用于水生病原菌的3种高通量活菌计数方法的比较
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摘要
以一株副溶血弧菌(Vibrioparahaemolyticus)作为研究对象,比较了MTT[3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐]比色法、ATP生物发光法和高通量生长曲线法在活细菌高通量计数上的应用效果。用96孔培养板进行不同浓度细菌活菌计数,确定了上述3种方法在副溶血弧菌活菌计数的标准曲线和线性范围。结果显示,副溶血弧菌的MTT比色法以DMSO溶解的MTT产物甲瓒在555 nm的吸光度(OD_(555 nm))为计数依据,活菌数的对数(Lg C)与Lg OD_(555 nm)线性关系的标准曲线为Lg C=(1.0439±0.0200)LgOD_(555 nm)+(8.0565±0.0125),相关系数R~2=0.9965,线性检测范围为7.8×10~6～2.5×10~8CFU/ml;ATP生物发光法以ATP产生的相对发光度值(RLU)为计数依据,Lg C与LgRUL线性关系的标准曲线为Lg C=(0.9590±0.0065)LgRLU+(0.9949±0.0366),相关系数R~2=0.9994,线性检测范围为1.0×10~4～3.0×10~8 CFU/ml;高通量生长曲线法以生长曲线达到拐点的时间(T_s)为计数依据,Lg C与T_s线性关系的标准曲线为Lg C=-(0.8727±0.0230)Ts+(9.0128±0.1572),相关系数R~2=0.9924,线性检测范围为1.0×10~0～1.0×10~7 CFU/ml。用3种方法对实际菌液测量并与平板计数法比较表明,ATP生物发光法与高通量生长曲线法有很好的准确性,MTT比色法准确度稍差,而高通量生长曲线法有最宽的线性范围,也最适合高通量测定。
Three viable counting methods, including MTT assay, ATP bioluminescence assay, and high-throughput growth curve assay, for live Vibrio parahaemolyticus were compared in a high-throughput manner on a 96-well culture plate. The standard curves and linear ranges of these three methods were determined. The results showed that the directly detected index of MTT assay is OD_(555 nm),which is the absorption at 555 nm of DMSO-dissolved formazan produced from MTT. The liner standard curve for viable V. parahaemolyticus between logarithmic viable counts(Lg C) and the logarithmic OD_(555 nm)(Lg OD_(555 nm)) was Lg C=(1.0439±0.0200)LgOD_(555 nm)+(8.0565±0.0125), with a correlation index R~2=0.9965, and the linear detection range is 7.8×10~6～2.5×10~8 CFU/ml. The directly detected index of ATP bioluminescence assay is the relative luminescence unit(RLU), which is produced by ATP of viable cells.The liner standard curve between Lg C and LgRUL was Lg C=(0.9590±0.0065)LgRLU+(0.9949±0.0366),with a correlation index R~2=0.9994, and the linear detection range is 1.0×10~4～3.0×10~8 CFU/ml. The directly detected index of the high-throughput growth curve method is the spinodal time(T_s), which is the time reaching the reflection point on the growth curve. The liner standard curve between Lg C and Ts was Lg C=–(0.8727±0.0230)T_s+(9.0128±0.1572), with a correlation index R~2=0.9924, and a linear detection range of 1.0×10~0～1.0×10~7 CFU/ml. These three methods were used to measure 10 broths of V. parahaemolyticus and comparison with the plate counting method, respectively. The results showed both the ATP bioluminescence assay and the high-throughput growth curve method had satisfying accuracy, but the MTT assay had lower accuracy. The high-throughput growth curve has the widest linear range and is the most suitable for high throughput measurement.
Three viable counting methods, including MTT assay, ATP bioluminescence assay, and high-throughput growth curve assay, for live Vibrio parahaemolyticus were compared in a high-throughput manner on a 96-well culture plate. The standard curves and linear ranges of these three methods were determined. The results showed that the directly detected index of MTT assay is OD_(555 nm),which is the absorption at 555 nm of DMSO-dissolved formazan produced from MTT. The liner standard curve for viable V. parahaemolyticus between logarithmic viable counts(Lg C) and the logarithmic OD_(555 nm)(Lg OD_(555 nm)) was Lg C=(1.0439±0.0200)LgOD_(555 nm)+(8.0565±0.0125), with a correlation index R~2=0.9965, and the linear detection range is 7.8×10~6～2.5×10~8 CFU/ml. The directly detected index of ATP bioluminescence assay is the relative luminescence unit(RLU), which is produced by ATP of viable cells.The liner standard curve between Lg C and LgRUL was Lg C=(0.9590±0.0065)LgRLU+(0.9949±0.0366),with a correlation index R~2=0.9994, and the linear detection range is 1.0×10~4～3.0×10~8 CFU/ml. The directly detected index of the high-throughput growth curve method is the spinodal time(T_s), which is the time reaching the reflection point on the growth curve. The liner standard curve between Lg C and Ts was Lg C=–(0.8727±0.0230)T_s+(9.0128±0.1572), with a correlation index R~2=0.9924, and a linear detection range of 1.0×10~0～1.0×10~7 CFU/ml. These three methods were used to measure 10 broths of V. parahaemolyticus and comparison with the plate counting method, respectively. The results showed both the ATP bioluminescence assay and the high-throughput growth curve method had satisfying accuracy, but the MTT assay had lower accuracy. The high-throughput growth curve has the widest linear range and is the most suitable for high throughput measurement.
引文
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Chen FC,Godwin SL.Comparison of a rapid ATPbioluminescence assay and standard plate count methods for assessing microbial contamination of consumers'refrigerators.Journal of Food Protection,2006,69(10):2534-2538
Dong X,Bi DX,Wang HL,et al.pir ABvp-bearing Vibrio parahaemolyticus and Vibrio campbellii pathogens isolated from the same AHPND-affected pond possess highly similar pathogenic plasmids.Frontiers in Microbiology,2017,8:1859
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Gerlier D,Thomasset N.Use of MTT colorimetric assay to measure cell activation.Journal of Immunological Methods,1986,94(1-2):57-63
Gong JL,Deng J,Wu HC,et al.Research on the optimum condition of MTT in yeast count.China Condiment,2016(1):10-14[龚加路,邓静,吴华昌,等.MTT法在酵母菌活菌计数中最佳条件的研究.中国调味品,2016(1):10-14]
Gr?nroos M,M?enp??J,Nieminen AL,et al.548 Correlation of steroid receptor contents with medroxyprogesterone and tamoxifen effects in endometrial cancer assayed by barin vitro ATP-bioluminescence method.Journal of Steroid Biochemistry,1983,19(S1):194
Hammes F,Goldschmidt F,Vital M,et al.Measurement and interpretation of microbial adenosine tri-phosphate(ATP)in aquatic environments.Water Research,2010,44(13):3915
Holm-Hansen O,Booth CR.The Measurement of adenosine triphosphate in the ocean and its ecological significance.Limnology and Oceanography,1966,11(4):510-519
Huang LK,Du P,Huo GC.Inquiring into the method of counting live germ with MTT.Food Industry,2008(3):62-65[黄立坤,杜鹏,霍贵成.MTT法测定乳酸菌活菌数的研究.食品工业,2008(3):62-65]
Ling Y,Wang LB,Shen H,et al.Uncertainty evaluation for plate counts of coliforms in foods.Food&Machinery,2010,26(5):75-77[凌云,王李宝,沈辉,等.食品中大肠菌群平板计数结果不确定度的评定.食品与机械,2010,26(5):75-77]
Mc Elroy WD,Strehler BL.Factors influencing the response of the bioluminescent reaction to adenosine triphosphate.Archives of Biochemistry,1949,22(3):420-433
Miller JN,Nawawi MB,Burgess C.Detection of bacterial ATPby reversed flow-injection analysis with luminescence detection.Analytica Chimica Acta,1992,266(2):339-343
Mosmann T.Rapid colorimetric assay for cellular growth and survival:Application to proliferation and cytotoxicity assays Journal of Immunological Methods,1983,65(1-2):55-63
Moyer JD,Henderson JF.Nucleoside triphosphate specificity of firefly luciferase.Analytical Biochemistry,1983,131(1):187-189
Nyrén P.Apyrase immobilized on paramagnetic beads used to improve detection limits in bioluminometric ATP monitoring.Luminescence,1994,9(1):29-34
Selan L,Berlutti F,Passariello C,et al.Reliability of a bioluminescence ATP assay for detection of bacteria.Journal of Clinical Microbiology,1992,30(7):1739-1742
Sun J,Song XL,Huang J.Optimization of fermentation conditions and analysis of the nutrition components of soybean meal fermented with Bacillus firmus.Progress in Fishery Sciences,2017,38(3):163-171[孙静,宋晓玲,黄倢.豆粕的坚强芽孢杆菌(Bacillus firmus)发酵工艺优化及其营养成分分析.渔业科学进展,2017,38(3):163-171]
Velten S,Hammes F,Boller M,et al.Rapid and direct estimation of active biomass on granular activated carbon through adenosine tri-phosphate(ATP)determination.Water Research,2007,41(9):1973-1983
Wang N,Wang HL,Bai N,et al.Isolation of lysogenic phage in Vibrio parahaemolyticus and its relationship with the pathogenicity of the host bacteria.Progress in Fishery Sciences,2016,37(2):105-110[王娜,王海亮,白楠,等.副溶血弧菌(Vibrio parahaemolyticus)中溶源噬菌体与其宿主菌致病力的相关性.渔业科学进展,2016,37(2):105-110
Wang TT,Deng TL,Liao MX.Discussions on the methodology of aquatic bacteria counting.World Sci-Tech R&D,2008,30(2):138-142[王婷婷,邓天龙,廖梦霞.水生细菌计数方法研究进展及展望.世界科技研究与发展,2008,30(2):138-142]
Wang X,Wu YC,Xia SP,et al.Inquiring into the method of counting live germ with MTT.Journal of Luzhou Medical College,2002,25(4):291-293[王栩,邬于川,夏世平,等MTT法进行活菌计数的方法学探讨.泸州医学院学报,2002,25(4):291-293]
Wang ZC,Fan LY,Cai W,et al.Study on counting of H.actinomycetemcomitans by MTT colorimetric method.Journal of Modern Medicine&Health,2010,26(8):1121-1123[王忠朝,范丽苑,蔡炜,等.MTT法用于检测伴放线菌嗜血菌的研究.现代医药卫生,2010,26(8):1121-1123]
Wang ZR,Gao Q,Ma CX,et al.Experimental study on the application of MTT colorimetric method in counting live Escherichia coli.Acta Scientiae Circumstantiae,2011,31(12):2642-2650[汪志荣,高琼,马传鑫,等.MTT法测定大肠杆菌活菌数实验研究.环境科学学报,2011,31(12):2642-2650]
Brewster JD.A simple micro-growth assay for enumerating bacteria.Journal of Microbiological Methods,2003,53(1):77-86
Chen FC,Godwin SL.Comparison of a rapid ATPbioluminescence assay and standard plate count methods for assessing microbial contamination of consumers'refrigerators.Journal of Food Protection,2006,69(10):2534-2538
Dong X,Bi DX,Wang HL,et al.pir ABvp-bearing Vibrio parahaemolyticus and Vibrio campbellii pathogens isolated from the same AHPND-affected pond possess highly similar pathogenic plasmids.Frontiers in Microbiology,2017,8:1859
Gao G,Zhu KL,Zhang QQ,et al.Simplified fermentation of a functional probiotics and the application in prawn(Litopenaeus vannamei)bio-floc breeding.Progress in Fishery Sciences,2017,38(3):140-147[高戈,朱开玲,张庆起,等.一株功能益生菌的简易发酵及其在凡纳滨对虾(Litopenaeus vannamei)生物絮团养殖中的应用.渔业科学进展,2017,38(3):140-147]
Gerlier D,Thomasset N.Use of MTT colorimetric assay to measure cell activation.Journal of Immunological Methods,1986,94(1-2):57-63
Gong JL,Deng J,Wu HC,et al.Research on the optimum condition of MTT in yeast count.China Condiment,2016(1):10-14[龚加路,邓静,吴华昌,等.MTT法在酵母菌活菌计数中最佳条件的研究.中国调味品,2016(1):10-14]
Gr?nroos M,M?enp??J,Nieminen AL,et al.548 Correlation of steroid receptor contents with medroxyprogesterone and tamoxifen effects in endometrial cancer assayed by barin vitro ATP-bioluminescence method.Journal of Steroid Biochemistry,1983,19(S1):194
Hammes F,Goldschmidt F,Vital M,et al.Measurement and interpretation of microbial adenosine tri-phosphate(ATP)in aquatic environments.Water Research,2010,44(13):3915
Holm-Hansen O,Booth CR.The Measurement of adenosine triphosphate in the ocean and its ecological significance.Limnology and Oceanography,1966,11(4):510-519
Huang LK,Du P,Huo GC.Inquiring into the method of counting live germ with MTT.Food Industry,2008(3):62-65[黄立坤,杜鹏,霍贵成.MTT法测定乳酸菌活菌数的研究.食品工业,2008(3):62-65]
Ling Y,Wang LB,Shen H,et al.Uncertainty evaluation for plate counts of coliforms in foods.Food&Machinery,2010,26(5):75-77[凌云,王李宝,沈辉,等.食品中大肠菌群平板计数结果不确定度的评定.食品与机械,2010,26(5):75-77]
Mc Elroy WD,Strehler BL.Factors influencing the response of the bioluminescent reaction to adenosine triphosphate.Archives of Biochemistry,1949,22(3):420-433
Miller JN,Nawawi MB,Burgess C.Detection of bacterial ATPby reversed flow-injection analysis with luminescence detection.Analytica Chimica Acta,1992,266(2):339-343
Mosmann T.Rapid colorimetric assay for cellular growth and survival:Application to proliferation and cytotoxicity assays Journal of Immunological Methods,1983,65(1-2):55-63
Moyer JD,Henderson JF.Nucleoside triphosphate specificity of firefly luciferase.Analytical Biochemistry,1983,131(1):187-189
Nyrén P.Apyrase immobilized on paramagnetic beads used to improve detection limits in bioluminometric ATP monitoring.Luminescence,1994,9(1):29-34
Selan L,Berlutti F,Passariello C,et al.Reliability of a bioluminescence ATP assay for detection of bacteria.Journal of Clinical Microbiology,1992,30(7):1739-1742
Sun J,Song XL,Huang J.Optimization of fermentation conditions and analysis of the nutrition components of soybean meal fermented with Bacillus firmus.Progress in Fishery Sciences,2017,38(3):163-171[孙静,宋晓玲,黄倢.豆粕的坚强芽孢杆菌(Bacillus firmus)发酵工艺优化及其营养成分分析.渔业科学进展,2017,38(3):163-171]
Velten S,Hammes F,Boller M,et al.Rapid and direct estimation of active biomass on granular activated carbon through adenosine tri-phosphate(ATP)determination.Water Research,2007,41(9):1973-1983
Wang N,Wang HL,Bai N,et al.Isolation of lysogenic phage in Vibrio parahaemolyticus and its relationship with the pathogenicity of the host bacteria.Progress in Fishery Sciences,2016,37(2):105-110[王娜,王海亮,白楠,等.副溶血弧菌(Vibrio parahaemolyticus)中溶源噬菌体与其宿主菌致病力的相关性.渔业科学进展,2016,37(2):105-110
Wang TT,Deng TL,Liao MX.Discussions on the methodology of aquatic bacteria counting.World Sci-Tech R&D,2008,30(2):138-142[王婷婷,邓天龙,廖梦霞.水生细菌计数方法研究进展及展望.世界科技研究与发展,2008,30(2):138-142]
Wang X,Wu YC,Xia SP,et al.Inquiring into the method of counting live germ with MTT.Journal of Luzhou Medical College,2002,25(4):291-293[王栩,邬于川,夏世平,等MTT法进行活菌计数的方法学探讨.泸州医学院学报,2002,25(4):291-293]
Wang ZC,Fan LY,Cai W,et al.Study on counting of H.actinomycetemcomitans by MTT colorimetric method.Journal of Modern Medicine&Health,2010,26(8):1121-1123[王忠朝,范丽苑,蔡炜,等.MTT法用于检测伴放线菌嗜血菌的研究.现代医药卫生,2010,26(8):1121-1123]
Wang ZR,Gao Q,Ma CX,et al.Experimental study on the application of MTT colorimetric method in counting live Escherichia coli.Acta Scientiae Circumstantiae,2011,31(12):2642-2650[汪志荣,高琼,马传鑫,等.MTT法测定大肠杆菌活菌数实验研究.环境科学学报,2011,31(12):2642-2650]