PMA-qPCR技术在发酵食品活菌计数中的应用
|
摘要
发酵食品具有丰富的口感和较高的营养价值,深受广大消费者喜爱。准确计数发酵食品生产加工与贮藏过程中的活菌,是对产品进行质量控制、评价以及工艺改进的基础。叠氮溴化丙锭(propidium monoazide,PMA)是一种具有高亲和力的光敏性DNA结合染料,用于处理样品后偶联使用实时荧光定量聚合酶链式反应(quantitative real-time PCR,q PCR)计数可极大缩短检测时长且灵敏度高、特异性强。该文综述PMA-qPCR技术的原理、影响因素及其在红酒、啤酒、酸奶等发酵食品活菌计数中的应用现状。将目标微生物及PMA处理条件列为此技术影响因素,以此为基础总结各类发酵食品中可检测微生物及检测线性范围及检测限,为扩大此方法使用范围,获取发酵食品中更为丰富和精准的活菌检测结果提供参考。
Fermented foods are favored by consumers because of their high nutritional values and tastes. Monitoring living microorganisms during processing and storage of fermented foods is important for quality control,production evaluation and process modification. Propidium monoazide(PMA) is a highly photosensitive DNA-binding dye that can be used with quantitative fluorescence real-time PCR(qPCR) for quick,accurate,and specific detection. The mechanisms and influencing factors of PMA-qPCR,as well as its applications in enumerating living microbes in wines,beers,and yoghurts were reviewed. It was found that the types of target microorganisms and PMA treatment conditions were the most important influencing factors. The linear ranges and detection limits of detectable microbes in various fermented foods were summarized. This review aimed to provide a reference for multi-faceted applications of PMA-qPCR.
Fermented foods are favored by consumers because of their high nutritional values and tastes. Monitoring living microorganisms during processing and storage of fermented foods is important for quality control,production evaluation and process modification. Propidium monoazide(PMA) is a highly photosensitive DNA-binding dye that can be used with quantitative fluorescence real-time PCR(qPCR) for quick,accurate,and specific detection. The mechanisms and influencing factors of PMA-qPCR,as well as its applications in enumerating living microbes in wines,beers,and yoghurts were reviewed. It was found that the types of target microorganisms and PMA treatment conditions were the most important influencing factors. The linear ranges and detection limits of detectable microbes in various fermented foods were summarized. This review aimed to provide a reference for multi-faceted applications of PMA-qPCR.
引文
[1]成黎.传统发酵食品营养保健功能与质量安全评价[J].食品科学,2012,33(1):280-284.
[2]姚粟,于学健,白飞荣,等.中国传统发酵食品用微生物菌种名单的研究[J].食品与发酵工业,2017,43(9):242-262.
[3]FONSECA S,CACHALDORA A,GOMEZ M,et al.Monitoring the bacterial population dynamics during the ripening of Galician chorizo,a traditional dry fermented Spanish sausage[J].Food Microbiology,2013,33(1):77-84.
[4]NOGVA H K,DROMTORP S M,NISSEN H,et al.Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR[J].Biotechniques,2003,34(4):804-813.
[5]赵晓祥,庞晓倩,庄惠生.荧光定量PCR技术在环境监测中的应用研究[J].环境科学与技术,2009,32(12):125-128.
[6]REYNEKE B,NDLOVU T,KHAN S,et al.Comparison of EMA-,PMA-and DNase q PCR for the determination of microbial cell viability[J].Applied Microbiology and Biotechnology,2017,101(19):7 371-7 383.
[7]NEBE-VON-CARON G,STEPHENS P J,HEWITT C J,et al.Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting[J].Journal of Microbiological Methods,2000,42(1):97-114.
[8]NOCKER A,CHEUNG C Y,CAMPER A K.Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs.dead bacteria by selective removal of DNA from dead cells[J].Journal of Microbiological Methods,2006,67(2):310-320.
[9]肖莉莉,张昭寰,娄阳,等.叠氮溴化丙锭(PMA)在食源性致病菌检测中的应用[J].中国食品学报,2016,16(6):187-194.
[10]ELIZAQUIVEL P,AZNAR R,SANCHEZ G.Recent developments in the use of viability dyes and quantitative PCR in the food microbiology field[J].Journal of Applied Microbiology,2013,116(1):1-13.
[11]LOVDAL T,HOVDA M B,BJORKBLOM B,et al.Propidium monoazide combined with real-time quantitative PCR underestimates heat-killed Listeria innocua[J].Journal of Microbiological Methods,2011,85(2):164-169.
[12]COCOLIN L,ALESSANDRIA V,DOLCI P,et al.Culture independent methods to assess the diversity and dynamics of microbiota during food fermentation[J].International Journal of Food Microbiology,2013,167(1):29-43.
[13]LAI C H,WU S R,PANG J C,et al.Designing primers and evaluation of the efficiency of propidium monoazidequantitative polymerase chain reaction for counting the viable cells of Lactobacillus gasseri and Lactobacillus salivarius.[J].Journal of Food&Drug Analysis,2017,25(3):533-542.
[14]LOPEZ I,RUIZ-LARREA F,COCOLIN L,et al.Design and evaluation of PCR primers for analysis of bacterial populations in wine by denaturing gradient gel electrophoresis[J].Applied and Environmental Microbiology,2003,69(11):6 801-6 807.
[15]GONZALEZA,HIERRO N,POBLET M,et al.Enumeration and detection of acetic acid bacteria by real-time PCR and nested PCR[J].FEMS Microbiology Letters,2006,254(1):123-128.
[16]BALDRIAN P,VEˇTROVSKY'T,CAJTHAML T,et al.Estimation of fungal biomass in forest litter and soil[J].Fungal Ecology,2013,6(1):1-11.
[17]HIERRO N,ESTEVE-ZARZOSO B,MAS A,et al.Monitoring of Saccharomyces and Hanseniaspora populations during alcoholic fermentation by real-time quantitative PCR[J].FEMS Yeast Research,2010,7(8):1 340-1 349.
[18]MARTORELL P,QUEROL A,FERNNDEZ-ESPINARM T.Rapid identification and enumeration of Saccharomyces cerevisiae cells in wine by Real-Time PCR[J].Applied and Environmental Microbiology,2005,71(11):6 823-6 830.
[19]VAN FRANKENHUYZEN J K,TREVORS J T,LEE H,et al.Molecular pathogen detection in biosolids with a focus on quantitative PCR using propidium monoazide for viable cell enumeration[J].Journal of Microbiological Methods,2011,87(3):263-272.
[20]TANTIKACHORNKIAT M,SAKAKIBARA S,NEUNERM,et al.The use of propidium monoazide in conjunction with q PCR and Illumina sequencing to identify and quantify live yeasts and bacteria.[J].International Journal of Food Microbiology,2016,234:53-59.
[21]ANDORRAI,ESTEVE-ZARZOSO B,GUILLAMON JM,et al.Determination of viable wine yeast using DNAbinding dyes and quantitative PCR[J].International Journal of Food Microbiology,2010,144(2):257-262.
[22]SHAO Y,WANG Z,BAO Q,et al.Application of propidium monoazide quantitative real-time PCR to quantify the viability of Lactobacillus delbrueckii ssp.bulgaricus[J].Journal of Dairy Science,2016,99(12):9 570-9 580.
[23]DING J,HUANG X,ZHANG L,et al.Tolerance and stress response to ethanol in the yeast Saccharomyces cerevisiae[J].Applied Microbiology&Biotechnology,2009,85(2):253-263.
[24]RIZZOTTI L,LEVAV N,FRACCHETTI F,et al.Effect of UV-C treatment on the microbial population of white and red wines,as revealed by conventional plating and PMA-qPCR methods[J].Food Control,2015,47:407-412.
[25]VENDRAME M,MANZANO M,COMI G,et al.Use of propidium monoazide for the enumeration of viable Brettanomyces bruxellensis in wine and beer by quantitative PCR.[J].Food Microbiology,2014,42:196-204.
[26]LV X C,LI Y,QIU W W,et al.Development of propidium monoazide combined with real-time quantitative PCR(PMA-qPCR)assays to quantify viable dominant microorganisms responsible for the traditional brewing of Hong Qu glutinous rice wine[J].Food Control,2016,66:69-78.
[27]杜鹏,霍贵成.国内外益生菌制品发展现状[J].食品科学,2004,25(5):194-198.
[28]Codex Alimentarius Commission.Codex standard 243-2003,Codex standard for fermented milks[S].Alimentarius Commission,2003.
[29]田其英.酸奶的研究进展[J].食品与发酵科技,2013,49(1):91-94.
[30]GARCIA-CAYUELA T,TABASCO R,PELAEZ C,et al.Simultaneous detection and enumeration of viable lactic acid bacteria and bifidobacteria in fermented milk by using propidium monoazide and real-time PCR[J].International Dairy Journal,2009,19(6-7):405-409.
[31]SCARIOT M C,VENTURELLI G L,PRUDNCIO E S,et al.Quantification of Lactobacillus paracasei viable cells in probiotic yoghurt by propidium monoazide combined with quantitative PCR[J].International Journal of Food Microbiology,2018,264:1-7.
[32]DESFOSSES-FOUCAULTE,DUSSAULT-LEPAGE V,LE BOUCHER C,et al.Assessment of probiotic viability during cheddar cheese manufacture and ripening using propidium monoazide-PCR quantification[J].Frontiers in Microbiology,2012,3:350.
[33]VILLARREAL M L M,PADILHA M,VIEIRA A D S,et al.Advantageous direct quantification of viable closely related probiotics in petit-suisse cheeses under in vitro gastrointestinal conditions by propidium monoazide-qPCR[J].Plos One,2013,79(4):1 414-1 417
[34]ERKUS O,DE JAGER V C L,GEENE R T C M,et al.Use of propidium monoazide for selective profiling of viable microbial cells during Gouda cheese ripening[J].International journal of food microbiology,2016,228:1-9.
[35]高洁,俞丹,陈頔,等.传统发酵乳开菲尔的研究进展[J].中国食品学报,2016,16(4):204-211.
[36]PORCELLATO D,MAGRI M,NARVHUS J.Viable cells differentiation improves microbial dynamics study of fermented milks[J].International Dairy Journal,2015,47(9):136-142.
[37]赵华杰,何炘,杨荣华,等.鱼露风味的研究进展[J].食品与发酵工业,2007,33(7):123-128.
[38]UDOMSIL N,CHEN S,RODTONG S,et al.Quantification of viable bacterial starter cultures of Virgibacillus sp.and Tetragenococcus halophilus in fish sauce fermentation by real-time quantitative PCR[J].Food Microbiology,2016,57(8):54-62.
[2]姚粟,于学健,白飞荣,等.中国传统发酵食品用微生物菌种名单的研究[J].食品与发酵工业,2017,43(9):242-262.
[3]FONSECA S,CACHALDORA A,GOMEZ M,et al.Monitoring the bacterial population dynamics during the ripening of Galician chorizo,a traditional dry fermented Spanish sausage[J].Food Microbiology,2013,33(1):77-84.
[4]NOGVA H K,DROMTORP S M,NISSEN H,et al.Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR[J].Biotechniques,2003,34(4):804-813.
[5]赵晓祥,庞晓倩,庄惠生.荧光定量PCR技术在环境监测中的应用研究[J].环境科学与技术,2009,32(12):125-128.
[6]REYNEKE B,NDLOVU T,KHAN S,et al.Comparison of EMA-,PMA-and DNase q PCR for the determination of microbial cell viability[J].Applied Microbiology and Biotechnology,2017,101(19):7 371-7 383.
[7]NEBE-VON-CARON G,STEPHENS P J,HEWITT C J,et al.Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting[J].Journal of Microbiological Methods,2000,42(1):97-114.
[8]NOCKER A,CHEUNG C Y,CAMPER A K.Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs.dead bacteria by selective removal of DNA from dead cells[J].Journal of Microbiological Methods,2006,67(2):310-320.
[9]肖莉莉,张昭寰,娄阳,等.叠氮溴化丙锭(PMA)在食源性致病菌检测中的应用[J].中国食品学报,2016,16(6):187-194.
[10]ELIZAQUIVEL P,AZNAR R,SANCHEZ G.Recent developments in the use of viability dyes and quantitative PCR in the food microbiology field[J].Journal of Applied Microbiology,2013,116(1):1-13.
[11]LOVDAL T,HOVDA M B,BJORKBLOM B,et al.Propidium monoazide combined with real-time quantitative PCR underestimates heat-killed Listeria innocua[J].Journal of Microbiological Methods,2011,85(2):164-169.
[12]COCOLIN L,ALESSANDRIA V,DOLCI P,et al.Culture independent methods to assess the diversity and dynamics of microbiota during food fermentation[J].International Journal of Food Microbiology,2013,167(1):29-43.
[13]LAI C H,WU S R,PANG J C,et al.Designing primers and evaluation of the efficiency of propidium monoazidequantitative polymerase chain reaction for counting the viable cells of Lactobacillus gasseri and Lactobacillus salivarius.[J].Journal of Food&Drug Analysis,2017,25(3):533-542.
[14]LOPEZ I,RUIZ-LARREA F,COCOLIN L,et al.Design and evaluation of PCR primers for analysis of bacterial populations in wine by denaturing gradient gel electrophoresis[J].Applied and Environmental Microbiology,2003,69(11):6 801-6 807.
[15]GONZALEZA,HIERRO N,POBLET M,et al.Enumeration and detection of acetic acid bacteria by real-time PCR and nested PCR[J].FEMS Microbiology Letters,2006,254(1):123-128.
[16]BALDRIAN P,VEˇTROVSKY'T,CAJTHAML T,et al.Estimation of fungal biomass in forest litter and soil[J].Fungal Ecology,2013,6(1):1-11.
[17]HIERRO N,ESTEVE-ZARZOSO B,MAS A,et al.Monitoring of Saccharomyces and Hanseniaspora populations during alcoholic fermentation by real-time quantitative PCR[J].FEMS Yeast Research,2010,7(8):1 340-1 349.
[18]MARTORELL P,QUEROL A,FERNNDEZ-ESPINARM T.Rapid identification and enumeration of Saccharomyces cerevisiae cells in wine by Real-Time PCR[J].Applied and Environmental Microbiology,2005,71(11):6 823-6 830.
[19]VAN FRANKENHUYZEN J K,TREVORS J T,LEE H,et al.Molecular pathogen detection in biosolids with a focus on quantitative PCR using propidium monoazide for viable cell enumeration[J].Journal of Microbiological Methods,2011,87(3):263-272.
[20]TANTIKACHORNKIAT M,SAKAKIBARA S,NEUNERM,et al.The use of propidium monoazide in conjunction with q PCR and Illumina sequencing to identify and quantify live yeasts and bacteria.[J].International Journal of Food Microbiology,2016,234:53-59.
[21]ANDORRAI,ESTEVE-ZARZOSO B,GUILLAMON JM,et al.Determination of viable wine yeast using DNAbinding dyes and quantitative PCR[J].International Journal of Food Microbiology,2010,144(2):257-262.
[22]SHAO Y,WANG Z,BAO Q,et al.Application of propidium monoazide quantitative real-time PCR to quantify the viability of Lactobacillus delbrueckii ssp.bulgaricus[J].Journal of Dairy Science,2016,99(12):9 570-9 580.
[23]DING J,HUANG X,ZHANG L,et al.Tolerance and stress response to ethanol in the yeast Saccharomyces cerevisiae[J].Applied Microbiology&Biotechnology,2009,85(2):253-263.
[24]RIZZOTTI L,LEVAV N,FRACCHETTI F,et al.Effect of UV-C treatment on the microbial population of white and red wines,as revealed by conventional plating and PMA-qPCR methods[J].Food Control,2015,47:407-412.
[25]VENDRAME M,MANZANO M,COMI G,et al.Use of propidium monoazide for the enumeration of viable Brettanomyces bruxellensis in wine and beer by quantitative PCR.[J].Food Microbiology,2014,42:196-204.
[26]LV X C,LI Y,QIU W W,et al.Development of propidium monoazide combined with real-time quantitative PCR(PMA-qPCR)assays to quantify viable dominant microorganisms responsible for the traditional brewing of Hong Qu glutinous rice wine[J].Food Control,2016,66:69-78.
[27]杜鹏,霍贵成.国内外益生菌制品发展现状[J].食品科学,2004,25(5):194-198.
[28]Codex Alimentarius Commission.Codex standard 243-2003,Codex standard for fermented milks[S].Alimentarius Commission,2003.
[29]田其英.酸奶的研究进展[J].食品与发酵科技,2013,49(1):91-94.
[30]GARCIA-CAYUELA T,TABASCO R,PELAEZ C,et al.Simultaneous detection and enumeration of viable lactic acid bacteria and bifidobacteria in fermented milk by using propidium monoazide and real-time PCR[J].International Dairy Journal,2009,19(6-7):405-409.
[31]SCARIOT M C,VENTURELLI G L,PRUDNCIO E S,et al.Quantification of Lactobacillus paracasei viable cells in probiotic yoghurt by propidium monoazide combined with quantitative PCR[J].International Journal of Food Microbiology,2018,264:1-7.
[32]DESFOSSES-FOUCAULTE,DUSSAULT-LEPAGE V,LE BOUCHER C,et al.Assessment of probiotic viability during cheddar cheese manufacture and ripening using propidium monoazide-PCR quantification[J].Frontiers in Microbiology,2012,3:350.
[33]VILLARREAL M L M,PADILHA M,VIEIRA A D S,et al.Advantageous direct quantification of viable closely related probiotics in petit-suisse cheeses under in vitro gastrointestinal conditions by propidium monoazide-qPCR[J].Plos One,2013,79(4):1 414-1 417
[34]ERKUS O,DE JAGER V C L,GEENE R T C M,et al.Use of propidium monoazide for selective profiling of viable microbial cells during Gouda cheese ripening[J].International journal of food microbiology,2016,228:1-9.
[35]高洁,俞丹,陈頔,等.传统发酵乳开菲尔的研究进展[J].中国食品学报,2016,16(4):204-211.
[36]PORCELLATO D,MAGRI M,NARVHUS J.Viable cells differentiation improves microbial dynamics study of fermented milks[J].International Dairy Journal,2015,47(9):136-142.
[37]赵华杰,何炘,杨荣华,等.鱼露风味的研究进展[J].食品与发酵工业,2007,33(7):123-128.
[38]UDOMSIL N,CHEN S,RODTONG S,et al.Quantification of viable bacterial starter cultures of Virgibacillus sp.and Tetragenococcus halophilus in fish sauce fermentation by real-time quantitative PCR[J].Food Microbiology,2016,57(8):54-62.