【作者】 王秋平；

【导师】 周小棉；

【作者基本信息】 广州医学院 ， 临床检验诊断学， 2011， 硕士

【摘要】 本论文工作利用微流控技术发展感染性疾病基因诊断方法,为微流控技术用于临床检验作出有益的尝试。论文研究内容包括:1.微流控芯片电泳用于乙型肝炎病毒YMDD基因变异检测目的:利用芯片电泳高分辨力和分析快速的优势,发展一种多重PCR与芯片电泳结合的方法,用于乙型肝炎病毒YMDD基因突变快速检测。方法:发展一种序列特异性多重PCR,用于YMDD变异基因:YVDD和YIDD的扩增。针对上述序列特异性多重PCR,发展一种芯片电泳DNA片段长度计算方法用于YVDD和YIDD基因扩增产物鉴定。将一定浓度的YMDD标准质粒与YVDD和YIDD标准质粒按不同比例混合,考察多重PCR-芯片电泳方法对上述混合样品中突变基因的检测灵敏度。使用本研究发展的YMDD基因变异检测方法分析86例经拉米夫定治疗慢性乙型肝炎患者血清样品中YMDD突变情况,并将其结果与荧光定量PCR比较。两种方法结果不一致的标本经测序鉴定。结果:芯片电泳法能够在3分钟内实现YMDD变异基因扩增产物的检测,并能够鉴定突变型(YVDD和YIDD)扩增产物。标准YVDD、YIDD质粒的检测限均达到101拷贝/μl;不同比例(V:M或I:M)混合质粒的分析结果显示,该方法可以检测含量为5%的YVDD变异以及含量10%的YIDD变异。86例经拉米夫定治疗的乙型肝炎患者标本中,芯片电泳法检测YMDD总变异65例突变率为75.6%(65/86),荧光定量PCR检出YMDD总变异62例,突变率为72.1%(62/86)。芯片电泳与荧光定量PCR法检测结果的χ2分析结果提示,两种方法所获得结果具有一致性(Kappa=0.609,P>0.05)。两种方法检测结果不一致的20例标本测序结果显示,芯片电泳法与测序结果符合率为95%。结论:本工作发展的结合多重PCR和芯片电泳的YMDD变异检测方法,不但具有准确、简单、快速、灵敏的特点,而且不需要昂贵的探针和费力测序程序,检测成本效益低。因此,该方法有潜力应用于乙型肝炎患者接受拉米夫定治疗的YMDD突变检测。2.基于毛细管直接PCR的新德里金属-β-内酰胺酶-1耐药基因快速和灵敏检测目的:利用毛细管直接PCR发展一种用于新德里金属-β-内酰胺酶-1(New Delthi Metallo-bata -Lactamase,NDM-1)耐药基因快速和灵敏检测的方法。方法:采用改良型高性能的DNA聚合酶,发展一种液体振荡流毛细管直接PCR,结合芯片电泳检测实现NDM-1基因阳性菌的快速鉴定。考察该方法进行NDM-1基因扩增的酶浓度、标本用量,以及扩增的退火温度和延伸时间。通过输入标准样品考察上述方法的灵敏度和特异性。利用毛细管直接PCR和常规PCR法对临床微生物检测的70株肠道杆菌和55株鲍曼不动杆菌(亚胺培南MIC≧64μg/ml)的NDM-1基因进行检测,并对阳性标本进行测序验证。结果:在优化的分析条件下,相比常规PCR,毛细管直接PCR反应所需试剂用量更少(只需3μL),完成DNA扩增时间更短(只需16 min)。毛细管直接PCR效果随着酶浓度的增加而提高,当酶加入量为0.5μL时趋向于饱和;最合适检测标本体积为2μL;每个循环中在68℃温区暂停10 s,将退火和延伸时间加长,芯片电泳荧光信号达最大值。结合芯片电泳检测,该方法检测最低限为1.15×102 CFU /mL。125例临床样本中,两种方法检测出2例NDM-1基因阳性。2例阳性标本基因扩增产物的测序结果与Genbank比对符合率为100%。结论毛细管直接PCR联合芯片电泳方法能实现NDM-1耐药基因的快速扩增和检测。该方法具有成本低廉、操作简单、反应快速、特异性强,高灵敏等特点,适合于临床NDM-1基因(+)耐药菌的早期快速检测。更多还原

【Abstract】 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.更多还原