Application Of Microfluidic Chip For Clinical Genetic Diagnosis

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.