构建染料显色RT-LAMP方法检测食源性HAV和HEV病毒的研究
|
摘要
目的构建稳定可靠的染料显色RT-LAMP方法用于食源性HAV和HEV病毒检测。方法根据HAV病毒的VP1/VP3衣壳蛋白基因的连接区和HEV病毒的ORF2区的保守序列分别设计和筛选出一套高效、特异的HAV和HEV RT-LAMP引物,结合LAMP扩增过程中dNTP反应析出的焦磷酸根离子、锰离子与钙黄绿素之间相互作用进而显色的原理优化和改进RT-LAMP体系。用优化的RT-LAMP体系进行HAV和HEV检测,评价方法的灵敏性和特异性。结果通用筛选的引物和优化的HAV、HEV RT-LAMP体系对4份HAV RNA阴性和17份HAV RNA阳性标本进行检测,敏感性100%,特异性100%;用优化的HEV RT-LAMP体系对4份HEV RNA阴性和12份HEV RNA阳性样本进行检测,敏感性100%,特异性100%。两种RT-LAMP体系的扩增结果均不受同属其他亚型病毒干扰。HAV RT-LAMP体系、HEV RT-LAMP体系敏感性好,扩增灵敏度均为10 copies/μl。检测灵敏度与传统Taqman荧光PCR法同功能试剂相近,但耗时减少,结果分析简便。结论建立的染料显色RT-LAMP检测食源性HAV和HEV病毒操作简便,检测结果易于分析、可靠,有望用于食源性HAV和HEV病毒监测和调查。
Objective To create a stable and reliable dye-based RT-LAMP technique for the detection of foodborne hepatitis A virus(HAV) and hepatitis E virus(HEV). Methods In accordance with the conserved sequence of the VP1 and VP3 capsid protein genes of the HAV and the ORF2 region of the HEV, a set of highly efficient and specific primers for HAV and HEV RT-LAMP were designed and screened. In conjunction with the principle of interaction between pyrophosphate ions, manganese ions, and calcein in a dNTP reaction during LAMP amplification, the RT-LAMP system was optimized and improved. Results Universally screened primers and optimized HAV and HEV RT-LAMP systems were used to test 4 HAV RNA-negative samples and 17 HAV RNA-positive samples. The systems had a sensitivity of 100% and a specificity of 100%. An optimized HEV RT-LAMP system was used to test 4 HEV RNA-negative samples and 12 HEV RNA-positive samples. The optimized system had a sensitivity of 100% and a specificity of 100%. Viruses of other subtypes did not interfere with amplification using the two RT-LAMP systems. The HAV RT-LAMP system and the HEV RT-LAMP system had good sensitivity and an amplification sensitivity of 10 copies/μl. The detection sensitivity was similar to that of the conventional Taqman fluorescent PCR, but the devised technique took less time and its results were easy to analyze. Conclusion The dye-based RT-LAMP technique created here to detect foodborne HAV and HEV is easy to perform and its results are easy to analyze. This technique has promise in the monitoring and study of foodborne HAV and HEV.
Objective To create a stable and reliable dye-based RT-LAMP technique for the detection of foodborne hepatitis A virus(HAV) and hepatitis E virus(HEV). Methods In accordance with the conserved sequence of the VP1 and VP3 capsid protein genes of the HAV and the ORF2 region of the HEV, a set of highly efficient and specific primers for HAV and HEV RT-LAMP were designed and screened. In conjunction with the principle of interaction between pyrophosphate ions, manganese ions, and calcein in a dNTP reaction during LAMP amplification, the RT-LAMP system was optimized and improved. Results Universally screened primers and optimized HAV and HEV RT-LAMP systems were used to test 4 HAV RNA-negative samples and 17 HAV RNA-positive samples. The systems had a sensitivity of 100% and a specificity of 100%. An optimized HEV RT-LAMP system was used to test 4 HEV RNA-negative samples and 12 HEV RNA-positive samples. The optimized system had a sensitivity of 100% and a specificity of 100%. Viruses of other subtypes did not interfere with amplification using the two RT-LAMP systems. The HAV RT-LAMP system and the HEV RT-LAMP system had good sensitivity and an amplification sensitivity of 10 copies/μl. The detection sensitivity was similar to that of the conventional Taqman fluorescent PCR, but the devised technique took less time and its results were easy to analyze. Conclusion The dye-based RT-LAMP technique created here to detect foodborne HAV and HEV is easy to perform and its results are easy to analyze. This technique has promise in the monitoring and study of foodborne HAV and HEV.
引文
[1] 黄建华,蒙世庭.甲型病毒性肝炎流行病学研究进展[J].实用预防医学,2012,19(5):799-801.
[2] 夏宁邵,张军,李少伟,等.戊型病毒性肝炎研究进展[J].厦门大学学报(自然科学版),2011,50(2):431-6.
[3] 周勃.戊型肝炎的流行病学进展[J].职业与健康,2016,32(1):138-42.
[4] 阮冰.戊型肝炎病毒感染的分子生物学检测[J].国外医学流行病学传染病学分册,1996,23(16):252-5.
[5] 阮冰,马亦林,左辉,等.散发性戊型肝炎的病原学和血清学研究[J].中华传染病杂志1999,17(1):7-10.
[6] Castello G,Scala S,Palmieri G,et al.HCV related hepatocellular carcinoma:from chronic in flammation to cancer[J].Clin Immunol,2010,134(3):237-50.
[7] Notomi T,Okayama H,Masubuchi H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Res,2000,28(12):E63.
[8] SNT2754.3-201.出口食品中致病菌环介导恒温扩增(LAMP)检测方法第3部分:志贺氏菌[S].北京:中国标准出版社,2011.
[9] SNT2754.4-2011.出口食品中致病菌环介导恒温扩增(LAMP)检测方法第4部分:单核细胞增生李斯特菌[S].北京:中国标准出版社,2011.
[10] SNT2754.5-2011.出口食品中致病菌环介导恒温扩增(LAMP)检测方法第5部分:副溶血性弧菌[S].北京:中国标准出版社,2011.
[11] 付红伟.戊型肝炎规范化实验室诊断模式的建立[D].天津:天津医科大学,2016.
[12] Foord A,Boyd V,Heine H.A Loop Mediated isothermal amplification (LAMP) assay for detection of hendra virus (HeV) in the field[J].Ecohealth,2012(7):S143.
[13] 邱丰,郭敏卓,丁军颖,等.一种改良的环介导恒温扩增技术在甲型肝炎病毒核酸检测上的应用[J].中华实验和临床病毒学杂志,2012,26(6):483-5.
[14] Parida M,Sannarangaiah S,Dash PK,et al.Loop mediated isothermal amplification (LAMP):a new generation of innovative gene amplification technique;perspectives in clinical diagnosis of infectious diseases[J].Rev Med Virol,2008,18(6):407-21.
[15] Mori Y,Nagamine K,Tomita N,et al.Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation[J].Biochem Biophys Res Commun,2001,289(1):150-4.
[16] Hong TC,Mai QL,Cuong DV,et al.Development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus[J].J Clin Microbiol,2004,42(5):1956-61.
[17] Tanimura N,Tsukamoto K,Okamatsu M,et al.Pathology of fatal highly pathogenic H5N1 avian influenza virus infection in large-billed crows (Corvus macrorhynchos) during the 2004 outbreak in Japan[J].Vet Pathol,2006,43(4):500-9.
[18] Fukuda S,Takao S,Kuwayama M,et al.Rapid detection of norovirus from fecal specimens by real-time reverse transcription-loop-mediated isothermal amplification assay[J].J Clin Microbiol,2006,44(4):1376-81.
[2] 夏宁邵,张军,李少伟,等.戊型病毒性肝炎研究进展[J].厦门大学学报(自然科学版),2011,50(2):431-6.
[3] 周勃.戊型肝炎的流行病学进展[J].职业与健康,2016,32(1):138-42.
[4] 阮冰.戊型肝炎病毒感染的分子生物学检测[J].国外医学流行病学传染病学分册,1996,23(16):252-5.
[5] 阮冰,马亦林,左辉,等.散发性戊型肝炎的病原学和血清学研究[J].中华传染病杂志1999,17(1):7-10.
[6] Castello G,Scala S,Palmieri G,et al.HCV related hepatocellular carcinoma:from chronic in flammation to cancer[J].Clin Immunol,2010,134(3):237-50.
[7] Notomi T,Okayama H,Masubuchi H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Res,2000,28(12):E63.
[8] SNT2754.3-201.出口食品中致病菌环介导恒温扩增(LAMP)检测方法第3部分:志贺氏菌[S].北京:中国标准出版社,2011.
[9] SNT2754.4-2011.出口食品中致病菌环介导恒温扩增(LAMP)检测方法第4部分:单核细胞增生李斯特菌[S].北京:中国标准出版社,2011.
[10] SNT2754.5-2011.出口食品中致病菌环介导恒温扩增(LAMP)检测方法第5部分:副溶血性弧菌[S].北京:中国标准出版社,2011.
[11] 付红伟.戊型肝炎规范化实验室诊断模式的建立[D].天津:天津医科大学,2016.
[12] Foord A,Boyd V,Heine H.A Loop Mediated isothermal amplification (LAMP) assay for detection of hendra virus (HeV) in the field[J].Ecohealth,2012(7):S143.
[13] 邱丰,郭敏卓,丁军颖,等.一种改良的环介导恒温扩增技术在甲型肝炎病毒核酸检测上的应用[J].中华实验和临床病毒学杂志,2012,26(6):483-5.
[14] Parida M,Sannarangaiah S,Dash PK,et al.Loop mediated isothermal amplification (LAMP):a new generation of innovative gene amplification technique;perspectives in clinical diagnosis of infectious diseases[J].Rev Med Virol,2008,18(6):407-21.
[15] Mori Y,Nagamine K,Tomita N,et al.Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation[J].Biochem Biophys Res Commun,2001,289(1):150-4.
[16] Hong TC,Mai QL,Cuong DV,et al.Development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus[J].J Clin Microbiol,2004,42(5):1956-61.
[17] Tanimura N,Tsukamoto K,Okamatsu M,et al.Pathology of fatal highly pathogenic H5N1 avian influenza virus infection in large-billed crows (Corvus macrorhynchos) during the 2004 outbreak in Japan[J].Vet Pathol,2006,43(4):500-9.
[18] Fukuda S,Takao S,Kuwayama M,et al.Rapid detection of norovirus from fecal specimens by real-time reverse transcription-loop-mediated isothermal amplification assay[J].J Clin Microbiol,2006,44(4):1376-81.