优化的外周血水痘—带状疱疹病毒DNA检测方法在VZV相关疾病中的临床应用

发布时间：2018-01-22 23:44

  本文关键词： 水痘—带状疱疹病毒 实时荧光定量多聚核苷酸链式反应 外周血单个核细胞 脑源性神经营养因子 S100B　出处：《西南医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景和目的:水痘-带状疱疹病毒(varicella zoster virus,VZV)感染后可导致水痘、带状疱疹(herpes zoster,HZ)及带状疱疹后遗神经痛(postherpetic neuralgia,PHN)等VZV相关疾病的发生。在VZV相关疾病中对VZV DNA进行检测,可作为感染、病毒血症存在的依据,以及对神经损伤程度、病情及预后作出相应的判断。目前国内对于外周血VZV DNA的检测,通常运用实时荧光定量多聚核苷酸链式反应(Real-timeQuantitativepolymerasechain reaction,qPCR)检测试剂盒。但因其价格昂贵,灵敏度不高,故在临床中尚不能推广。所以本文的目的之一在于优化DNA的提取方法,简化标准品的制作,设计特异性的引物,提高检测方法的敏感性、简单性、快速性、特异性及经济性。本文的另一个目的在于利用我们建立的qPCR方法,检测VZV相关疾病外周血病毒载量的差异;利用我们建立的qPCR方法,检测HZ外周血单个核细胞(peripheral blood mono-nuclear cells,PBMC)、血浆病毒载量的差异性;利用酶联免疫吸附测定(enzymelinkedimmunosorbent assay,ELISA)法,检测VZV相关疾病神经损伤评估因子脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)、S100B蛋白水平。方法实验一:qPCR方法检测HZ外周血患者VZV DNA的优化选择1.随机抽取10例HZ患者,分别利用煮沸法、离心柱法提取PBMC中dna。2.采用巢式pcr制备标准品。3.利用上海之江vzv检测试剂盒(qpcr方法)与本文建立的方法,分别对煮沸法及离心柱法提取的dna进行pbmc中vzvdna的定量检测。实验二:本文建立的qpcr方法在vzv相关疾病中的临床应用及检测血浆bdnf、s100b1.vzv相关疾病vzvdna载量差异性的分析:收集10例水痘、31例hz、11例phn及20例健康志愿者,分别设为水痘组、hz组、phn组及正常组,采用本文建立的qpcr方法对pbmc中vzvdna载量进行检测。2.hz患者治疗前、后pbmc与血浆vzvdna载量差异性的分析:在31例患者中,随机抽取22例hz患者,分别检测治疗前及治疗14天后pbmc、血浆中的vzvdna。3.vzv相关疾病神经损伤评估因子的分析:采用elisa法检测水痘组、hz组、phn组血浆中bdnf、s100b的水平。结果:实验一:1.标准品建立:巢式pcr所建立的标准品大小为385bp。2.提取方法比较:煮沸法、离心柱法分别联合上海之江vzv检测试剂盒(qpcr法),vzvdna的阳性检出率为10%、60%。离心柱法较煮沸法检出率高。3.引物比较:离心柱法分别联合上海之江vzv检测试剂盒(qpcr法)和本文建立的方法阳性检出率均为60%,离心柱法下,二者阳性检出一致。故本文建立的qpcr优化方法为:离心柱法+巢式pcr制作标准品+设计特异性引物。实验二:1.vzv相关疾病外周血pbmc中vzvdna阳性检出率、载量的比较:治疗前水痘组、hz组、phn组阳性检出率分别为:80%、70.97%、36.36%。治疗前pbmc水痘组的vzvdna载量高于hz组及phn组,hz组高于phn组,差异有统计学意义(z=-2.82,p0.05;z=-2.98,p0.05;z=-2.47,p0.05)。正常对照组vzvdna检出均为阴性。2.hz组治疗前后pbmc、血浆中vzvdna阳性检出率、载量的比较:在hz组随机抽取的22例患者中,治疗前pbmc、血浆中vzvdna的阳性检出率分别为:72.72%、63.64%,差异无统计学意义(χ2=1.33,df=1,p0.05)。治疗后分别为47.06%、35.29%,无明显的差异性(χ2=0.5,df=1,p0.05)。治疗前、后pbmc中的vzvdna载量较血浆高,两者有明显的差异(z=-4.107,p0.05;z=-2.432,p0.05)。治疗后pbmc、血浆中的vzvdna载量均较治疗前降低,差异有统计学意义(z=-4.11,p0.05;z=-3.03,p0.05)。3.vzv相关疾病血浆bdnf的比较:与正常组比较,治疗前血浆bdnf水痘组、hz组、phn组较高,差异有统计学意义(i-j=190.89,p0.05;i-j=232.49,p0.05;i-j=246.65,p0.05)。水痘组较hz组、phn组低,有明显的差异性(i-j=-46.60,p0.05;i-j=-55.75,p0.05)。HZ组与PHN组无明显的差异性(I-J=-14.15,P0.05)。4.VZV相关疾病S100B的比较:治疗前血浆S100B水痘组、HZ组、PHN组均较正常组高,差异有统计学意义(I-J=126.55,P0.05;I-J=164.85,P0.05;I-J=159.94,P0.05),水痘组较HZ组、PHN组低,具有明显的差异性(I-J=-38.30,P0.05;I-J=-33.38,P0.05),HZ组与PHN组无明显的差异性(I-J=4.96,P0.05)。5.HZ组血浆BDNF、S100B比较:HZ组治疗前、后BDNF、S100B均较正常组高,差异有统计学意义(I-J=232.50,27.38,I-J=164.84,21.46,P0.05),HZ组治疗后BDNF、S100B较治疗前明显降低,差异有统计学意义(I-J=205.11,P0.05;I-J=143.39,P0.05)。结论:1.离心柱法提取DNA用于qPCR优于煮沸法。2.巢式PCR制作的标准品具有更高的稳定性。3.本文建立的qPCR方法操作简单,灵敏度高,更经济,可作为优先选择。4.水痘患者PBMC中的VZV DNA阳性检出率、载量高于HZ及PHN患者,血浆BDNF、S100B低于HZ、PHN患者,提示水痘患者虽有较高的PBMC中VZV DNA载量,但神经损伤较HZ、PHN患者轻。5.HZ患者PBMC与血浆中的VZV DNA阳性率无明显差异性,血浆更方便用于临床筛查。
[Abstract]:BACKGROUND & OBJECTIVE : The detection of the DNA in peripheral blood mononuclear cells ( PBMC ) and human peripheral blood mononuclear cells ( PBMC ) by using the qPCR method , which can be used as the basis for the detection of the DNA in peripheral blood , and the sensitivity , simplicity , rapidity , specificity and economical efficiency of the detection method . The aim of this paper is to optimize the method of DNA extraction , to simplify the production of standard products , to design specific primers , to improve the sensitivity , simplicity , rapidity , specificity and economy of the detection method . the quantitative detection of vzvdna in pbmc by boiling method and centrifugal column method was carried out . The results were as follows : 1 . The positive detection rate of vzvdna in pbmc and plasma vzvdna was determined by using the method of qpcr . z = - 2.98 , p . 05 ; z = - 2.47 , p . 05 ) . The positive rates of vzvdna in pbmc and plasma were 72 . 72 % and 63 . 64 % , respectively , and no significant difference was found between the two groups ( 蠂2 = 1.33 , df = 1 , p . 05 ) . After treatment , 47.06 % and 35.29 % respectively , there was no significant difference ( 蠂2 = 0.5 , df = 1 , p0.05 ) . The vzvdna content in pbmc before and after treatment was higher than that in plasma ( z = - 4.107 , p0.05 ; z = - 2.432 , p . 05 ) . The levels of vzvdna in plasma of pbmc and plasma were lower before treatment ( z = - 4.11 , p0.05 ; z = - 3.03 , p . 05 ) . 3 . The plasma bdnf in the plasma bdnf group , hz group and phn group were higher in the treatment group than in the normal group ( i - j = 190.89 , p < 0.05 ; i - j = 232.49 , p < 0.05 ; i - j = 246.65 , p . 05 ) . There was a significant difference in phn group ( i - j = - 46.60 , p . 05 ; i - j = - 55.75 , p . 05 ) . 4.VZV鐩稿叧鐤剧梾S100B鐨勬瘮杈，

本文编号：1456060