基于微流控技术的感染性病原体基因诊断研究
|
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。
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 electrophoresis
Objective: 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.
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。
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 electrophoresis
Objective: 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.
引文
[1] Kim JK, Lee HJ, Lee YJ,et al. Direct detection of lamivudine-resistant hepatitis B virus mutants by a multiplex PCR using dual-priming oligonucleotide primers. J Virol Methods,2008,149(1):76-84
[2] Kobayashi M, Suzuki F, Akuta N, et al.Correlation of YMDD mutation and breakthrough hepatitis with hepatitis B virus DNA and serum ALT during lamivudine treatment. Hepatol Res,2010 ,40(2):125-134
[3] Dienstag,Jool dinR,Heatheote EJ,et al.Histologieal out come douring Long-term lamivudine therapy. Gastroenierology,2003,124:105-117
[4] Hosaka T, Suzuki F, Kobayashi M,et al. Development of HCC in patients receiving adefovir dipivoxil for lamivudine-resistant hepatitis B virus mutants. Hepatol Res, 2010,40(2): 145-152
[5] Selabe SG, Song E, Burnett RJ, Mphahlele MJ.Frequent detection of hepatitis B virus variants associated with lamivudine resistance in treated South African patients infected chronically with different HBV genotypes. J Med Virol,2009,81(6):996-1001
[6] Hashimoto Y, Suzuki F, Hirakawa M,et al.Clinical and virological effects of long-term (over 5 years) lamivudine therapy. J Med Virol, 2010 ,82(4):684-691
[7] Moucari R,Mackiewicz V,Ladaet O, et al . Early serum HBsAg drop: a strong predictor of sustained virological response to pegylated interferon alfa-2a in HBeAg-negative patients.Hepatology ,2009,49:1151-1157
[8] Libbrecht E,Doutreloigne J,Van H,et a1.Evolution of primary and compensatory lamivudine resistance mutations in chronic hepatitis B virus infected patients during long-term lamivudine treatment assessed by a line probe assay.Clin Microbiol, 2007, 45(12):3935-3941
[9] Kobayashi S, Ide T, Sata M.Detection of YMDD motif mutations in some lamivudine-untreated asymptomatic hepatitis B virus carriers. J Hepatol,2001,34(4):584-586
[10] Yoshida S, Hige S, Yoshida M, et al. Quantification of lamivudine-resistant hepatitis B virus mutants by type-specific TaqMan minor groove binder probe assay in patients with chronic hepatitis B. Ann Clin Biochem ,2008, 45: 59-64
[11] Wightman F, Walters T, Ayres A, et al. Bowden S,Comparison of sequence analysis and a novel discriminatory real-time PCR assay for detection and quantification of Lamivudine-resistant hepatitis B virus strains. J Clin Microbiol, 2004, 42(8): 3809-3812
[12] Wu D, Qin J, Lin B.Electrophoretic separations on microfluidic chips. J Chromatogr A, 2008,14:542-59
[13] Li SF, Kricka LJ.Clinical analysis by microchip capillary electrophoresis. Clin Chem, 2006, 52(1):37-45
[14] Kim JK, Lee HJ, Lee YJ,et al. Direct detection of lamivudine-resistant hepatitis B virus mutants by a multiplex PCR using dual-priming oligonucleotide primers. Journal of Virological Methods ,2008, 149(1):76-84
[15] ZhouX, LiuD, Zhong R,et al. Determination of SARS-coronavirus by a microfluidic chip system.Electrophoresis,2004,25(17),3032-3039
[16]周小棉,戴忠鹏,罗勇,等.注塑型聚甲基丙烯酸甲酯多通道微流控芯片的研制及其性能考察.高等学校化学学报,2005,26: 52-54
[17]周小棉,戴忠鹏,林炳承.通用型激光诱导荧光微流控芯片分析仪的研制与性能考察.高等学校化学学报,2004,3: 436-440
[18] Liu D, Zhou X, Zhong R, et al. Analysis of multiplex PCR fragments with PMMA microchip. Talanta,2006,68(3): 616-622
[19] Liu D, Chen B, Wang L, Zhou X. On-chip coupling of free-solution transient ITP and CGE for highly efficient separation of dsDNA with variable sample loading amounts. Electrophoresis,2009 ,30(24):4300-4305
[20] Shih Y,Yeh S,Chen P,et a1.Hepatitis B virus quantification and detection of YMDD mutants in a single reaction by real-time PCR and annealing curve analysis.Antivir Ther,2008,13(4):469-480
[21] Yoshida S,Hige S,Yoshida M,et a1.Quantification of lamivudine-resistant hepatitis B virus mutants by type-specific TaqMan minor groove binder probe assay in patients with chronic hepatitis B.Ann Clin Biochem,2008,45(1):59-64
[22]李永新,黎源倩,渠凌丽等.微流控芯片-激光诱导荧光快速检测4种食源性致病菌.分析化学,2008, 36, 12:1667-1671
[23] Bulent D, Munira H,et al. Sensitivity and accuracy of an updated line probe assay (HBV DR v.3) in detecting mutations associated with hepatitis B antiviral resistance. Journal of Hepatology ,2009,50:42-48
[24] Ntziora F, Paraskevis D, Haida C, et al. Detection of the M204V Hepatitis B Virus Minor Variants by Amplification Refractory Mutation System Real-Time PCR Combined with Molecular Beacon Technology, J Clin Microbiol,2009, 8:2544-2550
[25] Harrison DJ, Fluri K, Seiler K,et al. Micromachining a Miniaturized Capillary Electrophoresis-Based Chemical Analysis System on a Chip.Science,1993, 261(5123):895-897
[26] Chen L, Manz A,Day PJR.Total nucleic acid analysis integrated on microfluidic devices.Lab on a chip,2007,7(11):1413-1423
[27] Sinville R,Soper SA. High resolution DNA separations using microchip electrophoresis. J. Sep. Sci,2007, 30(11): 1714-1728
[28] Williams PE, Marino MA, Del Rio SA, et al. Analysis of DNA restriction fragments and polymerase chain reaction products by capillary electrophoresis. J Chromatogr A,1994,680(2):525-540
[1] Kumarasamy KK, Toleman MA, Walsh TR, et al. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis,2010,10(9):597-602
[2] Yong D, Toleman MA, Giske CG, et al. Characterization of a new metallo-beta -lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother,2009, 53: 5046-5054
[3] Muir A,Weinbren MJ.New Delhi metallo-beta-lactamase:a cautionary tale.J Hosp Infect,2010,75(3):239240
[4] Livermore DM, Walsh TR, Toleman M, Woodford N. Balkan.NDM-1: escape or transplant? Lancet Infect Dis 2011, 11: 164
[5] Rolain JM, Parola P, Cornaglia G.New Delhi metallo-b-lactamase(NDM-1): towards a new pandemia? Clin Microbiol Infect, 2010, 16:1699-701
[6] ChenY,ZhouZ, JiangY, YuY.Emergence of NDM-1-producing Acinetobacter baumannii in China. J Antimicrob Chemother,2011,3(10):1-5
[7] Karthikeyan K,Thirunarayan MA, Krishnan P.Coexistence of blaOXA-23 with blaNDM-1 and armA in clinical isolates of Acinetobacter baumannii from India. J Antimicrob Chemother, 2010,65:2253-2254
[8] Deshpande P, Rodrigues C, Shetty A, Kapadia F. New Delhi Metallo-beta lactamase (NDM-1) in Enterobacteriaceae:treatment options with carbapenems compromised.J Assoc Physicians India ,2010,58:147-149
[9] Wu D, Qin J, Lin B. Electrophoretic separations on microfluidic chips. J Chromatogr A, 2008,14, 184:542-559
[10] Liu P, Mathies RA.Integrated microfluidic systems for high-performance genetic analysis. Trends in Biotechnology,2009, 27(10):572-581
[11] Mir M, Homs A, Samitier J. Integrated electrochemical DNA biosensors for lab-on-a-chip devices. Electrophoresis, 2009,30(19): 3386-3397
[12] Chen L, Manz A, Day P.Total nucleic acid analysis integrated on microfluidic devices. Lab on a Chip, 2007, 7(11):1413-1423
[13] Horsman K, Bienvenue J, BlasierK, Landers, J. Forensic DNA analysis on microfluidic devices: A review.Journal of Forensic Sciences, 2007,52(4): 784-799
[14] Chiou J, Matsudaira P, Sonin A, Ehrlich D. A closed-cycle capillary polymerase chain reaction machine. Anal Chem ,2001,73(9):2018-21
[15] Chen L, West J, Auroux PA, Manz A, Day PJ. Ultrasensitive PCR and real-time detection from human genomic samples using a bidirectional flow microreactor. Anal Chem ,2007,79(23):9185-90
[16] Wang HY, Zhang CS, Li YY. Rapid Detection of Tobacco Mosaic Virus from Crude Samples on Oscillatory-flow Reverse Transcription Polymerase Chain Reaction Microfluidics. Chn. J. Anal Chem,2009,37(9):1286-1290
[17] Grisolia,AB. Targeted nucleotide exchange in bovine myostatin gene[J]. Anim Biotechnol,2009,20(1):15-27
[18] Oyama T.Atomic structures and functional implications of the archaeal RecQ-like helicase Hjm . BMC Struct Biol, 2009,22(1):92
[19] Kermekchiev MB, Kirilova LI, Vail EE, et al. Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples . Nucleic Acids Res,2009,7(5):40
[20] LiuD, et al.Analysis of multiplex PCR fragments with PMMA microchip. Talanta, 2006,68:616-622
[21] Liu D, et al. Simplified transient isotachophoresis/capillary gel electrophoresis method for highly sensitive analysis of polymerase chain reaction samples on a microchip with laser-induced fluorescence detection. J Chromatogr A, 2008, 12(14): 165-170
[22] LIANG Guang-Tie, WANG Qiu-Ping, WANG Wei,et al. Droplet-based Blood Hepatitis B Virus DNA extraction in a capillary .ChineseJ.Anal.Chem, 2011,39(5):670-674
[23] Da Yu L, Guang-Tie L, Xiao-Mian Z,et al. Precise prediction of DNA sizes with transient isotachophoresis-capillary gel electrophoresis analysis on a microchip. Chn J Anal Chem, 2010,38:15-20
[24] Walsh TR. Emerging carbapenemases: a global perspective.Int J Antimicrob Agents ,2010,36 (3):8-14
[25] Livermore DM. Has the era of untreatable infections arrived?J Antimicrob Chemother,2009,64 (1):29-36
[26] Krüttgen A, Razavi S, Imohl M, Ritter K.Real-time PCR assay and a synthetic positive control for the rapid and sensitive detection of the emerging resistance gene New Delhi Metallo-β-lactamase-1 (bla (NDM-1)).Med Microbiol Immunol,2011, 200(2):137-141
[1] Weigl B, Domingo G, LaBarre P, Gerlach J. Towards non- and minimally instrumented, microfluidics-based diagnostic devices. Lab on a Chip,2008,8:1999-2014
[2] Hay Burgess DC, Wasserman J, Dahl CA. Global health diagnostics.Nature, 2006, 11(23):1-2
[3] Schulze H, Giraud G, Crain J, Bachmann TT. Multiplexed optical pathogen detection with lab-on-a-chip devices. J Biophotonics,2009,2(4):199-211
[4]方肇伦,方群.微流控芯片发展与展望.现代科学仪器,2001,4(1):3-6
[5] [5]何晨光,杨春江,王馨.微流控芯片技术的研究进展.中国国境卫生检疫杂志,2010, 10(33):5
[6] Manz A.An international journal devoted to research and developmentof chemical transducers.Chromatogry,1992,593:253-258
[7] Woolley AT , Fuctional Intergration of PCR Amplification andCapillary Electrophoresis in a Microfabricated DNA Analysis Chips. Anal Chem,1996,68:4081-4086
[8] Anderson RC, Su X, Bogdan G, et al. A miniature integrated device for automated multistep genetic assays. Nucleic Acids Res,2000,15:60
[9] Auroux PA, Koc, et al. Miniaturized nucleicacid analysis. Lab Chip,2004, (4):534-546
[10] Roper MG., Easley, et al. Advancesin polymerase chain reactionon microuidic chips. Anal Chem, 2007,7:3887-3894
[11] ChoYK, Kim J, Lee Y, et al. Clinical evaluation of micro-scale chip-based PCR system for rapid detection of hepatitisB virus. Biosens Bioelectron,2006, (21):2161-2169
[12] GnanappaAK, Cathy K, Slattery O, Sheehan M. Thermal performance analysis of asilicon microreactor for rapid DNA analysis.In The Tenth Intersociety Conference on Thermaland Thermome chanical Phenomenain ElectronicsSystems, 2006:1330-1335
[13] Hataoka Y, Zhang LH, Yukimasa T, Baba Y. Rapid microvolume PCR of DNA conrmed by microchip electrophoresis.Anal Sci ,2005,(2):53-56
[14] Ke C, Kelleher AM, Berney H, Sheehan M. Single step cell lysis/PCR detection of Escherichia Coli inanin dependently control lable silicon microreactor. Sens. Actuators B Chem,2007,120:538-544
[15] Kim SH,Noh J, Jeon MK. Micro-Raman thermome try for measuring the temperature distribution inside the microchannel of apolymerase chain reaction chip. J Micromech Microeng ,2006,(16):526-530
[16] Lee JG.,Cheong KH,Huh N. Microchip-based one step DNA extraction and real-time PCR in one chamber for rapid pathogen identication.LabChip,2006(6):886-895
[17] Consolandi C,Sever M, Frosini A,et al. Polymerase chain reaction of 2-kb cyano bacterial geneand human anti-a-chymotrypsingene from genomic DNA on In-Check1 single-use microfabricate dsilicon chip. Anal Biochem, 2006,353:191-197
[18] Oh KW, Park C, Namkoong K, et al. World-to-chip microuidic interface with built-invalves form ultichamber chip-based PCR assays.LabChip, 2005,5:845-850
[19] Tewhey R, Warner JB, Nakano M, et al.Microdroplet-based PCR enrichment for large-scale targeted sequencing.Nat Biotechnol,2009, 27 (11):1025-1031
[20] Becker H, G?rtner C.Polymer microfabrication technologies for microfluidic systems. Analytical and Bioanalytical Chemistry, 2008, 390(1): 89-111
[21] Muck A, Svatos A.Chemical modification of polymeric microchip devices. Talanta, 2007,74(3): 333-341
[22] LuoY, Qin JH, Lin BC.Methods for pumping fluids on biomedical lab-on-a-chip. Frontiers in Bioscience, 2009, 14:3913-3924.
[23] Lin WY, Wang YJ, Wang ST, Tseng HR.Integrated microfluidic reactors. Nano Today, 2009,4(6):470-481
[24] Mansur EA, Ye MX, Wang YD, Dai YY. A state-of-the-art review of mixing in microfluidic mixers. Chinese Journal of Chemical Engineering, 2008, 16(4): 503-516.
[25] Wu DP, Qin JH, Lin BC.Electrophoretic separations on microfluidic chips. Journal of Chromatography A,2008,1184(1-2):542-559
[26] Teh SY, Lin R, Hung LH, Lee AP. Droplet microfluidics. Lab on a Chip, 2008, 8(2): 198-220
[27] Wang F, Burns MA.Performance of nanoliter-sized droplet-based microfluidic PCR. Biomedical Microdevices, 2009,11(5):1071-1080
[28] Xu T, Chakrabarty K.Functional testing of digital microfluidic biochips. 2007 Ieee International Test Conference, 2007: 505-514
[29] Atencia J, Beebe, DJ. Controlled microfluidic interfaces. Nature,2005, 437(7059): 648-655
[30] Liu P, Mathies RA. Integrated microfluidic systems for high-performance genetic analysis. Trends in Biotechnology,2009,27(10):572-581
[31] Duffy DC,Mcdonald JC,Schneller OJA, et al. Rapid Prototyping ofMicrofluidic Systems in Ploy (dimethylsiloxane). Anal Chem,1998,70:4974-4978
[32] Heyl P,Okchewski T,Wijnaendts RW.Microelectronic Manufacturing of 3D structures for microtools using laser ablation. Engineering,2001, 775:57-58
[33] Unger MA, Chou HP, Thorsen T, et al. Monolithic microfabricated valves and pumps by multilayer soft lithography.Science,2000,288(5463):113-116
[34] William H, Grovera, Alison M.,et al.Monolithic membrane valves and diaphragm pumps for practical large-scale integration into glass microfluidic devices.sensors and actuators B-chemica,2003,89(3):315-323
[35] Liu P, Mathies RA. Integrated microfluidic systems for high-performance genetic analysis. Trends in Biotechnology,2009, 27(10):572-581
[36] Mir M, Homs A, Samitier J. Integrated electrochemical DNA biosensors for lab-on-a-chip devices. Electrophoresis, 2009,30(19): 3386-3397
[37] Chen L, Manz A, Day PJR .Total nucleic acid analysis integrated on microfluidicdevices. Lab on a Chip, 2007, 7(11):1413-1423
[38] orsman KM, Bienvenue JM, Blasier K R, Landers JP. Forensic DNA analysis on microfluidic devices: A review.Journal of Forensic Sciences, 2007,52(4):784-799
[39] W. T. Liu and L. Zhu, Environmental microbiology on-a-chip and its future impacts. Trends in Biotechnology, 2005,23:174-179
[40] FS Ligler. Perspective on Optical Biosensors and Integrated Sensor Systems. Analytical Chemistry ,2009,81:519-526
[41] JW Hong, V Studer, G Hang.A nanoliter -scale nucleic acid processor with parallel architecture. Nature Biotechnology, 2004 ,22:435-439
[42] JG Lee, KH Cheong, N Huh, S Kim.Microchip-based one step DNA extraction and real-time PCR in one chamber for rapid pathogen identification. Lab on A Chip ,2006,6: 886-895
[43] Wan W, Yeow JT.Study of a novel cell lysis method with titanium dioxide for Lab-on-a-Chip devices. Biomed Microdevices, 2011,5(3)
[44] Ferguson BS, Buchsbaum SF, Swensen JS. Integrated Microfluidic Electrochemical DNA Sensor. Analytical Chemistry, 2009, 81(15): 6503-6508.
[45] Fujita SI, Yosizaki K, Ogushi T, et al. Rapid identification of Gram-negative bacteria with and without CTX-M extended-spectrum {beta}-lactamase from positive blood culture bottles by PCR followed by microchip gel electrophoresis. J Clin Microbiol, 2011,(2)
[46] Kang MH, Kim IW, Lee DW, et al. Development of a rapid detection method to detect tdh gene in Vibrio parahaemolyticus using 2-step ultrarapid real-time polymerase chain reaction. Diagn Microbiol Infect Dis,2011, 69(1):21-29
[47] CJ Easley, JM Karlinsey, JM Bienvenue, et al. A fully integrated microfluidic genetic analysis system with sample-in-answer-out capability. Proceedings of the National Academy of Sciences of the United States of America,2006, 103:19272-19277
[48] Yi Zhang, Seungkyung Park, Kelvin Liu, et al.A surface topography assisted droplet manipulation platform for biomarker detection and pathogenidentification,Lab Chip, 2011, 11:398-406
[49] Liu D, Shi M, Huang H, et al.Isotachophoresis preconcentration integrated microfluidic chip for highly sensitive genotyping of the hepatitis B virus. J Chromatogr B Analyt Technol Biomed Life Sci,2006, 844 (1):32-38
[50] Pal R.,Yang M, Lin R, et al. An integrated microfluidic device for influenza and other genetic analyses. Lab Chip,2005,5 (10):1024-1032
[51] Liu RH, Lodes MJ, Nguyen T,et al.Validation of a fully integrated microfluidic array device for influenza A subtype identification and sequencing. Anal Chem,2006, 78 (12):4184-4193
[52] WC Lee, KY Lien, GB Lee, and HY Lei. An integrated microfluidic system using magnetic beads for virus detection. Diagnostic Microbiology and Infectious Disease,2008, 60:51-58
[53] Kang-Yi Lien, Jr-Lung Lin, Cheng-Yu Liu ,et al.Purification and enrichment of virus samples utilizing magnetic beads on a microfluidic system.Lab Chip, 2007, 7:868-875
[54] Kristin A, Carmen R, Mari L, et al. An integrated valveless system for microfluidic purification and reverse transcription-PCR amplification of RNA for detection of infectious agents,Lab Chip,2011,11:957-961
[55] Keiichiro Yamanaka, Masato Saito, Kenji Kondoh, et al. Rapid detection for primary screening of influenza A virus: microfluidic RT-PCR chip and electrochemical DNA sensor.Analyst,2011, Advance Article
[56] Gerald M. Birnbaumer, Peter A. Lieberzeit, Lukas Richter, et al. Detection of viruses with molecularly imprinted polymers integrated on a microfluidic biochip using contact-less dielectric microsensors.Lab Chip,2009,9:3549-3556
[57] Yi Sun, Raghuram D, Dang DB, et al.A lab-on-a-chip device for rapid identification of avian influenza viral RNA by solid-phase PCR.Lab Chip, 2011,11:1457-1463
[58] Yao Lu, Weiwei Shi, Jianhua Qin , Bingcheng Lin Analytical.Fabrication and Characterization of Paper-Based Microfluidics Prepared in NitrocelluloseMembrane By Wax Printing,Chemistry ,2010,82 (1):329-335
[59] Wang CH, Kang YL, et al. A magnetic bead-based assay for the rapid detection of methicillin-resistant Staphylococcus aureus by using a microfluidic system with integrated loop-mediated isothermal amplification.Lab Chip, 2011, 11:1521-1531
[60] Weiwei Shi, Hui Wen, Yao Lu, et al. Droplet microfluidics for characterizing the neurotoxin-induced responses in individual Caenorhabditis elegans,Lab Chip,2010, 10:2855-2863
[61] PanaroNJ,Yuen PK, Sakazume T,et al.Evaluation of DNA fragment sizing and quantification by the agilent 2100 bioanalyzer.Clin Chem,2000,46 (11): 1851-1853
[62] http://www.chem.agilent.com/en-us/products/instruments/lab-on-a-chip/2100bioanalyzer/Pages/default.aspx
[63] Liu RH, Yang J, Lenigk R,et al. Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection. Anal Chem,2004,76 (7):1824-1831
[64] Ruano JM, Agirr M, Olabarria G, et al.The SmartBioPhone, a point of care vision under development through two European projects: OPTOLABCARD and LABONFOIL.Lab Chip,2009, 9(11):1495-1499
[2] Kobayashi M, Suzuki F, Akuta N, et al.Correlation of YMDD mutation and breakthrough hepatitis with hepatitis B virus DNA and serum ALT during lamivudine treatment. Hepatol Res,2010 ,40(2):125-134
[3] Dienstag,Jool dinR,Heatheote EJ,et al.Histologieal out come douring Long-term lamivudine therapy. Gastroenierology,2003,124:105-117
[4] Hosaka T, Suzuki F, Kobayashi M,et al. Development of HCC in patients receiving adefovir dipivoxil for lamivudine-resistant hepatitis B virus mutants. Hepatol Res, 2010,40(2): 145-152
[5] Selabe SG, Song E, Burnett RJ, Mphahlele MJ.Frequent detection of hepatitis B virus variants associated with lamivudine resistance in treated South African patients infected chronically with different HBV genotypes. J Med Virol,2009,81(6):996-1001
[6] Hashimoto Y, Suzuki F, Hirakawa M,et al.Clinical and virological effects of long-term (over 5 years) lamivudine therapy. J Med Virol, 2010 ,82(4):684-691
[7] Moucari R,Mackiewicz V,Ladaet O, et al . Early serum HBsAg drop: a strong predictor of sustained virological response to pegylated interferon alfa-2a in HBeAg-negative patients.Hepatology ,2009,49:1151-1157
[8] Libbrecht E,Doutreloigne J,Van H,et a1.Evolution of primary and compensatory lamivudine resistance mutations in chronic hepatitis B virus infected patients during long-term lamivudine treatment assessed by a line probe assay.Clin Microbiol, 2007, 45(12):3935-3941
[9] Kobayashi S, Ide T, Sata M.Detection of YMDD motif mutations in some lamivudine-untreated asymptomatic hepatitis B virus carriers. J Hepatol,2001,34(4):584-586
[10] Yoshida S, Hige S, Yoshida M, et al. Quantification of lamivudine-resistant hepatitis B virus mutants by type-specific TaqMan minor groove binder probe assay in patients with chronic hepatitis B. Ann Clin Biochem ,2008, 45: 59-64
[11] Wightman F, Walters T, Ayres A, et al. Bowden S,Comparison of sequence analysis and a novel discriminatory real-time PCR assay for detection and quantification of Lamivudine-resistant hepatitis B virus strains. J Clin Microbiol, 2004, 42(8): 3809-3812
[12] Wu D, Qin J, Lin B.Electrophoretic separations on microfluidic chips. J Chromatogr A, 2008,14:542-59
[13] Li SF, Kricka LJ.Clinical analysis by microchip capillary electrophoresis. Clin Chem, 2006, 52(1):37-45
[14] Kim JK, Lee HJ, Lee YJ,et al. Direct detection of lamivudine-resistant hepatitis B virus mutants by a multiplex PCR using dual-priming oligonucleotide primers. Journal of Virological Methods ,2008, 149(1):76-84
[15] ZhouX, LiuD, Zhong R,et al. Determination of SARS-coronavirus by a microfluidic chip system.Electrophoresis,2004,25(17),3032-3039
[16]周小棉,戴忠鹏,罗勇,等.注塑型聚甲基丙烯酸甲酯多通道微流控芯片的研制及其性能考察.高等学校化学学报,2005,26: 52-54
[17]周小棉,戴忠鹏,林炳承.通用型激光诱导荧光微流控芯片分析仪的研制与性能考察.高等学校化学学报,2004,3: 436-440
[18] Liu D, Zhou X, Zhong R, et al. Analysis of multiplex PCR fragments with PMMA microchip. Talanta,2006,68(3): 616-622
[19] Liu D, Chen B, Wang L, Zhou X. On-chip coupling of free-solution transient ITP and CGE for highly efficient separation of dsDNA with variable sample loading amounts. Electrophoresis,2009 ,30(24):4300-4305
[20] Shih Y,Yeh S,Chen P,et a1.Hepatitis B virus quantification and detection of YMDD mutants in a single reaction by real-time PCR and annealing curve analysis.Antivir Ther,2008,13(4):469-480
[21] Yoshida S,Hige S,Yoshida M,et a1.Quantification of lamivudine-resistant hepatitis B virus mutants by type-specific TaqMan minor groove binder probe assay in patients with chronic hepatitis B.Ann Clin Biochem,2008,45(1):59-64
[22]李永新,黎源倩,渠凌丽等.微流控芯片-激光诱导荧光快速检测4种食源性致病菌.分析化学,2008, 36, 12:1667-1671
[23] Bulent D, Munira H,et al. Sensitivity and accuracy of an updated line probe assay (HBV DR v.3) in detecting mutations associated with hepatitis B antiviral resistance. Journal of Hepatology ,2009,50:42-48
[24] Ntziora F, Paraskevis D, Haida C, et al. Detection of the M204V Hepatitis B Virus Minor Variants by Amplification Refractory Mutation System Real-Time PCR Combined with Molecular Beacon Technology, J Clin Microbiol,2009, 8:2544-2550
[25] Harrison DJ, Fluri K, Seiler K,et al. Micromachining a Miniaturized Capillary Electrophoresis-Based Chemical Analysis System on a Chip.Science,1993, 261(5123):895-897
[26] Chen L, Manz A,Day PJR.Total nucleic acid analysis integrated on microfluidic devices.Lab on a chip,2007,7(11):1413-1423
[27] Sinville R,Soper SA. High resolution DNA separations using microchip electrophoresis. J. Sep. Sci,2007, 30(11): 1714-1728
[28] Williams PE, Marino MA, Del Rio SA, et al. Analysis of DNA restriction fragments and polymerase chain reaction products by capillary electrophoresis. J Chromatogr A,1994,680(2):525-540
[1] Kumarasamy KK, Toleman MA, Walsh TR, et al. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis,2010,10(9):597-602
[2] Yong D, Toleman MA, Giske CG, et al. Characterization of a new metallo-beta -lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother,2009, 53: 5046-5054
[3] Muir A,Weinbren MJ.New Delhi metallo-beta-lactamase:a cautionary tale.J Hosp Infect,2010,75(3):239240
[4] Livermore DM, Walsh TR, Toleman M, Woodford N. Balkan.NDM-1: escape or transplant? Lancet Infect Dis 2011, 11: 164
[5] Rolain JM, Parola P, Cornaglia G.New Delhi metallo-b-lactamase(NDM-1): towards a new pandemia? Clin Microbiol Infect, 2010, 16:1699-701
[6] ChenY,ZhouZ, JiangY, YuY.Emergence of NDM-1-producing Acinetobacter baumannii in China. J Antimicrob Chemother,2011,3(10):1-5
[7] Karthikeyan K,Thirunarayan MA, Krishnan P.Coexistence of blaOXA-23 with blaNDM-1 and armA in clinical isolates of Acinetobacter baumannii from India. J Antimicrob Chemother, 2010,65:2253-2254
[8] Deshpande P, Rodrigues C, Shetty A, Kapadia F. New Delhi Metallo-beta lactamase (NDM-1) in Enterobacteriaceae:treatment options with carbapenems compromised.J Assoc Physicians India ,2010,58:147-149
[9] Wu D, Qin J, Lin B. Electrophoretic separations on microfluidic chips. J Chromatogr A, 2008,14, 184:542-559
[10] Liu P, Mathies RA.Integrated microfluidic systems for high-performance genetic analysis. Trends in Biotechnology,2009, 27(10):572-581
[11] Mir M, Homs A, Samitier J. Integrated electrochemical DNA biosensors for lab-on-a-chip devices. Electrophoresis, 2009,30(19): 3386-3397
[12] Chen L, Manz A, Day P.Total nucleic acid analysis integrated on microfluidic devices. Lab on a Chip, 2007, 7(11):1413-1423
[13] Horsman K, Bienvenue J, BlasierK, Landers, J. Forensic DNA analysis on microfluidic devices: A review.Journal of Forensic Sciences, 2007,52(4): 784-799
[14] Chiou J, Matsudaira P, Sonin A, Ehrlich D. A closed-cycle capillary polymerase chain reaction machine. Anal Chem ,2001,73(9):2018-21
[15] Chen L, West J, Auroux PA, Manz A, Day PJ. Ultrasensitive PCR and real-time detection from human genomic samples using a bidirectional flow microreactor. Anal Chem ,2007,79(23):9185-90
[16] Wang HY, Zhang CS, Li YY. Rapid Detection of Tobacco Mosaic Virus from Crude Samples on Oscillatory-flow Reverse Transcription Polymerase Chain Reaction Microfluidics. Chn. J. Anal Chem,2009,37(9):1286-1290
[17] Grisolia,AB. Targeted nucleotide exchange in bovine myostatin gene[J]. Anim Biotechnol,2009,20(1):15-27
[18] Oyama T.Atomic structures and functional implications of the archaeal RecQ-like helicase Hjm . BMC Struct Biol, 2009,22(1):92
[19] Kermekchiev MB, Kirilova LI, Vail EE, et al. Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples . Nucleic Acids Res,2009,7(5):40
[20] LiuD, et al.Analysis of multiplex PCR fragments with PMMA microchip. Talanta, 2006,68:616-622
[21] Liu D, et al. Simplified transient isotachophoresis/capillary gel electrophoresis method for highly sensitive analysis of polymerase chain reaction samples on a microchip with laser-induced fluorescence detection. J Chromatogr A, 2008, 12(14): 165-170
[22] LIANG Guang-Tie, WANG Qiu-Ping, WANG Wei,et al. Droplet-based Blood Hepatitis B Virus DNA extraction in a capillary .ChineseJ.Anal.Chem, 2011,39(5):670-674
[23] Da Yu L, Guang-Tie L, Xiao-Mian Z,et al. Precise prediction of DNA sizes with transient isotachophoresis-capillary gel electrophoresis analysis on a microchip. Chn J Anal Chem, 2010,38:15-20
[24] Walsh TR. Emerging carbapenemases: a global perspective.Int J Antimicrob Agents ,2010,36 (3):8-14
[25] Livermore DM. Has the era of untreatable infections arrived?J Antimicrob Chemother,2009,64 (1):29-36
[26] Krüttgen A, Razavi S, Imohl M, Ritter K.Real-time PCR assay and a synthetic positive control for the rapid and sensitive detection of the emerging resistance gene New Delhi Metallo-β-lactamase-1 (bla (NDM-1)).Med Microbiol Immunol,2011, 200(2):137-141
[1] Weigl B, Domingo G, LaBarre P, Gerlach J. Towards non- and minimally instrumented, microfluidics-based diagnostic devices. Lab on a Chip,2008,8:1999-2014
[2] Hay Burgess DC, Wasserman J, Dahl CA. Global health diagnostics.Nature, 2006, 11(23):1-2
[3] Schulze H, Giraud G, Crain J, Bachmann TT. Multiplexed optical pathogen detection with lab-on-a-chip devices. J Biophotonics,2009,2(4):199-211
[4]方肇伦,方群.微流控芯片发展与展望.现代科学仪器,2001,4(1):3-6
[5] [5]何晨光,杨春江,王馨.微流控芯片技术的研究进展.中国国境卫生检疫杂志,2010, 10(33):5
[6] Manz A.An international journal devoted to research and developmentof chemical transducers.Chromatogry,1992,593:253-258
[7] Woolley AT , Fuctional Intergration of PCR Amplification andCapillary Electrophoresis in a Microfabricated DNA Analysis Chips. Anal Chem,1996,68:4081-4086
[8] Anderson RC, Su X, Bogdan G, et al. A miniature integrated device for automated multistep genetic assays. Nucleic Acids Res,2000,15:60
[9] Auroux PA, Koc, et al. Miniaturized nucleicacid analysis. Lab Chip,2004, (4):534-546
[10] Roper MG., Easley, et al. Advancesin polymerase chain reactionon microuidic chips. Anal Chem, 2007,7:3887-3894
[11] ChoYK, Kim J, Lee Y, et al. Clinical evaluation of micro-scale chip-based PCR system for rapid detection of hepatitisB virus. Biosens Bioelectron,2006, (21):2161-2169
[12] GnanappaAK, Cathy K, Slattery O, Sheehan M. Thermal performance analysis of asilicon microreactor for rapid DNA analysis.In The Tenth Intersociety Conference on Thermaland Thermome chanical Phenomenain ElectronicsSystems, 2006:1330-1335
[13] Hataoka Y, Zhang LH, Yukimasa T, Baba Y. Rapid microvolume PCR of DNA conrmed by microchip electrophoresis.Anal Sci ,2005,(2):53-56
[14] Ke C, Kelleher AM, Berney H, Sheehan M. Single step cell lysis/PCR detection of Escherichia Coli inanin dependently control lable silicon microreactor. Sens. Actuators B Chem,2007,120:538-544
[15] Kim SH,Noh J, Jeon MK. Micro-Raman thermome try for measuring the temperature distribution inside the microchannel of apolymerase chain reaction chip. J Micromech Microeng ,2006,(16):526-530
[16] Lee JG.,Cheong KH,Huh N. Microchip-based one step DNA extraction and real-time PCR in one chamber for rapid pathogen identication.LabChip,2006(6):886-895
[17] Consolandi C,Sever M, Frosini A,et al. Polymerase chain reaction of 2-kb cyano bacterial geneand human anti-a-chymotrypsingene from genomic DNA on In-Check1 single-use microfabricate dsilicon chip. Anal Biochem, 2006,353:191-197
[18] Oh KW, Park C, Namkoong K, et al. World-to-chip microuidic interface with built-invalves form ultichamber chip-based PCR assays.LabChip, 2005,5:845-850
[19] Tewhey R, Warner JB, Nakano M, et al.Microdroplet-based PCR enrichment for large-scale targeted sequencing.Nat Biotechnol,2009, 27 (11):1025-1031
[20] Becker H, G?rtner C.Polymer microfabrication technologies for microfluidic systems. Analytical and Bioanalytical Chemistry, 2008, 390(1): 89-111
[21] Muck A, Svatos A.Chemical modification of polymeric microchip devices. Talanta, 2007,74(3): 333-341
[22] LuoY, Qin JH, Lin BC.Methods for pumping fluids on biomedical lab-on-a-chip. Frontiers in Bioscience, 2009, 14:3913-3924.
[23] Lin WY, Wang YJ, Wang ST, Tseng HR.Integrated microfluidic reactors. Nano Today, 2009,4(6):470-481
[24] Mansur EA, Ye MX, Wang YD, Dai YY. A state-of-the-art review of mixing in microfluidic mixers. Chinese Journal of Chemical Engineering, 2008, 16(4): 503-516.
[25] Wu DP, Qin JH, Lin BC.Electrophoretic separations on microfluidic chips. Journal of Chromatography A,2008,1184(1-2):542-559
[26] Teh SY, Lin R, Hung LH, Lee AP. Droplet microfluidics. Lab on a Chip, 2008, 8(2): 198-220
[27] Wang F, Burns MA.Performance of nanoliter-sized droplet-based microfluidic PCR. Biomedical Microdevices, 2009,11(5):1071-1080
[28] Xu T, Chakrabarty K.Functional testing of digital microfluidic biochips. 2007 Ieee International Test Conference, 2007: 505-514
[29] Atencia J, Beebe, DJ. Controlled microfluidic interfaces. Nature,2005, 437(7059): 648-655
[30] Liu P, Mathies RA. Integrated microfluidic systems for high-performance genetic analysis. Trends in Biotechnology,2009,27(10):572-581
[31] Duffy DC,Mcdonald JC,Schneller OJA, et al. Rapid Prototyping ofMicrofluidic Systems in Ploy (dimethylsiloxane). Anal Chem,1998,70:4974-4978
[32] Heyl P,Okchewski T,Wijnaendts RW.Microelectronic Manufacturing of 3D structures for microtools using laser ablation. Engineering,2001, 775:57-58
[33] Unger MA, Chou HP, Thorsen T, et al. Monolithic microfabricated valves and pumps by multilayer soft lithography.Science,2000,288(5463):113-116
[34] William H, Grovera, Alison M.,et al.Monolithic membrane valves and diaphragm pumps for practical large-scale integration into glass microfluidic devices.sensors and actuators B-chemica,2003,89(3):315-323
[35] Liu P, Mathies RA. Integrated microfluidic systems for high-performance genetic analysis. Trends in Biotechnology,2009, 27(10):572-581
[36] Mir M, Homs A, Samitier J. Integrated electrochemical DNA biosensors for lab-on-a-chip devices. Electrophoresis, 2009,30(19): 3386-3397
[37] Chen L, Manz A, Day PJR .Total nucleic acid analysis integrated on microfluidicdevices. Lab on a Chip, 2007, 7(11):1413-1423
[38] orsman KM, Bienvenue JM, Blasier K R, Landers JP. Forensic DNA analysis on microfluidic devices: A review.Journal of Forensic Sciences, 2007,52(4):784-799
[39] W. T. Liu and L. Zhu, Environmental microbiology on-a-chip and its future impacts. Trends in Biotechnology, 2005,23:174-179
[40] FS Ligler. Perspective on Optical Biosensors and Integrated Sensor Systems. Analytical Chemistry ,2009,81:519-526
[41] JW Hong, V Studer, G Hang.A nanoliter -scale nucleic acid processor with parallel architecture. Nature Biotechnology, 2004 ,22:435-439
[42] JG Lee, KH Cheong, N Huh, S Kim.Microchip-based one step DNA extraction and real-time PCR in one chamber for rapid pathogen identification. Lab on A Chip ,2006,6: 886-895
[43] Wan W, Yeow JT.Study of a novel cell lysis method with titanium dioxide for Lab-on-a-Chip devices. Biomed Microdevices, 2011,5(3)
[44] Ferguson BS, Buchsbaum SF, Swensen JS. Integrated Microfluidic Electrochemical DNA Sensor. Analytical Chemistry, 2009, 81(15): 6503-6508.
[45] Fujita SI, Yosizaki K, Ogushi T, et al. Rapid identification of Gram-negative bacteria with and without CTX-M extended-spectrum {beta}-lactamase from positive blood culture bottles by PCR followed by microchip gel electrophoresis. J Clin Microbiol, 2011,(2)
[46] Kang MH, Kim IW, Lee DW, et al. Development of a rapid detection method to detect tdh gene in Vibrio parahaemolyticus using 2-step ultrarapid real-time polymerase chain reaction. Diagn Microbiol Infect Dis,2011, 69(1):21-29
[47] CJ Easley, JM Karlinsey, JM Bienvenue, et al. A fully integrated microfluidic genetic analysis system with sample-in-answer-out capability. Proceedings of the National Academy of Sciences of the United States of America,2006, 103:19272-19277
[48] Yi Zhang, Seungkyung Park, Kelvin Liu, et al.A surface topography assisted droplet manipulation platform for biomarker detection and pathogenidentification,Lab Chip, 2011, 11:398-406
[49] Liu D, Shi M, Huang H, et al.Isotachophoresis preconcentration integrated microfluidic chip for highly sensitive genotyping of the hepatitis B virus. J Chromatogr B Analyt Technol Biomed Life Sci,2006, 844 (1):32-38
[50] Pal R.,Yang M, Lin R, et al. An integrated microfluidic device for influenza and other genetic analyses. Lab Chip,2005,5 (10):1024-1032
[51] Liu RH, Lodes MJ, Nguyen T,et al.Validation of a fully integrated microfluidic array device for influenza A subtype identification and sequencing. Anal Chem,2006, 78 (12):4184-4193
[52] WC Lee, KY Lien, GB Lee, and HY Lei. An integrated microfluidic system using magnetic beads for virus detection. Diagnostic Microbiology and Infectious Disease,2008, 60:51-58
[53] Kang-Yi Lien, Jr-Lung Lin, Cheng-Yu Liu ,et al.Purification and enrichment of virus samples utilizing magnetic beads on a microfluidic system.Lab Chip, 2007, 7:868-875
[54] Kristin A, Carmen R, Mari L, et al. An integrated valveless system for microfluidic purification and reverse transcription-PCR amplification of RNA for detection of infectious agents,Lab Chip,2011,11:957-961
[55] Keiichiro Yamanaka, Masato Saito, Kenji Kondoh, et al. Rapid detection for primary screening of influenza A virus: microfluidic RT-PCR chip and electrochemical DNA sensor.Analyst,2011, Advance Article
[56] Gerald M. Birnbaumer, Peter A. Lieberzeit, Lukas Richter, et al. Detection of viruses with molecularly imprinted polymers integrated on a microfluidic biochip using contact-less dielectric microsensors.Lab Chip,2009,9:3549-3556
[57] Yi Sun, Raghuram D, Dang DB, et al.A lab-on-a-chip device for rapid identification of avian influenza viral RNA by solid-phase PCR.Lab Chip, 2011,11:1457-1463
[58] Yao Lu, Weiwei Shi, Jianhua Qin , Bingcheng Lin Analytical.Fabrication and Characterization of Paper-Based Microfluidics Prepared in NitrocelluloseMembrane By Wax Printing,Chemistry ,2010,82 (1):329-335
[59] Wang CH, Kang YL, et al. A magnetic bead-based assay for the rapid detection of methicillin-resistant Staphylococcus aureus by using a microfluidic system with integrated loop-mediated isothermal amplification.Lab Chip, 2011, 11:1521-1531
[60] Weiwei Shi, Hui Wen, Yao Lu, et al. Droplet microfluidics for characterizing the neurotoxin-induced responses in individual Caenorhabditis elegans,Lab Chip,2010, 10:2855-2863
[61] PanaroNJ,Yuen PK, Sakazume T,et al.Evaluation of DNA fragment sizing and quantification by the agilent 2100 bioanalyzer.Clin Chem,2000,46 (11): 1851-1853
[62] http://www.chem.agilent.com/en-us/products/instruments/lab-on-a-chip/2100bioanalyzer/Pages/default.aspx
[63] Liu RH, Yang J, Lenigk R,et al. Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection. Anal Chem,2004,76 (7):1824-1831
[64] Ruano JM, Agirr M, Olabarria G, et al.The SmartBioPhone, a point of care vision under development through two European projects: OPTOLABCARD and LABONFOIL.Lab Chip,2009, 9(11):1495-1499