纳米技术检测乙肝病毒变异位点方法的建立及其应用

发布时间：2018-03-12 16:55

本文选题：乙肝病毒　切入点：纳米技术　出处：《首都医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:1.建立纳米磁珠提取血清乙型肝炎病毒脱氧核糖核酸(hepatitis B virus deoxyribonucleic acid,HBV DNA)联合量子点标记荧光探针检测HBV变异位点(M204I位点突变)的新方法。2.探讨新方法检测M204I位点突变在评价慢性乙型肝炎(chronic hepatitis B,CHB)核苷(酸)类(nucleos(t)ide analogues,NAs)药物治疗应答不佳患者中的意义。方法:将罗氏COBAS Taq Man检测的病毒载量为107IU/m L的血清样本,倍比稀释成不同浓度的HBV DNA作为血清标准品(理论值),分别用本方法(试剂1)、磁珠法(试剂2)、煮沸法(试剂3)三种方法提取HBV DNA,从检测结果与理论值的相关性以及可重复性方面比较本实验的纳米磁珠提取方法与国产磁珠法核酸自动提取法和传统煮沸法的提取效果。收集临床符合耐药,且直接测序法检测到有M204I位点突变的血清样本8例,不含有M204I位点突变的血清样本15例,设计并确定含有M204I位点突变的引物及探针序列,用量子点标记荧光探针杂交法检测HBV M204I变异位点,与直接测序法的检测结果进行比较,评价新方法检测位点变异的诊断效能,以及此方法在检测核苷(酸)类药物治疗应答不佳的慢乙肝患者HBV M204I位点变异时的意义。结果:纳米磁珠提取HBV DNA方法与理论值的相关性:试剂1、试剂2、试剂3提取HBV DNA定量与理论值相关性分析的r值分别为0.986(P0.001)、0.950(p=0.001)、0.979(P0.001),差异均具有统计学意义。三种方法检测的相对偏差均值分别为0.243±0.405(试剂1)、1.189±0.855(试剂2)、-0.439±0.618(试剂3)。在检测低水平HBV DNA定量时,理论值为101IU/ml,试剂1为3.520×101IU/ml,试剂2为9.123×103IU/ml,试剂3为6.195×101IU/ml。可重复性:试剂1对同一样本在不同时间检测结果的相对偏差均值为0.505±0.659。量子点标记荧光探针检测HBV M204I变异位点的检测下限是103IU/ml,且病毒水平越高,荧光光点越密集。利用量子点标记荧光探针法对临床中出现对核苷(酸)类药物治疗应答不佳的28例慢乙肝患者的HBV M204I位点进行检测,并与直接测序法比较,其中11例测序阳性样本中均检测到M204I位点的突变,17例测序阴性样本中均未检测到M204I位点的突变,两种方法检测结果一致。结论:利用纳米磁珠提取HBV DNA联合量子点探针杂交法检测病毒HBV DNA聚合酶基因变异的方法灵敏、准确,简便易行,为检测乙型肝炎病毒M204I位点变异提供了一个新的手段,且HBV DNA聚合酶位点变异可能是导致慢乙肝患者抗病毒治疗过程中发生应答不佳的原因之一。
[Abstract]:Objective 1. To establish a new method for the detection of mutation of HBV mutation site M204I by using nanomagnetic beads to extract hepatitis B virus deoxyribonucleic deoxyribonucleic DNA from serum. 2. To explore a new method for detection of M204I locus. The significance of mutagenesis in the evaluation of chronic hepatitis B hepatitis nucleostidine nucleostidine (analoguestide) in patients with poor response to drugs. Methods: serum samples with a viral load of 107 hepatitis / mL detected by COBAS Taq Man were used to evaluate the efficacy of the mutation in the treatment of chronic hepatitis B patients with adverse drug response. HBV DNA of different concentration was diluted into different times ratio as the standard serum (theoretical value). The method was used to extract HBV by three methods (reagent 1n, magnetic bead method (reagent 2N), boiling method (reagent 3)). The correlation between the results of detection and the theoretical value was analyzed. The results of extraction of nanomagnetic beads were compared with those of domestic magnetic beads and traditional boiling methods. The clinical results were consistent with drug resistance. In addition, 8 serum samples with M204I mutation and 15 serum samples without M204I mutation were detected by direct sequencing. Primers and probe sequences containing M204I mutation were designed and determined. The mutation sites of HBV M204I were detected by quantum dot labeled fluorescence probe hybridization and compared with the results of direct sequencing to evaluate the diagnostic effectiveness of the new method. And the significance of this method in detecting the variation of HBV M204I site in patients with chronic hepatitis B who have poor response to nucleoside (acid) drugs. Results: the correlation between the method of HBV DNA extraction by nanomagnetic beads and the theoretical value is as follows: reagent 1, reagent 2, reagent 3. The r values of quantitative and theoretical analysis of HBV DNA were 0.986p 0.001 ~ 0.950p 0.001C ~ (0.979) P 0.001 ~ (-1), respectively. The mean relative deviations of the three methods were 0.243 卤0.405 (reagents 1.189 卤0.855) (reagent 2-0.439 卤0.618) (reagents 2-0.439 卤0.618). The relative deviations of the three methods were 0.243 卤0.405 (reagent 2-0.439 卤0.618) and 0.43 卤0.405 (reagents 2-0.439 卤0.618), respectively. The theoretical value is 101 IUU / ml, reagent 1 is 3.520 脳 10 ~ (-1) IUP / ml, reagent 2 is 9.123 脳 10 ~ (3) IUP / ml, reagent 3 is 6.195 脳 10 ~ (-1) IUP / ml. Repeatability: the average relative deviation of reagent 1 to the same sample at different times is 0.505 卤0.659. The detection of HBV M204I variation site by fluorescence probe labeled by quantum dot is 0.505 卤0.659. The lower limit is 103 IU / ml, and the higher the virus level, The denser the fluorescent spot was, the more intense the fluorescent spot was. The HBV M204I loci in 28 patients with chronic hepatitis B who had a poor response to nucleoside (acid) drugs were detected by quantum dot labeling fluorescence probe method, and compared with the direct sequencing method. M204I locus mutations were detected in 11 sequencing positive samples and no M204I mutation was detected in 17 sequencing negative samples. Conclusion: the method of HBV DNA extraction and quantum dot hybridization is sensitive, accurate and easy to detect the mutation of virus HBV DNA polymerase gene. It provides a new method to detect the mutation of M204I site of hepatitis B virus, and the mutation of HBV DNA polymerase site may be one of the causes of poor response to antiviral therapy in patients with chronic hepatitis B.
【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.62

【参考文献】