实时可视化LAMP快速检测高危亚型HPV病毒方法的建立
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摘要
目的:建立实时可视化LAMP快速检测高危亚型人乳头瘤病毒的方法。方法:基于HPV病毒基因中的L1区保守序列,构建重组标准质粒。应用钙黄绿素作为可视化荧光染料,通过环介导等温扩增技术手段,检测结果可由肉眼观测和荧光检测信号来判读。结果:应用LAMP技术检测HPV病毒,大部分高危亚型的检测限可达到103copies/μL,其中HPV16,HPV33,HPV59,HPV73亚型灵敏度可达101copies/μL;15种高危亚型样品的检测结果显示不存在交叉反应,表明其特异性良好;针对318例临床样本,LAMP的阳性检出率达到79.56%(253/318),与反向杂交试剂盒检测结果基本持平。结论:本研究针对15种高危亚型HPV病毒,建立高效快捷的实时可视化LAMP检测方法,其临床样本符合率高达91.20%,因而具有广泛的市场推广前景和潜在的临床应用意义。
Aims:A real-time visual loop-mediated isothermal amplification assay was established for the rapid detection of human papillomavirus in high-risk subtypes.Methods:In this paper,conserved sequences(L1 region)of HPV virus genes were selected to construct recombinant standard plasmids.Combined with calcein,a visualized fluorescent dye,the results were determined by visual observation and fluorescence detection signals.Results:The detection limit of most high-risk subtypes could reach 103 copies/μL by LAMP assay,among which the sensitivity of HPV16,HPV33,HPV59 and HPV73 subtypes could reach 101 copies/μL.It indicated good specificity that there is no cross reaction among the 15 high-risk subtypes.For 318 clinical samples,the positive detection rate could reach 79.56%(253/318),which was almost the same as the reverse hybridization kit.Conclusions:In view of 15 high-risk subtypes,a fast and efficient real-time visual LAMP method was established,and the coincidence rate of clinical samples was as high as 91.20%.It has broad marketing prospects and potential clinical applications.
Aims:A real-time visual loop-mediated isothermal amplification assay was established for the rapid detection of human papillomavirus in high-risk subtypes.Methods:In this paper,conserved sequences(L1 region)of HPV virus genes were selected to construct recombinant standard plasmids.Combined with calcein,a visualized fluorescent dye,the results were determined by visual observation and fluorescence detection signals.Results:The detection limit of most high-risk subtypes could reach 103 copies/μL by LAMP assay,among which the sensitivity of HPV16,HPV33,HPV59 and HPV73 subtypes could reach 101 copies/μL.It indicated good specificity that there is no cross reaction among the 15 high-risk subtypes.For 318 clinical samples,the positive detection rate could reach 79.56%(253/318),which was almost the same as the reverse hybridization kit.Conclusions:In view of 15 high-risk subtypes,a fast and efficient real-time visual LAMP method was established,and the coincidence rate of clinical samples was as high as 91.20%.It has broad marketing prospects and potential clinical applications.
引文
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[5]马骉.HPV分型环介导核酸等温扩增检测技术研究[D].杭州:中国计量大学,2016.MA B.Research on Loop-Mediated Isothermal Amplification of Nucleic Acid Detection Technology of HPV Genotypes[D].Hangzhou:China Jiliang University,2016.
[6]吕蓓,程海荣,严庆丰,等.体外核酸快速扩增技术的发展和不断创新[J].中国生物工程杂志,2011,31(3):91-96.LYU B,CHENG H R,YAN Q F,et al.The development and recent improvements of in vitro nucleic acid amplification technology[J].China Biotechnology,2011,31(3):91-96.
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[9]马骉,吴莹莹,戴明雁,等.副溶血性弧菌PMA-LAMP方法的建立[J].现代食品科技,2016,32(7):205-213.MA B,WU Y Y,DAI M Y,et al.Development of loop-mediated isothermal(LAMP)assay based on propidium monoazide(PMA)for detecting Vibrio parahemolyticus[J].Modern Food Science and Technology,2016,32(7):205-213.
[10]LIN J,MA B,FANG J,et al.Colorimetric detection of23human papillomavirus genotypes by loop-Mediated isothermal amplification[J].Clinical Laboratory,2017,63(3):495-505.
[11]徐淑菲,孔繁德,苗丽,等.LAMP技术研究进展及其在动物疫病检测中的应用[J].中国动物检疫,2017,34(1):75-80.XU S F,KONG F D,MIAO L,et al.Research progress of LAMP technology and its application in animal diseases detection[J].China Animal Health Inspection,2017,34(1):75-80.
[12]李森,王源升,余红伟,等.多重环介导等温扩增技术的研究进展[J].生物技术通讯,2017,28(2):217-221.LI S,WANG Y S,YU H W,et al.Development ofthe multiplex loop-mediated isothermal amplification technology[J].Letters in Biotechnology,2017,28(2):217-221.
[13]张婧,陶霞.HPV疫苗研究进展及认知度、接受度现状[J].中国妇产科临床杂志,2017,18(1):84-86.ZHANG J,TAO X.Advances in HPV vaccine research and current status ofcognition and acceptance[J].Chinese Journal of Clinical Obstetrics and Gynecology,2017,18(1):84-86.
[14]ELENA S,ROMERO M P,BERNARDINO J I,et al.Human papillomavirus mRNA testing for the detection of anal high-grade squamous intraepithelial lesions in men who have sex with men infected with HIV[J].Journal of Medical Virology,2015,87(8):1397-1403.
[15]YAMAMOTO S P,ATSUSHI K,ATSUSHI O,et al.Detection and characterization of a human G9P4rotavirus strain in Japan[J].Journal of Medical Virology,2015,87(8):1311-1318.
[2]朱佳妮,王东卓,刘宗唐.宫颈癌筛查技术研究进展[J].中国实用妇科与产科杂志,2016(6):597-600.ZHU J N,WANG D Z,LIU Z T.Advances in screening technology for cervical cancer[J].Chinese Journal of Practical Gynecology and Obstetrics,2016(6):597-600.
[3]吕攀攀,邢志芳.人乳头瘤病毒实验室检测方法的研究进展[J].国际检验医学杂志,2017,38(5):660-664.LYU P P,XING Z F.Progress in laboratory detection of human papillomavirus[J].International Journal of Laboratory Medicine,2017,38(5):660-664.
[4]CHEN W,JERONIMO J,ZHAO F H,et al.The concordance of HPV DNA detection by Hybrid Capture 2and care HPV on clinician-and self-collected specimens[J].Journal of Clinical Virology,2014,61(4):553-557.
[5]马骉.HPV分型环介导核酸等温扩增检测技术研究[D].杭州:中国计量大学,2016.MA B.Research on Loop-Mediated Isothermal Amplification of Nucleic Acid Detection Technology of HPV Genotypes[D].Hangzhou:China Jiliang University,2016.
[6]吕蓓,程海荣,严庆丰,等.体外核酸快速扩增技术的发展和不断创新[J].中国生物工程杂志,2011,31(3):91-96.LYU B,CHENG H R,YAN Q F,et al.The development and recent improvements of in vitro nucleic acid amplification technology[J].China Biotechnology,2011,31(3):91-96.
[7]NOTOMI T,OKAYAMA H,MASUBUCHI H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Research,2000,28(12):63.
[8]NORIHIRO T,YASUYOSHI M,HIDETOSHI K,et al.Loop-mediated isothermal amplification(LAMP)of gene sequences and simple visual detection of products[J].Nature Protocol,2008,3(5):877-882.
[9]马骉,吴莹莹,戴明雁,等.副溶血性弧菌PMA-LAMP方法的建立[J].现代食品科技,2016,32(7):205-213.MA B,WU Y Y,DAI M Y,et al.Development of loop-mediated isothermal(LAMP)assay based on propidium monoazide(PMA)for detecting Vibrio parahemolyticus[J].Modern Food Science and Technology,2016,32(7):205-213.
[10]LIN J,MA B,FANG J,et al.Colorimetric detection of23human papillomavirus genotypes by loop-Mediated isothermal amplification[J].Clinical Laboratory,2017,63(3):495-505.
[11]徐淑菲,孔繁德,苗丽,等.LAMP技术研究进展及其在动物疫病检测中的应用[J].中国动物检疫,2017,34(1):75-80.XU S F,KONG F D,MIAO L,et al.Research progress of LAMP technology and its application in animal diseases detection[J].China Animal Health Inspection,2017,34(1):75-80.
[12]李森,王源升,余红伟,等.多重环介导等温扩增技术的研究进展[J].生物技术通讯,2017,28(2):217-221.LI S,WANG Y S,YU H W,et al.Development ofthe multiplex loop-mediated isothermal amplification technology[J].Letters in Biotechnology,2017,28(2):217-221.
[13]张婧,陶霞.HPV疫苗研究进展及认知度、接受度现状[J].中国妇产科临床杂志,2017,18(1):84-86.ZHANG J,TAO X.Advances in HPV vaccine research and current status ofcognition and acceptance[J].Chinese Journal of Clinical Obstetrics and Gynecology,2017,18(1):84-86.
[14]ELENA S,ROMERO M P,BERNARDINO J I,et al.Human papillomavirus mRNA testing for the detection of anal high-grade squamous intraepithelial lesions in men who have sex with men infected with HIV[J].Journal of Medical Virology,2015,87(8):1397-1403.
[15]YAMAMOTO S P,ATSUSHI K,ATSUSHI O,et al.Detection and characterization of a human G9P4rotavirus strain in Japan[J].Journal of Medical Virology,2015,87(8):1311-1318.