASM press,washington,1998,2:25-31
[6]Lechner M S,Laimins L A.Inhibition of p53 DNA binding by human papillmavurus E6 proteins.J virol[J],1984,68:4262-73
[7] APt D,Chong T,Liu Y.Nuclear factor 1 and epithelial cell-specific transcription of human papillomavirus type 16.J Virol[J] ,1993,67:4455-4463
[8] Um SJ.Abrogation of IRF-1 respense by high-risk HPV E7 Protein in vivo.Cancer Letters[J],2000,179:205-212
[9] Harald zur Hausen.Papillomaviruses Causing Cancer:Evasion from Host- Cell Control in Early Events in Carcinogenesis Journal of the National Cancer[J].Insititute,2000,92(9):690-698
[10]Fausch S C, Da Silva D M,Rudolf M P,etal.Human Papillomavirus virus-like Particles do not activaet Langerhans cells: a possible immune escape mechanism uesd by human papillomaviruses.J Immunol,2002,169(6):3242-3249
[11]Frazer I H.The role of the immune system in anogenital human papillomavirous.Aust J Dermatol [J], 1998, 39 (9):55-57
[12]Parkin DM, Bray F, Ferlay J, ct al.Estimating the world cancer burden: global cancer 2000.Int J cancer [M], 2001, 94-153
[13]World Health Organization.Cytological screening in the control of cervical cancer:Technical guidelines [J].Geneva: World Health Organization, 1988
[14]Anttila A,Pukkala E,Sodeman B,et al.Effect of organized screening on cervical cancer incidence and moratality in Finland,1963~1995:Recent increase in cervical cancer incidence.Int J Cancer[J],1999,83-89
[15]Lombard I,Vincent-salomon A,Validire P,et al.Human papillomavirus genotype as a major determinant of the course of cervical cancer.J Clin Oncol[J],1998,16:216
[16]Mclachlin CM.Human papillomavirus in cervical neoplasia: Role, risk, and implications.Clin Lab Med [J], 2000, 20(2):257
[17]Phelps W C, Barnes J A, Lobe D C.Molecular tagets for human papillomaviruses: prospects for antiviral therapy.Antiviral Chemistry & Chemo therapy [J], 1998, 9: 359-377
[18]Williams H C, Pottier A, Strachan D.The descriptive epidemiology of warts in British school children.Br J Dermato [J], 1993, 128:504-511
[19]Larsson P A,Liden S.Prevalance of skin diseases among adolescents 12-16 years of age.Acta Derm Venerol[J],1980,60:415-423
[20]Phelps W C, Barnes J A, Lobe D C.Molecular tagets for human papillomaviruses: prospects for antiviral therapy. Antiviral Chemistry & Chemotherapy [J], 1998, 9: 359-377
[21]Bauknecht T,Randelzhofer B,Schmitt B,et al.Response to IL-6 of HPV-18 cervical carcinoma cell lines Virology[J],1999,258:344-354
[22]Kirwan J M, Herrington C S.Human papillomavirus and cervical cancer: where are you now? Br J Obstet Gynaecol [J], 2001, 108:1204-13
[23]Cuschieri K S,Cubie H A,Whitley M W,et al.Multiple high risk HPV infections are common in cervical neoplasia and young women in a cervical screening population.J.Clin.Pathol[J],2004,57:68-72
[24]Peyton C L,Gravitt P E,Hunt W C,et al.Determinants of genital human papillomavirus detection in a US population.J Infect Dis[J],2001,183:1554-1564
[25]Molano M,Posso H,Weiderpass E,et al.Prevalence and determinants of HPV infection among Columbion women with normal cytology.Br J Cancer,2002,87:324-333
[26]Lidia Rosi Medeiros,Anaelena Braganca,de Moraes Ethur,et al.Roselaine Ruviaro Zanini 1 Otavio Berwanger, Mary Clarisse Bozzetti,Luciane Calil Mylius Cad.Saude Publica,Rio de Janeiro,2005,21(4):1006-1015
[27]Robet E, Bristow F J.Montz Human Papillomavirous: Moleclar biology and screening applications in cervical neoplasia-a primer for primary care physician primcare update ob/gyns [J], 1998, 5(5):238-246
[28]金奇,医学分子病毒学[M].北京:科学出版社,2002,500-502
[29]Syrjanen K, Syrjanen S.Papaillomavirus infections in human pathology. Chichester [J]: Wiley, 2000, 58:118-121
[30]Matsukura T.Sugase M.Relationships between 80 human papillomavirus genotypes and different gredes of cervical intraepithelial neoplasia: association and causality.Viology [J], 2001, 283:139-147
[31]Chan S Y,Delius H,Halpern AL,et al.Analysis of genomic sequences of 95 papillomavirus types:uniting typing,phylogeny,and taxonomy.J Virol[J],1995,69(5)3074-3083
[32]Hesselink A T, van-den-Brule AJ, Brink AA, ET al.Comparison of hybrid capture 2 with in situ hybridization for the detection of high-risk human papillomavirus in liquid-based cervical samples.Cancer [M], 2004, 102(1):11-18
[33]Pretet JL,Dalstein V,Monnier-Benoit S,et al.High risk HPV load estimated by hybrid captureⅡcorrelates with HPV16 load measured by real-time PCR in cervical smears of HPV16-infected women.J Clin Virol[J],2004,31(2):140-147
[34]Castle PE, Schiffman M, Wheeler CM.Hybrid capture 2 viral load and the 2-year cumulative risk of cervical intraepithelial neoplasia grade 3 or caner.Am J Obstet Gynecol[J], 2004, 191(5):1590-1597
[35]Castle PE, Wheeler CM, Solomon D, ET al.Interlaboratory reliability of hybrid capture 2.Am J Clin Pathol [J], 2004, 122(2):238-245
[36]Castle PE,Schiffman M,Burk RD,et al.Restricted cross-reactivity of hybrid capture2 with nononcogenic human papinomavirus types.Cancer Epidemiol Biomarkers Prev[J],2002,11(11):1394-1399
[37]Castle PE,Lorincz AT,Scott DR,et al.Comparison between prototype hybridcapture 3 and hybrid capture 2 and human papillonavirus DNA assays for detection of high-grade cervical intraepithelial neoplasia and cancer.J Clin Microbiol[J],2003,41(9):4022-4030
[38]Gravitt PE,Peyton CL,Alessi TQ,et al.Improved amplification of genital human papillomaviruses.J Clin Microbiol[J],2000,38(1):357-361
[39]Jacpbs MV,Snijders PJ,van den Brule AJ,et al.A general primer GP5+/GP6(+)-mediated PCR-enzyme immunoassay method for rapid detection of 14 highrisk and 6 low-risk human papillomavirus genotypes in cervical scrapings.J Clin Microbiol[J],1997,35(3):791-795
[40]Kleter B,van Doorn LJ,ter Schegget J,et al.A novel short-fragment PCR assay for highly sensitive broad-spectrum detection of anogenital human papillomaviruses.Am J Pathol[J],1998,153(6):1731-1739
[41]Moberg M,Gustavsson I,Gyllensten U.Real-time PCR-based system for simultaneous quantification of human papillomavirus type associated with high risk of cervical cancer.J Clin Microbiol,2003,41(7):3221-3228
[42]Cubie HA,Seagar AL,Mc Googan E,et al.Rapid real-time PCR to distinguish between human papillomavirus types 16 and 18.J Clin Pathol Mol Pathol[J],2001,54(1):24-29
[43]Hart KW,Williams OM,Thelwell N,et al.Novel method for detection,typing,and quantification of human papillomavirus in clinical samples.J Clin Microbiol[J],2001,39(9):3204-3212
[44]Lamarcq L,Deeds J,Ginzinger D,et al.Quantitation of human papillomavirus transcripts by real time quantitative reverse transcription-polymerase chain reaction in samples collected for cervical cancer screening,J Mol Diagn[J],2002,4(2):97-102
[45]Wang JF,Lu DW,Wang Y,et al.Quantitation of human papillomavirus 16 E6 and E7 DNA in residual material from ThinPrep papanicolaou tests using realtime polymerase chain reaction analysis.Cancer[J],2002,94(8):2199-2210
[46]Kraus I,Molden T,Erno LE,et al.Human papillomavirus oncogenic expression in the dysplastic portio;an investigation of biopsies from 190 cervical cones.Br J Cancer[J],2004,90(7):1407-1413
[47]Hwang TS,Jeong JK,Park M,et al.Detection and typing of HPV genotypes in various cervical lesions by HPV oligonucleotide microarray.Gynecol Oncol[J],2003,90(1):51-56
[48]Kim KH,Yoon MS,Na YJ,et al.Development and evaluation of a highly sensitive human papillomavirus genotyping DNA chip.Gynecol Oncol[J],2006,100(1):38-43
[49]Wallace J,Woda BA,Pihan G.Facile,comprehensive,high-throughput genotyping of human genital papillomaviruses using spectrally addressable liquid bead microarrays. J Mol Diagn [J], 2005, 7(1):72-80
[50]Valkirs GE and Barton R.Immuno concentrationa new format for solid -phace immunoassays. Clin Chem [J], 1985; 31:1427– 1431
[51]Spielberg F, Kabeya CM, Ryder RW, ET al.Field testing and comparative evaluation of rapid visually read screening assays for antibody to human immuneodeficiency virus.Lancet [J], 1989, 1:580-584
[52]Huang Q, Lan X, Tong T, ET al.Dotimmuneogold filtration assay as a screening test for syphilis.J Clin Microbiol [J], 1996, 34(8):201l-2013
[53]刘瑛.快速斑点免疫结合试验的研究进展[J].微生物学与免疫学进展1998,26 (4):84-86
[54]Genaro O, Melo MA, Costa RT da, ET al.Evaluation of the Immunochromatography assay for the diagnosis of canine visceral leishmaniasis.Aprospective study on experimentally infected dogs with leishmania chagasi.Memorias do Instituto Oswaldo Cruz [J], 1996, 91:192-193
[55]Kwok S, Higuchi R.Avoiding false positives with PCR.Nature.1989 May 18; 339(6221):237-8. Erratum in: Nature [J], 1989 Jun 8; 339(6224):490