【作者】 陈斌；

【导师】 周小棉；

【作者基本信息】 广州医学院 ， 临床检验诊断学， 2010， 硕士

【摘要】 本论文工作以微流控芯片为基本研究平台,发展用于疾病诊断的基因检测技术,为微流控芯片技术用于临床检测作出有益的尝试。论文研究内容包括:1.联合多重聚合酶链反应与微流控芯片电泳对人乳头瘤病毒进行基因分型。目的:建立人乳头瘤病毒的芯片电泳基因分型方法,初步探讨其对于宫颈高度病变诊断的临床价值。方法:利用多重聚合酶链反应联合芯片电泳检测114例妇女宫颈上皮细胞10种HPV感染状况,采用相对迁移时间比例的方法能够精确计算待测DNA片段长度,实现HPV基因型的准确判断。结果与导流杂交法进行比较,参考组织病理学检测结果,评价其针对宫颈高度病变(≥宫颈上皮内瘤变2级)诊断的临床价值。结果:芯片电泳方法能够在3分钟内实现10种HPV基因亚型特异性扩增片段的分离。114例宫颈上皮细胞样本中,芯片电泳法检测HPV总感染率为66.7%(76/114),其与导流杂交法检测结果总符合率为90.4%(103/114),(Kappa=0.795,P>0.05)。10种常见的HPV基因型按检出率递顺序依次为:58、16、52、11、33、31、18、59、6、45。诊断宫颈高度病变的敏感性为87.5%,特异性为37.8%。结论:芯片电泳法用于人乳头瘤病毒基因分型检测快速、可靠,适合于临床样本筛查。2. CD式微流控芯片用于苯丙酮尿症基因突变检测。目的:发展一种离心力驱动往复流式DNA杂交微流控芯片技术,用于苯丙酮尿症快速筛查。方法:设计并加工一种具有12个微通道的CD式微流控芯片。芯片采用聚二甲基硅氧烷-玻璃双层结构,分别含有微通道和水凝胶结合探针区。探针区包括R243Q、V245V和阴性对照。将苯丙氨酸羟化酶基因外显子7扩增产物加入芯片的进样孔,在离心力的作用下进入杂交通道区。芯片在离心机中交替旋转或暂停,通道内液体由于离心力与毛细管作用力形成往复流以提高DNA杂交速度。杂交后将芯片置于荧光显微镜下观察结果,阳性结果与测序结果进行比对。结果:利用离心力驱动往复流式DNA杂交芯片只需1.5μl样品,并能够将杂交时间缩短至15分钟。对30例疑似PKU孕妇样本进行检测,筛查出4例携带V245V突变,1例携带R243Q突变。通过测序结果验证了该方法的可靠性。结论:建立了基于离心力驱动往复流CD式DNA杂交芯片检测苯丙酮尿症基因突变,该方法简便、快速、高灵敏,可以用于苯丙酮尿症产前筛查。更多还原

【Abstract】 This thesis focuses on the application of microfluidic chip for clinical genetic diagnosis .The research work includes:1. Genotyping of Human Papillomavirus Using Combined Multiplex Polymerase Chain Reaction And Microchip Electrophoresis Method.Objective: To establish a microchip electrophoresis (MCE) method for fast genotyping of human papillomaviruses (HPV) and to discuss its clinical significance in laboratory diagnosis of high-grade cervical intraepithelial neoplasia.Methods: Cervical squamous cell specimens from 114 women were selected for the genotyping assay that combined multiplex PCR and MCE. A DNA sizing method based on relative migration-time proportion (RMP) was also developed for precise prediction of the sizes of amplification fragments of 10 HPV genotypes. The detection rates of 10 HPV types were compared with those obtained with flow-through hybridation and gene chip (HybrMax). The value of MCE based HPV genotyping for clinical diagnosis of high-grade cervical intraepithelial neoplasia (≥CIN2) was validated by histopathologic diagosis.Results: The amplified fragments of 10 HPV genotypes were separated in 3 minutes by MCE. The total detection rate of the 10 HPV subtypes using MCE was 66.7% (76/114). The accordance between HybrMax and MCE analysis was 90.4% (Kappa=0.795). According to descending prevalence rates, the 10 genotypes were 58, 16, 52, 11, 33, 31, 18, 59, 6 and 45. The sensitivity and specificity of high-grade cervical intraepithelial neoplasia prediction with MCE based HPV genotyping were 83.3% and 37.8%, respectively. Conclusions: The MCE based HPV genotyping assay is fast and reliable, thus well suited to the screening of HPV infection.2. Rapid Screening of Phenylketonuria with Reciprocating Flow DNA Hybridization on a Compact Disk Type Microfluidic chip.Objective: To develop a compact disk type microfluidic chip capable of generating reciprocating flow within the microchannels that facilitate rapid DNA hybridization. Based on the microchip, a phenylketonuria (PKU) screening method was developed.Methods: This microfluidic device consisted of a two-layer structure: a polydimethylsiloxane (PDMS) top layer containing 12 DNA hybridization microchannels, and a bottom glass layer with immobilized hydrogel conjugated DNA arrays. The DNA arrays included R243Q, V245V and the negative control probes. When the CD device was spun, the PCR products were driven into the hybridization channel by centrifugal force. When the rotation of the CD device was stopped, capillary force pulled the PCR products solution to flow back to the channel. After the on-chip hybridization, the hybridization signals were imaged on a fluorescence microscope. The positive samples were identified by DNA sequencing.Results: With the compact disk type microfluidic chip, the hybridization time could be reduced to 15 min, and sample consume could be as low as 1.5μL. With the chip based method, 4 cases of V245V mutation and 1 case of R243Q mutation were detected in 30 samples from suspected PKU carried pregnant women. The microfluidichip detection results were further confirmed by DNA sequencing results.Conclusions: The compact disk microfluidic device has advantages of simple, rapid and highly sensitive, thus is well suited to PKU screening.更多还原