数字聚合酶链反应及其在感染性疾病中的应用
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摘要
数字聚合酶链反应(polymerase chain reaction,PCR)采用与定量PCR相同的荧光化学原理和不同的数学原理来实现对靶标核酸序列的绝对定量,其摒弃了对外部参照的依赖,同时具有更高的数据精密度,提高了重复性和再现性。数字PCR的应用涵盖生命科学众多领域,特别是在医学检验领域,其对疾病相关核酸分子标记的准确分析,为疾病的早期诊断、进展监测、疗效评估提供了动态量化指标。数字PCR的出现将推动基于核酸扩增技术的分子生物学检测迈入精准定量阶段。本文就数字PCR尤其是微滴式数字PCR在感染性疾病中的应用进展及前沿进行综述。
Digital polymerase chain reaction(PCR) is an emerging technology for quantitative analysis of nucleic acids, employing the same fluorescence chemistry as quantitative PCR(qPCR) but different mathematical principles to achieve absolute quantification of target nucleic acid sequences, without the reliance on external references such as standard curve. This technology is both conceptually simple and extremely robust in terms of assay performance, including increased sensitivity, precision, reproducibility and repeatability. Digital PCR technology has been widely used in life science research and applications. In the field of medical testing, digital PCR provides accurate quantification of disease-related nucleic acid biomarkers, enabling early diagnosis of diseases, monitoring of disease progression and evaluation of therapeutic efficacy as well. The emergence of digital PCR will push the medical testing into the stage of precise quantification. Here we review the progress and cutting edge of digital PCR technology, especially droplet digital PCR technology in the field of infectious diseases.
Digital polymerase chain reaction(PCR) is an emerging technology for quantitative analysis of nucleic acids, employing the same fluorescence chemistry as quantitative PCR(qPCR) but different mathematical principles to achieve absolute quantification of target nucleic acid sequences, without the reliance on external references such as standard curve. This technology is both conceptually simple and extremely robust in terms of assay performance, including increased sensitivity, precision, reproducibility and repeatability. Digital PCR technology has been widely used in life science research and applications. In the field of medical testing, digital PCR provides accurate quantification of disease-related nucleic acid biomarkers, enabling early diagnosis of diseases, monitoring of disease progression and evaluation of therapeutic efficacy as well. The emergence of digital PCR will push the medical testing into the stage of precise quantification. Here we review the progress and cutting edge of digital PCR technology, especially droplet digital PCR technology in the field of infectious diseases.
引文
[1] Diaz LJ Jr,Bardelli A.Liquid biopsies:genotyping circulating tumor DNA [J].J Clin Oncol,2014,32(6):579-586.
[2] Weaver S,Dube S,Mir A,Qin J,Sun G,Ramakrishnan R,Jones RC,Livak KJ.Taking qPCR to a higher level:analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution [J].Methods,2010,50(4):271-276.
[3] Higuchi R,Fockler C,Dollinger G,Watson R.Kinetic PCR analysis:real-time monitoring of DNA amplification reactions [J].Biotechnology (NY),1993,11(9):1026-1030.
[4] Hindson CM,Chevillet JR,Briggs HA,Gallichotte EN,Ruf IK,Hindson BJ,Vessella RL,Tewari M.Absolute quantification by droplet digital PCR versus analog real-time PCR [J].Nat Methods,2013,10(10):1003-1005.
[5] Sykes PJ,Neoh SH,Brisco MJ,Hughes E,Condon J,Morley AA.Quantitation of targets for PCR by use of limiting dilution [J].Biotechniques,1992,13(3):444-449.
[6] Brisco MJ,Codon J,Hughes E,Neoh SH,Sykes PJ,Seshadri R,Morley AA,Toogood I,Waters K,Tauro G.Ekert H.Outcome prediction in childhood acute lymphoblastic leukaemia by molecular quantification of residual disease at the end of induction [J].Lancet,1994,343(8891):196-200.
[7] Vogelstein B,Kinzler KW.Digital PCR [J].Proc Natl Acad Sci USA,1999,96(16):9236-9241.
[8] Nakano M,Komatsu J,Matsuura S,Takashima K,Katsura S,Mizuno A.Single-molecule PCR using water-in-oil emulsion [J].J Biotechnol,2003,102(2):117-124.
[9] Pinheiro LB,Coleman VA,Hindson CM,Herrmann J,Hindson BJ,Bhat S,Emslie KR.Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification [J].Anal Chem,2012,84(2):1003-1011.
[10] Hindson BJ,Ness KD,Masquelier DA,Belgrader P,Heredia NJ,Makarewicz AJ,Bright IJ,Lucero MY,Hiddessen AL,Legler TC,Kitano TK,Hodel MR,Petersen JF,Wyatt PW,Steenblock ER,Shah PH,Bousse LJ,Troup CB,Mellen JC,Wittmann DK,Erndt NG,Cauley TH,Koehler RT,So AP,Dube S,Rose KA,Montesclaros L,Wang S,Stumbo DP,Hodges SP,Romine S,Milanovich FP,White HE,Regan JF,Karlin-Neumann GA,Hindson CM,Saxonov S,Colston BW.High-throughput droplet digital PCR system for absolute quantitation of DNA copy number [J].Anal Chem,2011,83(22):8604-8610.
[11] Whale AS,Bushell CA,Grant PR,Cowen S,Gutierrez-Aguirre I,O’Sullivan DM,?el J,Milavec M,Foy CA,Nastouli E,Garson JA,Huggett JF.Detection of rare drug resistance mutations by digital PCR in a human influenza A virus model system and clinical samples [J].J Clin Microbiol,2016,54(2):392-400.
[12] Persuad D,Gay H,Ziemniak C,Chen YH,Piatak M Jr,Chun TW,Strain M,Richman D,Luzuriaga K.Absence of detectable HIV-1 viremia after treatment cessation in an infant [J].N Engl J Med,2013,369(19):1828-1835.
[13] Bharuthram A,Paximadis M,Picton AC,Tiemessen CT.Comparison of a quantitative real-time PCR assay and droplet digital PCR for copy number analysis of the CCL4L genes [J].Infect Genet Evol,2014,25:28-35.
[14] de Smith AJ,Walsh KM,Hansen HM,Endicott AA,Wiencke JK,Metayer C,Wiemels JL.Somatic mutation allelic ratio test using ddPCR (SMART-ddPCR):an accurate method for assessment of preferential allelic imbalance in tumor DNA [J].PLoS One,2015,10(11):e0143343.
[15] Whale AS,Devonshire AS,Karlin-Neumann G,Regan J,Javier L,Cowen S,Fernandez-Gonzalez A,Jones GM,Redshaw N,Beck J,Berger AW,Combaret V,Dahl Kjersgaard N,Davis L,Fina F,Forshew T,Fredslund Andersen R,Galbiati S,González Hernández á,Haynes CA,Janku F,Lacave R,Lee J,Mistry V,Pender A,Pradines A,Proudhon C,Saal LH,Stieglitz E,Ulrich B,Foy CA,Parkes H,Tzonev S,Huggett JF.International interlaboratory digital PCR study demonstrating high reproducibility for the measurement of a rare sequence variant [J].Anal Chem,2017,89(3):1724-1733.
[16] Perkel MJ.Life science technologies:The digital PCR revolution [J].Science,2014,344(6180).doi:10.1126/science.344.6180.212.
[17] Dong L,Meng Y,Sui Z,Wang J,Wu L,Fu B.Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material [J].Sci Rep,2015,5:13174.doi:10.1038/srep13174.
[18] Dobnik D,Dem■ analysis of selected genetically modified plant reference materials with digital PCR [J].Anal Bioanal Chem,2018,410(1):211-221.
[19] Haynes RJ,Kline MC,Toman B,Scott C,Wallace P,Butler JM,Holden MJ.Standard reference material 2366 for measurement of human cytomegalovirus DNA [J].J Mol Diagn,2013,15(2):177-185.
[20] Strain MC,Lada SM,Luong T,Rought SE,Gianella S,Terry VH,Spina CA,Woelk CH,Richman DD.Highly precise measurement of HIV DNA by droplet digital PCR [J].PLoS One,2013,8(4):e55943.
[21] Taylor SC,Carbonneau J,Shelton DN,Boivin G.Optimization of droplet digital PCR from RNA and DNA extracts with direct comparison to RT-qPCR:Clinical implications for quantification of Oseltamivir-resistant subpopulations [J].J Virol Methods,2015,224:58-66.
[22] Dingle TC,Sedlak RH,Cook L,Jerome KR.Tolerance of droplet-digital PCR vs real-time quantitative PCR to inhibitory substances [J].Clin Chem,2013,59(11):1670-1672.
[23] Sedlak RH,Kuypers J,Jerome KR.A multiplexed droplet digital PCR assay performs better than qPCR on inhibition prone samples [J].Diagn Microbiol Infect Dis,2014,80(4):285-286.
[24] Cao Y,Raith MR,Griffith JF.Droplet digital PCR for simultaneous quantification of general and human-associated fecal indicators for water quality assessment [J].Water Res,2015,70:337-349.
[25] Pav■i■ J,?el J,Milavec M.Digital PCR for direct quantification of viruses without DNA extraction [J].Anal Bioanal Chem,2016,408(1):67-75.
[26] Hoffman NG,Cook L,Atienza EE,Limaye AP,Jerome KR.Marked variability of BK virus load measurement using quantitative real-time PCR among commonly used assays [J].J Clin Microbiol,2008,46(8):2671-2680.
[27] Mukaide M,Sugiyama M,Korenaga M,Murata K,Kanto T,Masaki N,Mizokami M.High-throughput and sensitive next-generation droplet digital PCR assay for the quantitation of the hepatitis C virus mutation at core amino acid 70 [J].J Virol Methods,2014,207:169-177.
[28] Bloom K,Ely A,Mussolino C,Cathomen T,Arbuthnot P.Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases [J].Mol Ther,2013,21(10):1889-1897.
[29] Sedlak RH,Liang S,Niyonzima N,De Silva Feelixge HS,Roychoudhury P,Greninger AL,Weber ND,Boissel S,Scharenberg AM,Cheng A,Magaret A,Bumgarner R,Stone D,Jerome KR.Digital detection of endonuclease mediated gene disruption in the HIV provirus [J].Sci Rep,2016,6:20064.doi:10.1038/srep20064.
[30] Mock U,Machowicz R,Hauber I,Horn S,Abramowski P,Berdien B,Hauber J,Fehse B.mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5 [J].Nucleic Acids Res,2015,43(11):5560-5571.
[31] Provasi E,Genovese P,Lombardo A,Magnani Z,Liu PQ,Reik A,Chu V,Paschon DE,Zhang L,Kuball J,Camisa B,Bondanza A,Casorati G,Ponzoni M,Ciceri F,Bordignon C,Greenberg PD,Holmes MC,Gregory PD,Naldini L,Bonini C.Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer [J].Nat Med,2012,18(5):807-815.
[32] Berdien B,Mock U,Atanackovic D,Fehse B.TALEN-mediated editing of endogenous T-cell receptors facilitates efficient reprogramming of T lymphocytes by lentiviral gene transfer [J].Gene Ther,2014,21(6):539-548.
[33] Weber ND,Stone D,Sedlak RH,De Silva Feelixge HS,Roychoudhury P,Schiffer JT,Aubert M,Jerome KR.AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication [J].PLoS One,2014,9(5):e97579.
[34] Findlay SD,Vincent KM,Berman JR,Postovit LM.A digital PCR-based method for efficient and highly specific screening of genome edited cells [J].PLoS One,2016,11(4):e0153901.
[35] Miyaoka Y,Chan AH,Conklin BR.Detecting single-nucleotide substitutions induced by genome editing [J].Cold Spring Harb Protoc,2016.doi:10.1101/pdb.top090845.
[36] Mock U,Hauber I,Fehse B.Digital PCR to assess gene-editing frequencies (GEF-dPCR) mediated by designer nucleases [J].Nat Protoc,2016,11(3):598-615.
[37] Miyaoka Y,Chan AH,Judge LM,Yoo J,Huang M,Nguyen TD,Lizarraga PP,So PL,Conklin BR.Isolation of single-base genome-edited human iPS cells without antibiotic selection [J].Nat Methods,2014,11(3):291-293.
[38] McMahon TC,Blais BW,Wong A,Carrillo CD.Multiplexed single intact cell droplet digital PCR (MuSIC ddPCR) method for specific detection of enterohemorrhagic E.coli (EHEC) in food enrichment cultures [J].Front Microbiol,2017,8:332.doi:10.3389/fmicb.2017.00332.
[39] Yucha RW,Hobbs KS,Hanhauser E,Hogan LE,Nieves W,Ozen MO,Inci F,York V,Gibson EA,Thanh C,Shafiee H,El Assal R,Kiselinova M,Robles YP,Bae H,Leadabrand KS,Wang S,Deeks SG,Kuritzkes DR,Demirci U,Henrich TJ.High-throughput characterization of HIV-1 reservoir reactivation using a single-cell-in-droplet PCR assay [J].EBioMedicine,2017,20:217-229.
[2] Weaver S,Dube S,Mir A,Qin J,Sun G,Ramakrishnan R,Jones RC,Livak KJ.Taking qPCR to a higher level:analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution [J].Methods,2010,50(4):271-276.
[3] Higuchi R,Fockler C,Dollinger G,Watson R.Kinetic PCR analysis:real-time monitoring of DNA amplification reactions [J].Biotechnology (NY),1993,11(9):1026-1030.
[4] Hindson CM,Chevillet JR,Briggs HA,Gallichotte EN,Ruf IK,Hindson BJ,Vessella RL,Tewari M.Absolute quantification by droplet digital PCR versus analog real-time PCR [J].Nat Methods,2013,10(10):1003-1005.
[5] Sykes PJ,Neoh SH,Brisco MJ,Hughes E,Condon J,Morley AA.Quantitation of targets for PCR by use of limiting dilution [J].Biotechniques,1992,13(3):444-449.
[6] Brisco MJ,Codon J,Hughes E,Neoh SH,Sykes PJ,Seshadri R,Morley AA,Toogood I,Waters K,Tauro G.Ekert H.Outcome prediction in childhood acute lymphoblastic leukaemia by molecular quantification of residual disease at the end of induction [J].Lancet,1994,343(8891):196-200.
[7] Vogelstein B,Kinzler KW.Digital PCR [J].Proc Natl Acad Sci USA,1999,96(16):9236-9241.
[8] Nakano M,Komatsu J,Matsuura S,Takashima K,Katsura S,Mizuno A.Single-molecule PCR using water-in-oil emulsion [J].J Biotechnol,2003,102(2):117-124.
[9] Pinheiro LB,Coleman VA,Hindson CM,Herrmann J,Hindson BJ,Bhat S,Emslie KR.Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification [J].Anal Chem,2012,84(2):1003-1011.
[10] Hindson BJ,Ness KD,Masquelier DA,Belgrader P,Heredia NJ,Makarewicz AJ,Bright IJ,Lucero MY,Hiddessen AL,Legler TC,Kitano TK,Hodel MR,Petersen JF,Wyatt PW,Steenblock ER,Shah PH,Bousse LJ,Troup CB,Mellen JC,Wittmann DK,Erndt NG,Cauley TH,Koehler RT,So AP,Dube S,Rose KA,Montesclaros L,Wang S,Stumbo DP,Hodges SP,Romine S,Milanovich FP,White HE,Regan JF,Karlin-Neumann GA,Hindson CM,Saxonov S,Colston BW.High-throughput droplet digital PCR system for absolute quantitation of DNA copy number [J].Anal Chem,2011,83(22):8604-8610.
[11] Whale AS,Bushell CA,Grant PR,Cowen S,Gutierrez-Aguirre I,O’Sullivan DM,?el J,Milavec M,Foy CA,Nastouli E,Garson JA,Huggett JF.Detection of rare drug resistance mutations by digital PCR in a human influenza A virus model system and clinical samples [J].J Clin Microbiol,2016,54(2):392-400.
[12] Persuad D,Gay H,Ziemniak C,Chen YH,Piatak M Jr,Chun TW,Strain M,Richman D,Luzuriaga K.Absence of detectable HIV-1 viremia after treatment cessation in an infant [J].N Engl J Med,2013,369(19):1828-1835.
[13] Bharuthram A,Paximadis M,Picton AC,Tiemessen CT.Comparison of a quantitative real-time PCR assay and droplet digital PCR for copy number analysis of the CCL4L genes [J].Infect Genet Evol,2014,25:28-35.
[14] de Smith AJ,Walsh KM,Hansen HM,Endicott AA,Wiencke JK,Metayer C,Wiemels JL.Somatic mutation allelic ratio test using ddPCR (SMART-ddPCR):an accurate method for assessment of preferential allelic imbalance in tumor DNA [J].PLoS One,2015,10(11):e0143343.
[15] Whale AS,Devonshire AS,Karlin-Neumann G,Regan J,Javier L,Cowen S,Fernandez-Gonzalez A,Jones GM,Redshaw N,Beck J,Berger AW,Combaret V,Dahl Kjersgaard N,Davis L,Fina F,Forshew T,Fredslund Andersen R,Galbiati S,González Hernández á,Haynes CA,Janku F,Lacave R,Lee J,Mistry V,Pender A,Pradines A,Proudhon C,Saal LH,Stieglitz E,Ulrich B,Foy CA,Parkes H,Tzonev S,Huggett JF.International interlaboratory digital PCR study demonstrating high reproducibility for the measurement of a rare sequence variant [J].Anal Chem,2017,89(3):1724-1733.
[16] Perkel MJ.Life science technologies:The digital PCR revolution [J].Science,2014,344(6180).doi:10.1126/science.344.6180.212.
[17] Dong L,Meng Y,Sui Z,Wang J,Wu L,Fu B.Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material [J].Sci Rep,2015,5:13174.doi:10.1038/srep13174.
[18] Dobnik D,Dem■ analysis of selected genetically modified plant reference materials with digital PCR [J].Anal Bioanal Chem,2018,410(1):211-221.
[19] Haynes RJ,Kline MC,Toman B,Scott C,Wallace P,Butler JM,Holden MJ.Standard reference material 2366 for measurement of human cytomegalovirus DNA [J].J Mol Diagn,2013,15(2):177-185.
[20] Strain MC,Lada SM,Luong T,Rought SE,Gianella S,Terry VH,Spina CA,Woelk CH,Richman DD.Highly precise measurement of HIV DNA by droplet digital PCR [J].PLoS One,2013,8(4):e55943.
[21] Taylor SC,Carbonneau J,Shelton DN,Boivin G.Optimization of droplet digital PCR from RNA and DNA extracts with direct comparison to RT-qPCR:Clinical implications for quantification of Oseltamivir-resistant subpopulations [J].J Virol Methods,2015,224:58-66.
[22] Dingle TC,Sedlak RH,Cook L,Jerome KR.Tolerance of droplet-digital PCR vs real-time quantitative PCR to inhibitory substances [J].Clin Chem,2013,59(11):1670-1672.
[23] Sedlak RH,Kuypers J,Jerome KR.A multiplexed droplet digital PCR assay performs better than qPCR on inhibition prone samples [J].Diagn Microbiol Infect Dis,2014,80(4):285-286.
[24] Cao Y,Raith MR,Griffith JF.Droplet digital PCR for simultaneous quantification of general and human-associated fecal indicators for water quality assessment [J].Water Res,2015,70:337-349.
[25] Pav■i■ J,?el J,Milavec M.Digital PCR for direct quantification of viruses without DNA extraction [J].Anal Bioanal Chem,2016,408(1):67-75.
[26] Hoffman NG,Cook L,Atienza EE,Limaye AP,Jerome KR.Marked variability of BK virus load measurement using quantitative real-time PCR among commonly used assays [J].J Clin Microbiol,2008,46(8):2671-2680.
[27] Mukaide M,Sugiyama M,Korenaga M,Murata K,Kanto T,Masaki N,Mizokami M.High-throughput and sensitive next-generation droplet digital PCR assay for the quantitation of the hepatitis C virus mutation at core amino acid 70 [J].J Virol Methods,2014,207:169-177.
[28] Bloom K,Ely A,Mussolino C,Cathomen T,Arbuthnot P.Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases [J].Mol Ther,2013,21(10):1889-1897.
[29] Sedlak RH,Liang S,Niyonzima N,De Silva Feelixge HS,Roychoudhury P,Greninger AL,Weber ND,Boissel S,Scharenberg AM,Cheng A,Magaret A,Bumgarner R,Stone D,Jerome KR.Digital detection of endonuclease mediated gene disruption in the HIV provirus [J].Sci Rep,2016,6:20064.doi:10.1038/srep20064.
[30] Mock U,Machowicz R,Hauber I,Horn S,Abramowski P,Berdien B,Hauber J,Fehse B.mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5 [J].Nucleic Acids Res,2015,43(11):5560-5571.
[31] Provasi E,Genovese P,Lombardo A,Magnani Z,Liu PQ,Reik A,Chu V,Paschon DE,Zhang L,Kuball J,Camisa B,Bondanza A,Casorati G,Ponzoni M,Ciceri F,Bordignon C,Greenberg PD,Holmes MC,Gregory PD,Naldini L,Bonini C.Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer [J].Nat Med,2012,18(5):807-815.
[32] Berdien B,Mock U,Atanackovic D,Fehse B.TALEN-mediated editing of endogenous T-cell receptors facilitates efficient reprogramming of T lymphocytes by lentiviral gene transfer [J].Gene Ther,2014,21(6):539-548.
[33] Weber ND,Stone D,Sedlak RH,De Silva Feelixge HS,Roychoudhury P,Schiffer JT,Aubert M,Jerome KR.AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication [J].PLoS One,2014,9(5):e97579.
[34] Findlay SD,Vincent KM,Berman JR,Postovit LM.A digital PCR-based method for efficient and highly specific screening of genome edited cells [J].PLoS One,2016,11(4):e0153901.
[35] Miyaoka Y,Chan AH,Conklin BR.Detecting single-nucleotide substitutions induced by genome editing [J].Cold Spring Harb Protoc,2016.doi:10.1101/pdb.top090845.
[36] Mock U,Hauber I,Fehse B.Digital PCR to assess gene-editing frequencies (GEF-dPCR) mediated by designer nucleases [J].Nat Protoc,2016,11(3):598-615.
[37] Miyaoka Y,Chan AH,Judge LM,Yoo J,Huang M,Nguyen TD,Lizarraga PP,So PL,Conklin BR.Isolation of single-base genome-edited human iPS cells without antibiotic selection [J].Nat Methods,2014,11(3):291-293.
[38] McMahon TC,Blais BW,Wong A,Carrillo CD.Multiplexed single intact cell droplet digital PCR (MuSIC ddPCR) method for specific detection of enterohemorrhagic E.coli (EHEC) in food enrichment cultures [J].Front Microbiol,2017,8:332.doi:10.3389/fmicb.2017.00332.
[39] Yucha RW,Hobbs KS,Hanhauser E,Hogan LE,Nieves W,Ozen MO,Inci F,York V,Gibson EA,Thanh C,Shafiee H,El Assal R,Kiselinova M,Robles YP,Bae H,Leadabrand KS,Wang S,Deeks SG,Kuritzkes DR,Demirci U,Henrich TJ.High-throughput characterization of HIV-1 reservoir reactivation using a single-cell-in-droplet PCR assay [J].EBioMedicine,2017,20:217-229.