环介导等温扩增技术可视化检测乙、丙型肝炎病毒的研究

发布时间：2018-04-22 01:07

本文选题：环介导等温扩增 + 乙型肝炎病毒　；参考：《承德医学院》2017年硕士论文

【摘要】：乙型肝炎病毒(Hepatitis B virus,HBV)和丙型肝炎病毒(Hepatitis C virus,HCV)感染是全球公共卫生问题,尤其是在实验设备匮乏的发展中国家和地区。HBV和HCV核酸检测是诊断和疗效检测的最佳方法,国内大、中型医院普遍使用荧光定量PCR(Fluorescence quantitative PCR,FQ-PCR),然而,FQ-PCR操作流程烦琐,试剂、仪器均较昂贵,对操作人员的技术要求高,不适用于在医疗设备匮乏的场所进行操作。本研究建立了依赖钙黄绿素和羟基萘酚蓝(hydroxynaphthol blue,HNB)的可视化环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术,以利于在实验设备匮乏的环境下进行HBV和HCV检测。第一部分应用环介导等温扩增技术可视化检测乙型肝炎病毒目的:建立可视化检测HBV的LAMP方法。方法:首先,根据NCBI网站上公布的HBV S区段保守序列设计LAMP引物。第二,收集临床血清样本,分别按照试剂盒法和煮沸法提取DNA,用FQ-PCR对病毒进行定量。第三,对反应条件进行优化,通过对非特异模板扩增实验和内切酶消化实验以验证扩增特异性;通过检测试剂盒提取后倍比稀释的HBV模板以评价LAMP的灵敏性;通过LAMP和PCR扩增煮沸血清后结果的比较,以评价LAMP的抗干扰性。同时,根据电泳结果比较依赖SYBR Green I的和依赖HNB的可视化检测效果。最后,用LAMP和PCR扩增临床样本,用SPSS 17.0统计学软件分别与FQ-PCR(金标准)进行比较。结果:经过一系列预实验,建立了最适LAMP反应条件,LAMP特异性高,没有产生非特异性扩增。无论使用哪种核酸提取方法,LAMP灵敏性均为10拷贝/管,由此可得出LAMP的抗干扰性较好。依赖HNB可视化检测的效果和SYBR Green I、电泳分析相当,却不似染料SYBR Green I需要开盖加入而造成气溶胶污染。另外,lamp对于煮沸血清的检测结果和fq-pcr没有统计学差异(p0.05),且一致性较好(kappa=0.762)。然而,pcr对于煮沸血清的检测结果和fq-pcr有统计学差异(p0.05),且一致性较差(kappa=0.186)。结论:本研究针对hbvs区段,设计了针对我国主要的四种hbv基因型lamp通用引物,通过设计环引物,提高了扩增效率。另外,使用煮沸血清为模板,简化了操作流程,从而建立了依赖hnb可视化检测hbv的lamp技术。第二部分应用反转录环介导等温扩增技术可视化检测丙型肝炎病毒目的:建立可视化检测hcv的rt-lamp方法。方法:首先,收集并用fq-pcr(金标准)检测75例临床样本。根据9种hcv基因亚型的5’非编码区(5’utr)设计通用rt-lamp引物。然后,通过比较传统rt-lamp系统和加入taqdna聚合酶的系统以优化体系,通过非特异模板扩增实验以评价其特异性,通过rt-lamp和rt-pcr同时检测倍比稀释的hcv模板以评价其灵敏性。用电泳分析扩增情况,同时用钙黄绿素和hnb可视化检测产物。最后,用rt-lamp和rt-pcr检测临床样本,用spss17.0统计学软件分别与fq-pcr进行比较。结果:结果表明向传统rt-lamp体系中加入taqdna聚合酶可提高20min的扩增效率。rt-lamp的特异性好,没有非特异性扩增现象。经检测稀释模板,rt-lamp的灵敏性为10iu/管,比rt-pcr高10倍。另外,钙黄绿素和hnb的可视效果与电泳分析相一致。经检测75例临床样本,rt-lamp显示了和fq-pcr较好的一致性(p0.05,kappa=0.762)。结论:本研究针对hcv5’utr区段,设计了检测hcv9种基因亚型的rt-lamp通用引物,建立了依赖钙黄绿素或hnb可视化检测hcv的rt-lamp技术。第三部分应用反转录等温扩增技术对丙型肝炎病毒1b和2a基因型检测目的:建立对HCV 1b和2a基因型分型的RT-LAMP方法。方法:首先,收集74例阳性临床采样标本并用FQ-PCR方法分型和定量。第二,分别根据HCV 1b和2a序列各自的5’UTR设计相应的RT-LAMP引物。第三,利用与非特异模板扩增实验和酶切实验以评价各引物的特异性。第四,扩增倍比稀释的模板以评价各引物的灵敏性,并用依赖钙黄绿素的可视化方法判定结果。最后,对所有临床样本分别用HCV 1b和2a分型引物进行两组平行反应,应用SPSS 17.0统计学软件比较RT-LAMP和FQ-PCR的一致性。结果:结果表明RT-LAMP引物特异性好,于非特异模板无交叉反应且HCV1b和2a型酶切产物大小与预期相一致。RT-LAMP法最低检测HCV 1b和2a的灵敏度为100 IU/mL,且依赖钙黄绿素的可视化方法和电泳分析相一致。经对临床样本进行平行实验,RT-LAMP检测HCV 1b型的阳性率为97.37%,检测HCV 2a型的阳性率为94.44%。SPSS 17.0统计学软件表明RT-LAMP和FQ-PCR没有统计学差异(P0.05)。结论:本研究通过比较多组HCV基因序列,设计了两套分型引物,建立了依赖钙黄绿素可视化分型检测HCV的RT-LAMP技术。
[Abstract]:Hepatitis B virus (Hepatitis B virus, HBV) and hepatitis C virus (Hepatitis C virus, HCV) infection are the global public health problems, especially in the developing countries and regions with lack of experimental equipment,.HBV and HCV nucleic acid detection is the best method for diagnosis and efficacy detection. Large and medium-sized hospitals are widely used in medium size hospitals with fluorescent quantitative PCR (Fluorescenc). E quantitative PCR, FQ-PCR), however, the FQ-PCR operation process is tedious, the reagent, the instrument are all expensive, the technical requirement of the operator is high, it is not suitable for the operation in the place where the medical equipment is scarce. This study established the visual loop mediated isothermal amplification (loop-mediat) depending on the calc and the hydroxyl naphthol blue (hydroxynaphthol blue, HNB). Ed isothermal amplification, LAMP) technology to facilitate the detection of HBV and HCV in the environment of lack of experimental equipment. The first part uses ring mediated isothermal amplification to visualize hepatitis B virus purpose: to establish a LAMP method for visual detection of HBV. Method: first, to design L according to the HBV S section published on NCBI site. AMP primers. Second, the clinical serum samples were collected, the DNA was extracted according to the kit method and the boiling method respectively. The virus was quantified by FQ-PCR. Third, the reaction conditions were optimized. The non specific template amplification experiment and the digestion experiment of endonuclease were used to verify the specificity. The sensitivity of valence LAMP; comparing the results of boiling serum after LAMP and PCR amplification to evaluate the anti-interference of LAMP. At the same time, according to the results of electrophoresis, the results of SYBR Green I and HNB depend on the visual detection results. Finally, the clinical samples were amplified by LAMP and PCR and compared with FQ-PCR (gold standard) by SPSS 17 system software. Fruit: after a series of pre experiments, the optimum LAMP reaction conditions were established, and the specificity of LAMP was high and no non specific amplification was produced. No matter which nucleic acid extraction method was used, the sensitivity of LAMP was 10 copies / tubes. Thus, the anti-interference of LAMP was better. The effect of HNB visual detection and SYBR Green I, electrophoretic analysis, were not similar to dyed. SYBR Green I needed open cover to cause aerosol pollution. In addition, there was no statistical difference between the results of detection of boiling serum by lamp and FQ-PCR (P0.05), and the consistency was better (kappa=0.762). However, the results of PCR for boiling serum were statistically different from FQ-PCR (P0.05), and the consistency was poor (kappa=0.186). Conclusion: This study According to the HBVs section, we designed four main HBV genotypes lamp universal primers for our country. By designing ring primers, the amplification efficiency was improved. In addition, using boiling serum as a template, the operation process was simplified, and the lamp technology that depended on the visual detection of HBV by HNB was established. The second part applied inversion ring mediated isothermal amplification technology to be visible. Detection of hepatitis C virus Objective: to establish a RT-LAMP method for visual detection of HCV. Method: first, collect and use FQ-PCR (gold standard) to detect 75 clinical samples. General RT-LAMP primers are designed according to the 5 'non coding region (5' UTR) of the 9 HCV subtypes. Then, the system is superior to the traditional RT-LAMP system and the system adding taqdna polymerase. The system was used to evaluate its specificity by nonspecific template amplification experiments. The sensitivity was evaluated by simultaneous detection of double dilution HCV templates by RT-LAMP and RT-PCR. The amplification conditions were analyzed by electrophoretic analysis. At the same time, calcium yellow green and HNB were used to visualize the products. Finally, the clinical samples were detected by RT-LAMP and RT-PCR, and the SPSS17.0 statistical software was used respectively. Results compared with FQ-PCR. Results: the results showed that adding taqdna polymerase to the traditional RT-LAMP system could improve the specificity of 20min amplification efficiency.Rt-lamp without non specific amplification. The sensitivity of RT-LAMP was 10 times higher than that of RT-PCR by detecting the dilution template. Besides, the visual effect and electrophoresis analysis of calcein and HNB Consistent. After testing 75 clinical samples, RT-LAMP showed good consistency with FQ-PCR (P0.05, kappa=0.762). Conclusion: This study designed a RT-LAMP universal primer for detection of hcv9 gene subtypes for hcv5 'UTR section, and established a RT-LAMP technique for the visualization of HCV by the visualization of calcein or HNB. The third part was used for reverse transcription isothermal. Detection of hepatitis C virus 1b and 2A genotypes by amplification: a RT-LAMP method for typing HCV 1b and 2A genotypes. Methods: first, 74 samples of positive clinical samples were collected and classified and quantified by FQ-PCR method. Second, the corresponding RT-LAMP primers were designed according to the 5 'UTR HCV 1b and 2A sequences. Third. Specific template amplification experiments and enzyme cutting experiments were used to evaluate the specificity of the primers. Fourth, the amplification of double dilution templates was used to evaluate the sensitivity of the primers, and the results were determined by the visualization of calcein dependence. Finally, two groups of parallel reactions were performed with HCV 1b and 2A types, respectively, and SPSS 17 statistics were applied to all clinical samples. The software compared the consistency between RT-LAMP and FQ-PCR. Results: the results showed that the specificity of RT-LAMP primers was good, the non specific template was no cross reaction and the sensitivity of.RT-LAMP method to detect HCV 1b and 2a was 100 IU/mL with the same.RT-LAMP method, and the visual method and electrophoresis analysis were the same. Parallel experiments on clinical samples showed that the positive rate of RT-LAMP HCV 1b was 97.37%. The positive rate of HCV 2 A was 94.44%.SPSS 17 statistics software showed that there was no statistical difference between RT-LAMP and FQ-PCR (P0.05). Conclusion: This study set up two sets of type primers by comparing multiple groups of HCV gene sequences, and established the dependence on calcium yellow green. The RT-LAMP technology of HCV was detected by stereotyping.

【学位授予单位】：承德医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.6;R440

【参考文献】