Genetic Diagnostic Research Of Infectious Pathogens Using Microfluidic Technologies

This thesis focuses on the application of microfluidic technologies for genetic detection of drug-resistance related gene of pathogens.he research work includes:1. Detection of hepatitis B virus YMDD mutants using microchip electrophoresisObjective: To establish an YMDD mutant detection method based on multiplex PCR and microchip electrophoresis with the high-resolution and rapid separation features. Methods: A sequence-specific multiplex PCR was developed for YMDD mutants (YVDD and YIDD) amplification. The PCR products were analyzed using microchip electrophoresis. The DNA sizing method was developed to identify YVDD and YIDD amplicons. Detection sensitivity of the microchip electrophoresis was investigated by analyzing YVDD and YIDD DNAs mixed with different concentrations of YMDD plasmid. Serum DNA samples from 86 chronic hepatitis B cases that accepted lamivudine therapy were analyzed using the microchip electrophoresis, and the results were compared with that obtained from the commercial fluorescent quantitative PCR. Those inconsistent cases were further analyzed by DNA sequencing.Results: Microchip electrophoresis analysis was completed within 3 minutes, and reached a detection sensitivity of 101 copies /μl of standard DNA template. The minimum mutation detection rate of YVDD and YIDD was 5% and 10%, respectively. Among the 86 samples analyzed, the microchip electrophoresis method detected 65 mutants(75.6%), the fluorescentquantitative PCR assay detected 62 mutants(72.1%). Theχ2 test results of two methods suggested that the two methods showed no statistically significant defference,χ2 test (Kappa =0.609, P> 0.05). The assay results of 20 patient samples were compared with DNA sequencing. Finally, the overall agreement between DNA sequencing and the microchip electrophoresis assay was 95%.Conclusions: A method that combined multiplex PCR with microchip electrophoresis was developed for the detection of YMDD mutants. This assay is not only highly accurate and sensitive, but is also simple and cost-effective, requiring no expensive probes, laborious sequencing procedures. Accordingly, the assay has the potential to be applied in detecting YMDD mutants in patients with chronic hepatitis B receiving lamivudine therapy.2. Capillary direct PCR for rapid and sensitive detection of resistance gene New Delthi Metallo-bata-lactamase (NDM-1) Objective: To develope a rapid and sensitive capillary direct PCR molecular assays for detecting blaNDM-1 in carbapenem-resistant Enterobacteriaceae from clinical isolates. Methods: The capillary direct PCR was developed using the high-performance improved DNA polymerase, and combined with microchip electrophoresis to rapidly identificate NDM-1 gene positive bacteria. The enzyme,samples reagents, and the annealing temperature and extension time in NDM-1 gene amplification were optimized. The sensitivity and specificity of the capillary direct PCR assay were studied by adding the inner standard controls. The NDM-1 genes of 70 strains Enterobacteriaceae and 55 strains Acinetobacter baumannii ( imipenem MIC≥64μg / ml) were tested by both the capillary direct PCR and the conventional PCR. The positive samples all were analyzed by DNA sequencing.Results: Under the optimum analysis conditions, the capillary direct PCR assay requires low reaction volume (3μL) and short NDM-1 gene amplification time (16 min). The effect of the capillary direct PCR was increasing with the concentration of the high-performance improved DNA polymerase. When the addition volume of enzyme was 0.5μL, the PCR effect was best. The most suitable sample volume was 2μL; When the each cycle suspended 10 s at the temperature of 68℃, to extended the annealing and extension time, that will achieve the maximum of fluorescence signal. Combined with microchip electrophoresis testing methods, the minimum bacterial concentration as blaNDM-1 was 1.15×102 CFU /mL. Two of 125 clinical samples were identified to be NDM-1 gene positive by the two assays. To compare with the sequence of NDM-1 gene at Genbank, two results of DNA sequencing was accordant (100% of coincidence rate ).Conclusions: The method combined capillary direct PCR with microfluidic electrophoresis is able to rapid amplify and detect NDM-1 gene. The method is cost-effective, sample, rapid, accurate and sensitive. Therefore, it is suitable for rapid detection drug-resistant gene of clinical samples.