水中大肠菌群微滴数字PCR定量检测方法的建立
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摘要
以大肠菌群的lacZ基因为靶基因,建立一种快速、稳定、灵敏、特异的微滴数字聚合酶链式反应(Droplet Digital Polymerase Chain Reaction,ddPCR)定量检测方法。对ddPCR反应体系中试剂浓度、退火温度等条件优化筛选的同时,考察了方法的线性范围、精密度和定量限。最终确定反应体系中的最佳引物和探针浓度分别为0.2和0.5μmol/L,最佳退火温度为56℃。大肠菌群lacZ基因组DNA浓度范围为3.95～7.80×10~4拷贝(copies)/20μL ddPCR反应液时,方法的线性关系良好(R~2=0.999)。文章所建立的方法可检出每微升单个拷贝数的大肠菌群lacZ基因组DNA,且具有快速测定、灵敏度高、特异性强、重复性良好等特点,为建立水污染早期应急机制提供了重要的参考价值。
In this research,the ddPCR detection method for absolute quantification of total coliforms was established by selection of lacZ gene as the target gene for coliform group detection.The experimental conditions such as the added reagent concentrations,annealing temperatures have been well optimized.In addition,the linear range,precision and limit of quantitation have been investigated and evaluated.The results showed that the optimal primer and probe concentrations in each ddPCR reaction system were 0.2 μmol/L and 0.5 μmol/L,respectively.The optimal annealing temperature was 56 ℃.The linear regression was quite good(R~2=0.999) with the total coliform genomic DNA concentrations from 3.95 to7.80×10~4 copies/20 μL ddPCR reaction system.The limit of detection for total coliforms was single copy in each microliter of coliform template.Our innovative ddPCR detection method had the characteristics of fast detection,good repeatability,high sensitivity and specificity.This is probably the 1 st research using ddPCR technology for absolute quantification of total coliforms in water which provides with the significant reference value for setting-up the emergency mechanisms of water pollution in the early stage.
In this research,the ddPCR detection method for absolute quantification of total coliforms was established by selection of lacZ gene as the target gene for coliform group detection.The experimental conditions such as the added reagent concentrations,annealing temperatures have been well optimized.In addition,the linear range,precision and limit of quantitation have been investigated and evaluated.The results showed that the optimal primer and probe concentrations in each ddPCR reaction system were 0.2 μmol/L and 0.5 μmol/L,respectively.The optimal annealing temperature was 56 ℃.The linear regression was quite good(R~2=0.999) with the total coliform genomic DNA concentrations from 3.95 to7.80×10~4 copies/20 μL ddPCR reaction system.The limit of detection for total coliforms was single copy in each microliter of coliform template.Our innovative ddPCR detection method had the characteristics of fast detection,good repeatability,high sensitivity and specificity.This is probably the 1 st research using ddPCR technology for absolute quantification of total coliforms in water which provides with the significant reference value for setting-up the emergency mechanisms of water pollution in the early stage.
引文
[1] FLOR R C,ABRAHAM L M,MARIO J,et al.Waterborne pathogens:detection methods and challenges[J].Pathogens,2015,4(2):307-334.
[2]MCGINNIS S,SPENCER S,FIRNSTAHL A,et al.Human bacteroidesand total coliforms as indicators of recent combined sewer overflows and rain events in urban creeks[J].Science of the total environment,2018,630:967-976.
[3]MOHAMMED H,HAMEED I A,SEIDU R.Comparative predictive modelling of the occurrence of faecal indicator bacteria in a drinking water source in Norway[J].Science of the total environment,2018,628-629:1178-1190.
[4]MESSNER M J,BERGER P,JAVIER J.Total coliform and E.coli in public water systems using undisinfected ground water in the United Sates[J].International journal of hygiene and environmental health,2017,220(4):736-743.
[5]陈亚楠,王亚炜,魏源送,等.不同功能地表水体中病原微生物指示物的标准比较[J].环境科学学报,2015,35(2):337-351.
[6]NOBLE R T,MOORE D F,LEECASTER M K,et al.Comparison of total coliform,fecal coliform and enterococcus bacterial indicator response for ocean recreational water quality testing[J].Water research,2003,37(7):1637-1643.
[7]MAHEUX A F,DION-DUPONT V,BISSON M A,et al.Detection of Escherichia Coli colonies on confluent plates of chromogenic media used in membrane filtration[J].Journal of microbiological methods,2014,97:51-55.
[8]WANG D L and FIESSEL W.Evaluation of media for simultaneous enumeration of total coliform and Escherichia Coli in drinking water supplies by membrane filtration techniques[J].Journal of environmental sciences,2008,20(3):273-277.
[9]崔希,熊齐荣,熊勇华,等.免疫磁分离结合胶体金免疫层析法快速检测大肠杆菌O157∶H7[J].分析化学,2013,41(12):1812-1816.
[10]SHELTON D R,HIGGINS J A,VAN KESSEL J A S,et al.Estimation of viable Escherichia Coli O157 in surface waters using enrichment in conjunction with immunological detection[J].Journal of microbiological methods,2004,58(2):223-231.
[11]TAKAHASHI H,SAITO R,MIYA S,et al.Development of quantitative real-time PCR for detection and enumeration of enterobacteriaceae[J].International journal of food microbiology,2017,246:92-97.
[12]ASHRAF A,IMRAN M,YAQUB T,et al.A novel multiplex PCR assay for simultaneous detection of nine clinically significant bacterial pathogens associated with Bovine Mastitis[J].Molecular and cellular probes,2017,33:57-64.
[13]CARNELLI A,MAURI F,DEMARTA A.Characterization of genetic determinants involved in antibiotic resistance in Aeromonas spp.and fecal coliforms isolated from different aquatic environments[J].Research in microbiology,2017,168(5):461-471.
[14]SILVA D M and DOMINGUES L.On the track for an efficient detection of Escherichia Coli in water:A review on PCR-based methods[J].Ecotoxicology and environmental safety,2015,113:400-411.
[15]EDEN R.Enterobacteriaceae,coliforms and E.coli:classical and modern methods for detection and enumeration[M].2nd ed.California:Encyclopedia of food microbiology,2014,667-673.
[16]WANG D L and FIESSEL W.Evaluation of media for simultaneous enumeration of total coliform and Escherichia Coli in drinking water supplies by membrane filtration techniques[J].Journal of environmental sciences,2008,20(3):273-277.
[17]MAHEUX A F,HUPPE V,BOISSINOT M,et al.Analytical limits of four β-glucuronidase and β-galactosidase-based commercial culture methods used to detect Escherichia Coli and total coliforms[J].Journal of microbiological methods,2008,75(3):506-514.
[18] ROMPRE A,SERVAIS P,BAUDART J,et al.Detection and enumeration of coliforms in drinking water:current methods and emerging approaches[J].Journal of microbiological methods,2002,49(1):31-54.
[19] DESHMUKH R A,JOSHI K,BHAND S,et al.Recent development in detection and enumeration of waterborne bacteria:A retrospective minireview[J].MicrobiologyOpen,2016,5(6):901-922.
[20] HUBNER I,STEINMETZ I,OBST U,et al.Rapid determination of members of the family enterobacteriaceae in drinking water by an immunological assay using a monoclonal antibody against enterobacterial common antigen[J].Applied and environmental microbiology,1992,58(9):3187-3191.
[21]LIU Q,ZHANG X L,YAO Y H,et al.A Novel microfluidic module for rapid detection of airborne and waterborne pathogens[J].Sensors and actuators B:chemical,2018,258:1138-1145.
[22]ELS?βER D,HO J,NIESSNER R,et al.Heterogeneous asymmetric recombinase polymerase amplification (haRPA) for rapid hygiene control of large-volume water samples[J].Analytical biochemistry,2018,546:58-64.
[23] DAO T N T,LEE E Y,KOO B,et al.A microfluidic enrichment platform with a recombinase polymerase amplification sensor for pathogen diagnosis[J].Analytical biochemistry,2018,544:87-92.
[24] MARTZY R,KOLM C,BRUNNER K,et al.A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Enterococcus spp.in water[J].Water research,2017,122:62-69.
[25] LACOUT A,MONE Y,FRANCK M,et al.Blood cell disruption to significantly improve the borrelia PCR detection sensitivity in borreliosis in humans[J].Medical hypothesis,2018,116:1-3.
[26] GAROFALO C,BANCALARI E,MILANOVIC V,et al.Study of the bacterial diversity of foods:PCR-DGGE versus LH-PCR[J].International journal of food microbiology,2017,242:24-36.
[27]WANG Y J,ZHU S Y,HONG W M,et al.A multiplex PCR for detection of six viruses in ducks[J].Journal of virological methods,2017,248:172-176.
[28]GUNAWARDANA M,CHANG S,JIMENEZ A,et al.Isolation of PCR quality microbial community DNA from heavily contaminated environments[J].Journal of microbiological methods,2014,102:1-7.
[29]TAKAHASHI H,SAITO R,MIYA S,et al.Development of quantitative real-time PCR for detection and enumeration of Enterobacteriaceae[J].International journal of food microbiology,2017(246):92-97.
[30] MAHEUX A F,BOUDREAU D K,BISSON M A,et al.Molecular method for detection of total coliforms in drinking water samples[J].Applied and environmental microbiology,2014,80(14):4074-4084.
[31] K?MPFER P,NIENHüSER A,PACKROFF G,et al.Molecular identification of coliform bacteria isolated from drinking water reservoirs with traditional methods and the Colilert-18 system[J].International journal of hygiene and environmental health,2008,211(3-4):374-384.
[32]LIU L,CLOUTIER M,CRAIOVAN E,et al.Quantitative real-time PCR-based assessment of tile drainage management influences on bacterial pathogens in the tile drainage and groundwater[J].Science of the total environment,2018,624:1586-1597.
[33]NIKALJE G C,SRIVASTAVA A K,SABLOK G,et al.Identification and validation of reference genes for quantitative real-time PCR under salt stress in a halophyte,Sesuvium portulacastrum[J].Plant gene,2018,13:18-24.
[34] MARCIAL-QUINO J,FIERRO F,DE LA MORA-DE LA MORA I,et al.Validation of housekeeping genes as an internal control for gene expression studies in Giardia Lamblia using quantitative real-time PCR[J].Gene,2016,581(1):21-30.
[35]LU S,SMITH A P,MOORE D,et al.Different real-time PCR yield different gene expression values[J].Molecular and cellular probes,2010,24(5):315-320.
[36]刘津,李婷,冼钰茵,等.转基因大豆MON89788双重数字PCR通用定量检测方法的建立[J].食品科学,2018,39(4):312-319.
[37]WANG M,YANG J J,GAI Z T,et al.Comparison between digital PCR and real-time PCR in detection of Salmonella typhimurium in Milk[J].International journal of food microbiology,2018,266:251-256.
[38]IWOBI A,GERDES L,BUSCH U,et al.Droplet digital PCR for routine analysis of genetically modified foods (GMO) – A comparison with real-time quantitative PCR[J].Food control,2016,69:205-213.
[39] RAJESWARI P K P,SODERBERG L M,YACOUB A,et al.Multiple pathogen biomarker detection using an encoded bead array in droplet PCR[J].Journal of microbiological methods,2017,129:22-28.
[40]MEMON A A,Z?LLER B,HEDELIUS A,et al.Quantification of mitochondrial DNA copy number in suspected cancer patients by a well optimized ddPCR method[J].Biomolecular detection and quantification,2017,13:32-39.
[41] SCOLLO F,EGEA L A,GENTILE A,et al.Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR):comparison of isolation and amplification methodologies[J].Food chemistry,2016 (213):388-394.
[42] FLOREN C,WIEDEMANN I,BRENIG B,et al.Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR)[J].Food chemistry,2015,173:1054-1058.
[43]姜志军,江颖,徐摇光,等.利用微滴数字PCR方法快速分析转基因玉米中外源基因的拷贝数[J].生物技术进展,2016,6(4):288-294.
[44] EASTBURN D J,HUANG Y,PELLEGRINO M,et al.Microfluidic droplet enrichment for targeted sequencing[J].Nucleic acids research,2015,43(13):e86.
[45]JOENSSON H N and ADERSSON S H.Droplet microfluidics – A tool for single-cell analysis[J].Angewandte chemie international edition,2012,51(49):12176-12192.
[46]刘渠,李涌,陈应坚,等.饮用水大肠菌群Taqman-MGB荧光定量PCR检测方法研究[J].现代预防医学,2010,37(12):2312-2317.
[47]王建龙.PCR技术检测水体中大肠菌群的研究[J].中国生物工程杂志,2004,24(7):60-64.
[48]International organization for standardization.ISO/TS 12869:2012[S/OL].[2012-10-12].https://www.iso.org/standard/52079.html.
[49]董莲华,张玲,姜君,等.大肠杆菌O157:H7微滴数字PCR定量检测方法的建立[J].分析化学,2015,43(3):319-324.
[50] DONG L,MENG Y,WANG J,et al.Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers[J].Analytical and bioanalytical chemistry,2014,406(6):1701-1712.
[2]MCGINNIS S,SPENCER S,FIRNSTAHL A,et al.Human bacteroidesand total coliforms as indicators of recent combined sewer overflows and rain events in urban creeks[J].Science of the total environment,2018,630:967-976.
[3]MOHAMMED H,HAMEED I A,SEIDU R.Comparative predictive modelling of the occurrence of faecal indicator bacteria in a drinking water source in Norway[J].Science of the total environment,2018,628-629:1178-1190.
[4]MESSNER M J,BERGER P,JAVIER J.Total coliform and E.coli in public water systems using undisinfected ground water in the United Sates[J].International journal of hygiene and environmental health,2017,220(4):736-743.
[5]陈亚楠,王亚炜,魏源送,等.不同功能地表水体中病原微生物指示物的标准比较[J].环境科学学报,2015,35(2):337-351.
[6]NOBLE R T,MOORE D F,LEECASTER M K,et al.Comparison of total coliform,fecal coliform and enterococcus bacterial indicator response for ocean recreational water quality testing[J].Water research,2003,37(7):1637-1643.
[7]MAHEUX A F,DION-DUPONT V,BISSON M A,et al.Detection of Escherichia Coli colonies on confluent plates of chromogenic media used in membrane filtration[J].Journal of microbiological methods,2014,97:51-55.
[8]WANG D L and FIESSEL W.Evaluation of media for simultaneous enumeration of total coliform and Escherichia Coli in drinking water supplies by membrane filtration techniques[J].Journal of environmental sciences,2008,20(3):273-277.
[9]崔希,熊齐荣,熊勇华,等.免疫磁分离结合胶体金免疫层析法快速检测大肠杆菌O157∶H7[J].分析化学,2013,41(12):1812-1816.
[10]SHELTON D R,HIGGINS J A,VAN KESSEL J A S,et al.Estimation of viable Escherichia Coli O157 in surface waters using enrichment in conjunction with immunological detection[J].Journal of microbiological methods,2004,58(2):223-231.
[11]TAKAHASHI H,SAITO R,MIYA S,et al.Development of quantitative real-time PCR for detection and enumeration of enterobacteriaceae[J].International journal of food microbiology,2017,246:92-97.
[12]ASHRAF A,IMRAN M,YAQUB T,et al.A novel multiplex PCR assay for simultaneous detection of nine clinically significant bacterial pathogens associated with Bovine Mastitis[J].Molecular and cellular probes,2017,33:57-64.
[13]CARNELLI A,MAURI F,DEMARTA A.Characterization of genetic determinants involved in antibiotic resistance in Aeromonas spp.and fecal coliforms isolated from different aquatic environments[J].Research in microbiology,2017,168(5):461-471.
[14]SILVA D M and DOMINGUES L.On the track for an efficient detection of Escherichia Coli in water:A review on PCR-based methods[J].Ecotoxicology and environmental safety,2015,113:400-411.
[15]EDEN R.Enterobacteriaceae,coliforms and E.coli:classical and modern methods for detection and enumeration[M].2nd ed.California:Encyclopedia of food microbiology,2014,667-673.
[16]WANG D L and FIESSEL W.Evaluation of media for simultaneous enumeration of total coliform and Escherichia Coli in drinking water supplies by membrane filtration techniques[J].Journal of environmental sciences,2008,20(3):273-277.
[17]MAHEUX A F,HUPPE V,BOISSINOT M,et al.Analytical limits of four β-glucuronidase and β-galactosidase-based commercial culture methods used to detect Escherichia Coli and total coliforms[J].Journal of microbiological methods,2008,75(3):506-514.
[18] ROMPRE A,SERVAIS P,BAUDART J,et al.Detection and enumeration of coliforms in drinking water:current methods and emerging approaches[J].Journal of microbiological methods,2002,49(1):31-54.
[19] DESHMUKH R A,JOSHI K,BHAND S,et al.Recent development in detection and enumeration of waterborne bacteria:A retrospective minireview[J].MicrobiologyOpen,2016,5(6):901-922.
[20] HUBNER I,STEINMETZ I,OBST U,et al.Rapid determination of members of the family enterobacteriaceae in drinking water by an immunological assay using a monoclonal antibody against enterobacterial common antigen[J].Applied and environmental microbiology,1992,58(9):3187-3191.
[21]LIU Q,ZHANG X L,YAO Y H,et al.A Novel microfluidic module for rapid detection of airborne and waterborne pathogens[J].Sensors and actuators B:chemical,2018,258:1138-1145.
[22]ELS?βER D,HO J,NIESSNER R,et al.Heterogeneous asymmetric recombinase polymerase amplification (haRPA) for rapid hygiene control of large-volume water samples[J].Analytical biochemistry,2018,546:58-64.
[23] DAO T N T,LEE E Y,KOO B,et al.A microfluidic enrichment platform with a recombinase polymerase amplification sensor for pathogen diagnosis[J].Analytical biochemistry,2018,544:87-92.
[24] MARTZY R,KOLM C,BRUNNER K,et al.A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Enterococcus spp.in water[J].Water research,2017,122:62-69.
[25] LACOUT A,MONE Y,FRANCK M,et al.Blood cell disruption to significantly improve the borrelia PCR detection sensitivity in borreliosis in humans[J].Medical hypothesis,2018,116:1-3.
[26] GAROFALO C,BANCALARI E,MILANOVIC V,et al.Study of the bacterial diversity of foods:PCR-DGGE versus LH-PCR[J].International journal of food microbiology,2017,242:24-36.
[27]WANG Y J,ZHU S Y,HONG W M,et al.A multiplex PCR for detection of six viruses in ducks[J].Journal of virological methods,2017,248:172-176.
[28]GUNAWARDANA M,CHANG S,JIMENEZ A,et al.Isolation of PCR quality microbial community DNA from heavily contaminated environments[J].Journal of microbiological methods,2014,102:1-7.
[29]TAKAHASHI H,SAITO R,MIYA S,et al.Development of quantitative real-time PCR for detection and enumeration of Enterobacteriaceae[J].International journal of food microbiology,2017(246):92-97.
[30] MAHEUX A F,BOUDREAU D K,BISSON M A,et al.Molecular method for detection of total coliforms in drinking water samples[J].Applied and environmental microbiology,2014,80(14):4074-4084.
[31] K?MPFER P,NIENHüSER A,PACKROFF G,et al.Molecular identification of coliform bacteria isolated from drinking water reservoirs with traditional methods and the Colilert-18 system[J].International journal of hygiene and environmental health,2008,211(3-4):374-384.
[32]LIU L,CLOUTIER M,CRAIOVAN E,et al.Quantitative real-time PCR-based assessment of tile drainage management influences on bacterial pathogens in the tile drainage and groundwater[J].Science of the total environment,2018,624:1586-1597.
[33]NIKALJE G C,SRIVASTAVA A K,SABLOK G,et al.Identification and validation of reference genes for quantitative real-time PCR under salt stress in a halophyte,Sesuvium portulacastrum[J].Plant gene,2018,13:18-24.
[34] MARCIAL-QUINO J,FIERRO F,DE LA MORA-DE LA MORA I,et al.Validation of housekeeping genes as an internal control for gene expression studies in Giardia Lamblia using quantitative real-time PCR[J].Gene,2016,581(1):21-30.
[35]LU S,SMITH A P,MOORE D,et al.Different real-time PCR yield different gene expression values[J].Molecular and cellular probes,2010,24(5):315-320.
[36]刘津,李婷,冼钰茵,等.转基因大豆MON89788双重数字PCR通用定量检测方法的建立[J].食品科学,2018,39(4):312-319.
[37]WANG M,YANG J J,GAI Z T,et al.Comparison between digital PCR and real-time PCR in detection of Salmonella typhimurium in Milk[J].International journal of food microbiology,2018,266:251-256.
[38]IWOBI A,GERDES L,BUSCH U,et al.Droplet digital PCR for routine analysis of genetically modified foods (GMO) – A comparison with real-time quantitative PCR[J].Food control,2016,69:205-213.
[39] RAJESWARI P K P,SODERBERG L M,YACOUB A,et al.Multiple pathogen biomarker detection using an encoded bead array in droplet PCR[J].Journal of microbiological methods,2017,129:22-28.
[40]MEMON A A,Z?LLER B,HEDELIUS A,et al.Quantification of mitochondrial DNA copy number in suspected cancer patients by a well optimized ddPCR method[J].Biomolecular detection and quantification,2017,13:32-39.
[41] SCOLLO F,EGEA L A,GENTILE A,et al.Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR):comparison of isolation and amplification methodologies[J].Food chemistry,2016 (213):388-394.
[42] FLOREN C,WIEDEMANN I,BRENIG B,et al.Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR)[J].Food chemistry,2015,173:1054-1058.
[43]姜志军,江颖,徐摇光,等.利用微滴数字PCR方法快速分析转基因玉米中外源基因的拷贝数[J].生物技术进展,2016,6(4):288-294.
[44] EASTBURN D J,HUANG Y,PELLEGRINO M,et al.Microfluidic droplet enrichment for targeted sequencing[J].Nucleic acids research,2015,43(13):e86.
[45]JOENSSON H N and ADERSSON S H.Droplet microfluidics – A tool for single-cell analysis[J].Angewandte chemie international edition,2012,51(49):12176-12192.
[46]刘渠,李涌,陈应坚,等.饮用水大肠菌群Taqman-MGB荧光定量PCR检测方法研究[J].现代预防医学,2010,37(12):2312-2317.
[47]王建龙.PCR技术检测水体中大肠菌群的研究[J].中国生物工程杂志,2004,24(7):60-64.
[48]International organization for standardization.ISO/TS 12869:2012[S/OL].[2012-10-12].https://www.iso.org/standard/52079.html.
[49]董莲华,张玲,姜君,等.大肠杆菌O157:H7微滴数字PCR定量检测方法的建立[J].分析化学,2015,43(3):319-324.
[50] DONG L,MENG Y,WANG J,et al.Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers[J].Analytical and bioanalytical chemistry,2014,406(6):1701-1712.