dPCR的发展历史、原理及其应用(3),生物化学论文.docx

《dPCR的发展历史、原理及其应用(3),生物化学论文.docx》由会员分享，可在线阅读，更多相关《dPCR的发展历史、原理及其应用(3),生物化学论文.docx（8页珍藏版）》请在淘文阁 - 分享文档赚钱的网站上搜索。

1、dPCR的发展历史、原理及其应用(3),生物化学论文传统的qPCR方式方法能够对食品中的转基因成分进行测定32,33,由于该方式方法需要建立标准曲线，加上食品与作物等复杂样品中抑制剂对PCR扩增效率的影响，使qPCR对于低丰度的转基因成分的检测有很大的局限34.有研究利用dPCR定量转基因MON810与参考基因hmg基因拷贝数的比值，定量结果与qPCR一样35.2020年，Morisset等36利用微滴式dPCR双重检测转基因MON810和参考基因hmg拷贝数比值，微滴式dPCR检测hmg和MON810的线性范围分别为：5118 000拷贝/反响和64 340拷贝/反响，检测的线性范围较宽，能

2、知足常规转基因成分检测的要求。有研究报道dPCR检测转基因成分的灵敏度为0.1%,低 于 欧 盟 规 定 转 基 因 成 分 的 检 出 限0.9%37.研究表示清楚dPCR能够作为常规的检测技术应用于转基因成分鉴定。 4结束语与瞻望。 dPCR作为核酸定量的新技术，与此前的核酸定量方式方法相比，方式方法的灵敏度、准确度更高层次。dPCR为分子生物学、微生物学等领域提供了新的检测方式方法和实验思路。从应用的范围、实验的成本角度来看，dPCR不可能完全取代qPCR,但是dPCR方式方法能够进行精准的绝对定量分析，极好的数据重现性，而且样本需求较低，在核酸检测与定量等方面有非常重要的补充。在整个d

3、PCR的发展经过中，应用越来越广泛，在核酸检测定量、抗病毒治疗监测、预后判定具有重要的意义。随着dPCR技术和商品化平台的发展，dPCR的通量将会更高层次，成本更低，在科学研究领域将会发挥更重要的作用。 以下为参考文献： 1Sykes P J,Neoh S H,Brisco M J,et al.Quantitation oftargets for PCR by use of limiting dilutionJ.Biotech-niques,1992,133：444-449. 2Olga K,Irina L,James B,et al.Nanoliter scale PCRwith TaqMa

4、n detectionJ.Nucleic Acids Res,1997,2510：1999-2004. 3Vogelstein B,Kinzler K W.Digital PCRJ.PNAS,1999,9616：9236-9241. 4Devin D,Hai Y,Giovanni T,et al.Transforming sin-gle DNA molecules into fluorescent magnetic particlesfor detection and enumeration of genetic variationsJ.PNAS,2003,10015：8817-8822. 5

5、Dingle TC,Sedlak R H,Cook L,et al.Tolerance ofdroplet-digital PCR versus real-time quantitative PCR toinhibitory substancesJ.Clin Chem,2020,5911：1670-1672. 6Bosman K J,Nijhuis M,Van Ham P M,et al.Compar-ison of digital PCR platforms and semi-nested qPCR as atool to determine the size of the HIV rese

6、rvoirJ.Sci-entific Reports,2021,5:13811. 7Nicot F,Cazabat M,Lhomme S,et al.Quantificationof HEV RNA by Droplet Digital PCRJ.Viruses,2021,8:233. 8Sanders R,Huggett J F,Bushell C A,et al.Evaluationof digital PCR for absolute DNA quantificationJ.A-nal Chem,2018,83,6474-6484. 9Sanders R,Mason D J,Foy C

7、A,et al.Evaluation ofdigital PCR for absolute RNA quantificationJ/OL.PLoS One,2020,89：e75296. 10Pinheiro L B,Coleman V A,Hindson C M,et al.E-valuation of a droplet digital polymerase chain reactionformat for DNA copy number quantificationJ.AnalChem,2020,84:1003-1011. 11White R A,Quake S R,Curr K.Dig

8、ital PCR providesabsolute quantitation of viral load for an occult RNA vi-rusJ.J Virol Methods,2020,179:45-50. 12Racki N,Morisset D,Gutierrez A I,et al.One-stepRT-droplet digital PCR:a breakthrough in the quanti-fication of waterborne RNA virusesJ.Anal BioanalChem 2020,406:661-667. 13Yan Yong,Jia X

9、J,Wang H H,et al.Dynamic quan-tification of avian influenza H7N9Avirus in a humaninfection during clinical treatment using droplet digitalPCRJ.J Virol Methods,2021,234:22-27. 14Lo Y M D,Corbetta N,Chamberlain P F,et al.Pres-ence of fetal DNA in maternal plasma and serumJ.The Lancet,3509076：485-487.

10、15Lo Y M D,Fiona M F,Chan K C A,et al.DigitalPCR for the molecular detection of fetal chromosomalaneuploidyJ.Proc Natl Acad Sci USA,2007,10432：13116-13121. 16Lun F M F,Allen C K,Yeung L T,et al.Microfluid-ics digital PCR reveals a higher than expected fractionof fetal DNA in maternal plasmaJ.Clin Ch

11、em,2008,5410：1664-1672. 17Barrett A N,McDonnell T C,Chan K C et al.DigitalPCR analysis of maternal plasma for noninvasive detec-tion of sickle cell anemiaJ.Clinical Chemistry,2020,58:6 1026-1032. 18Tsui N B,Kadir R A,Chan K C,et al.Noninvasiveprenatal diagnosis of hemophilia by microfluidics digital

12、PCR analysis of maternal plasma DNAJ.Blood,2018,11713：3684-3691. 19Tsui N B,Hyland C A,Gardener G J,et al.Noninva-sive fetal RHD genotyping by microfluidics digital PCRusing maternal plasma from two alloimmunized withthe variant RHDIVS3+1G AalleleJ.Prenat Di-agn,2020,3312：1214-1216. 20Lu J,Getz G,Mi

13、ska E A,et al.MicroRNA expres-sion profiles classify human cancers.JNature,2005,4357043：834-838. 21Yanaihara N,Caplen N,Bowman E,et al.Unique mi-croRNA molecular profiles in lung cancerdiagnosis andprognosisJ.Cancer Cell,2006,93：189-198. 22Hindson C M,Chevillet J R,Briggs H A,et al.Abso-lute quantif

14、ication by droplet digital PCR versus analogreal-time PCRJ.Nat Methods,2020,1010：1003-1005. 23Ma J,Li N,Guarnera M,et al.Quantification of plas-ma miRNAs by digital PCR for cancer diagnosisJ.Biomarker Insights,2020,8:127-136. 24Conte D,Verri C,Borzi C et al.Novel method to de-tect microRNAs using ch

15、ip-based QuantStudio 3Ddig-ital PCRJ.BMC Genomics,2021,16:849. 25Li N,Ma J,Guarnera M,et al.Digital PCR quantifi-cation of miRNAs in sputum for diagnosis of lung canc-erJ.J Cancer Res Clin Oncol,2020,1401：145-150. 26Yung T K,Chan K A,Mok T S,et al.Sing-moleculedetection of epidermal growth factor re

16、ceptor mutationsin plasma by microfluidics digital PCR in non-small celllung cancer patientsJ.Clinical Cancer Research,2018,156：2076-2084. 27Wang J,Ramakrishnan R,Tang Z,et al.QuantifyingEGFR alterations in the lung cancer genome withnanofluidic digital PCR arraysJ.Clin Chem,2018,564：623-632. 28Taly

17、 V,Pekin D,Benhaim L,et al.Multiplex picodr-oplet digital PCR to detect KRAS mutation in circulat-ing DNA from the plasma of colorectal cancer patientsJ.Clin Chem,2020,5912：1722-1731. 29Jacob H J.Next-generation sequencing for clinical di-agnosticsJ.N Engl J Med,2020,369:1557-1558. 30White R A,Blain

18、ey P C,Fan H C,et al.Digital PCRprovides sensitive and absolute calibration for highthroughput sequencingJ.BMC Genomics,2018,10:116. 31Robin J D,Ludlow A T,LaRanger R,et al.Compari-son of DNA quantification methods for Next generationsequencingJ.Scientific Reports,2021,6:24067. 32Holst-Jensen A.Test

19、ing for genetically modified or-ganismsGMOs：Past,present and future perspec-tivesJ.Biotechnol Adv,2018,27:1071-1082. 33Settanni L,Corsetti A.The use of multiplex PCR todetect and differentiate food-and beverage-associatedmicroorganisms:A reviewJ.Journal of Microbiolog-ical Methods,2007,69:1-22. 34Ca

20、nkar K,Stebih D,Dreo T,et al.Critical points ofDNA quantification by real-time PCR-effects of DNAextraction method and sample matrix on quantificationof genetically modified organismsJ.BMC Biotechnol,2006,6:37. 35Corbisier P,Bhat S,Partis L,et al.Absolute quantifi-cation of genetically modified MON8

21、10maize by digitalpolymerase chain reactionJ.Analytical and Bioana-lytical Chenistry,2018,3966：2143-2150. 36Morisset D,Stebih D,Milavec M,et al.Quantitativeanalysis of food and feed samples with droplet digitalPCRJ/OL.PLoS One,2020,85：e62583. 37Fu W,Zhu P Y,Wang C G,et al.A highly sensitiveand specific method for the screening detection of ge-netically modified organisms based on digital PCR with-out pretreatmentJ.Scientific Reports,2021,5:12715