利用MTT建立一种快速检测青枯菌活菌的方法
|
摘要
利用四氮唑蓝(MTT),优化了检测波长、菌浓度、MTT用量、MTT反应时间、三联液的用量和溶解时间等,建立一种有效快速检测青枯菌活菌的方法。结果发现,MTT反应的最佳检测波长为512 nm,青枯菌浓度A_(600)为0. 3,MTT用量为30μL,MTT还原反应控制在20 min以内,三联液溶解甲臜晶体的用量为3 m L,溶解8 h,甲臜溶液在10～120μL以内与A512的数值线性关系良好,平板计数印证了这一结果。实验结果表明,MTT法可以用于快速检测青枯菌等植物病原菌活菌,对农产品进出口检验检疫具有一定的应用价值。
Method for assay of the viable cells of Ralstonia solanacearum was established by using methylthiazolyldiphenyl-tetrazolium bromide( MTT) and the parameters like the detection wavelength,bacterial concentration,MTT dosage and incubation time,triplex solution dosage and dissolution time were optimized. Results showed that the best detection wavelength for the reaction of MTT was 512 nm,Ralstonia solanacearum concentration was 0. 3 in A600,the dosage of MTT was 30 μL,the reduction reaction of MTT was within 20 min,the dosage of triplex solution for dissolving formazan crystal was 3 m L,dissolved 8 h,and the A512 value was linear with formazan solutions between 10 μL and 120 μL. The study would provide the MTT method for rapid detection of plant pathogenic viable bacteria such as Ralstonia solanacearum,and have potential application value in agricultural products import and export detections.
Method for assay of the viable cells of Ralstonia solanacearum was established by using methylthiazolyldiphenyl-tetrazolium bromide( MTT) and the parameters like the detection wavelength,bacterial concentration,MTT dosage and incubation time,triplex solution dosage and dissolution time were optimized. Results showed that the best detection wavelength for the reaction of MTT was 512 nm,Ralstonia solanacearum concentration was 0. 3 in A600,the dosage of MTT was 30 μL,the reduction reaction of MTT was within 20 min,the dosage of triplex solution for dissolving formazan crystal was 3 m L,dissolved 8 h,and the A512 value was linear with formazan solutions between 10 μL and 120 μL. The study would provide the MTT method for rapid detection of plant pathogenic viable bacteria such as Ralstonia solanacearum,and have potential application value in agricultural products import and export detections.
引文
[1]RIGOTTI R T,CORRA J A F,MAIA N J L,et al. Combination of natural antimicrobials and sodium dodecyl sulfate for disruption of biofilms formed by contaminant bacteria isolated from sugarcane mills[J]. Innovative Food Science&Emerging Technologies,2017,41:26-33.
[2]RAHMAN M,KHN I,RAHMAN M,et al. Evaluation of a scanner-assisted colorimetric MIC method for susceptibility testing of gram-negative fermentative bacteria[J]. Applied and Environmental Microbiology,2004,70(4):2398-2403.
[3]CAMPANELLA D,RIZZELLO C G,FASCIANO C,et al. Exploitation of grape marc as functional substrate for lactic acid bacteria and bifidobacteria growth and enhanced antioxidant activity[J]. Food Microbiology,2017,65:25-35.
[4]SHOKRGOZAR M A,ZALI H,REZAEI TAVIRANI M,et al. Comparison of two staining assays trypan blue and mtt in vitro evaluation of human calprotectin proliferation inhibition on human gastric cancer cells[J]. Kowsar Medical Journal,2007,12(2):127-137.
[5]SELESTINO NETA M C,VITTORAZZI C,GUIMARES A C,et al. Effects ofβ-caryophyllene and Murraya paniculata essential oil in the murine hepatoma cells and in the bacteria and fungi 24-h time-kill curve studies[J]. Pharmaceutical Biology,2017,55(1):190-197.
[6]AZIM A A,AKSEL H,ZHUANG T,et al. Efficacy of 4 irrigation protocols in killing bacteria colonized in dentinal tubules examined by a novel confocal laser scanning microscope analysis[J]. Journal of Endodontics,2016,42(6):928-934.
[7]QI J,DONG Z,LIU J,et al. Effective targeting of the survivin dimerization interface with small-molecule inhibitors[J]. Cancer Research,2016,76(2):453-462.
[8]WANG H,CHENG H,WANG F,et al. An improved 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)reduction assay for evaluating the viability of Escherichia coli cells[J]. Journal of Microbiological Methods,2010,82(3):330-333.
[9]FU L,WU H,CHENG S Y. Set7 mediated Gli3 methylation plays a positive role in the activation of Sonic Hedgehog pathway in mammals[J]. Elife,2016,5:e15690.
[10]GHANWATE N,THAKARE P,BHISE P R,et al. Colorimetric method for rapid detection of oxacillin resistance in Staphylococcus aureusand its comparison with PCR formec A gene[J]. Scientific Reports,2016,6:23013.
[11]SUN M,ZHOU Z,DONG J,et al. Antibacterial and antibiofilm activities of docosahexaenoic acid(DHA)and eicosapentaenoic acid(EPA)against periodontopathic bacteria[J]. Microbial Pathogenesis,2016,99:196-203.
[12]TAKAHASHI S,ABE T,GOTOH J,et al. Substrate-dependence of reduction of MTT:a tetrazolium dye differs in cultured astroglia and neurons[J]. Neurochemistry International,2002,40(5):441-448.
[13]FISCHER J,PROSENC M H,WOLFF M,et al. Interference of magnesium corrosion with tetrazolium-based cytotoxicity assays[J].Acta Biomaterialia,2010,6(5):1813-1823.
[14]LAAKSONEN T,SANTOS H,VIHOLA H,et al. Failure of MTT as a toxicity testing agent for mesoporous silicon microparticles[J].Chemical Research in Toxicology,2007,20(12):1913-1918.
[15]TSUKATANI T,SUENAGA H,HIGUCHI T,et al. Colorimetric cell proliferation assay for microorganisms in microtiter plate using water-soluble tetrazolium salts[J]. Journal of Microbiological Methods,2008,75(1):109-116.
[2]RAHMAN M,KHN I,RAHMAN M,et al. Evaluation of a scanner-assisted colorimetric MIC method for susceptibility testing of gram-negative fermentative bacteria[J]. Applied and Environmental Microbiology,2004,70(4):2398-2403.
[3]CAMPANELLA D,RIZZELLO C G,FASCIANO C,et al. Exploitation of grape marc as functional substrate for lactic acid bacteria and bifidobacteria growth and enhanced antioxidant activity[J]. Food Microbiology,2017,65:25-35.
[4]SHOKRGOZAR M A,ZALI H,REZAEI TAVIRANI M,et al. Comparison of two staining assays trypan blue and mtt in vitro evaluation of human calprotectin proliferation inhibition on human gastric cancer cells[J]. Kowsar Medical Journal,2007,12(2):127-137.
[5]SELESTINO NETA M C,VITTORAZZI C,GUIMARES A C,et al. Effects ofβ-caryophyllene and Murraya paniculata essential oil in the murine hepatoma cells and in the bacteria and fungi 24-h time-kill curve studies[J]. Pharmaceutical Biology,2017,55(1):190-197.
[6]AZIM A A,AKSEL H,ZHUANG T,et al. Efficacy of 4 irrigation protocols in killing bacteria colonized in dentinal tubules examined by a novel confocal laser scanning microscope analysis[J]. Journal of Endodontics,2016,42(6):928-934.
[7]QI J,DONG Z,LIU J,et al. Effective targeting of the survivin dimerization interface with small-molecule inhibitors[J]. Cancer Research,2016,76(2):453-462.
[8]WANG H,CHENG H,WANG F,et al. An improved 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)reduction assay for evaluating the viability of Escherichia coli cells[J]. Journal of Microbiological Methods,2010,82(3):330-333.
[9]FU L,WU H,CHENG S Y. Set7 mediated Gli3 methylation plays a positive role in the activation of Sonic Hedgehog pathway in mammals[J]. Elife,2016,5:e15690.
[10]GHANWATE N,THAKARE P,BHISE P R,et al. Colorimetric method for rapid detection of oxacillin resistance in Staphylococcus aureusand its comparison with PCR formec A gene[J]. Scientific Reports,2016,6:23013.
[11]SUN M,ZHOU Z,DONG J,et al. Antibacterial and antibiofilm activities of docosahexaenoic acid(DHA)and eicosapentaenoic acid(EPA)against periodontopathic bacteria[J]. Microbial Pathogenesis,2016,99:196-203.
[12]TAKAHASHI S,ABE T,GOTOH J,et al. Substrate-dependence of reduction of MTT:a tetrazolium dye differs in cultured astroglia and neurons[J]. Neurochemistry International,2002,40(5):441-448.
[13]FISCHER J,PROSENC M H,WOLFF M,et al. Interference of magnesium corrosion with tetrazolium-based cytotoxicity assays[J].Acta Biomaterialia,2010,6(5):1813-1823.
[14]LAAKSONEN T,SANTOS H,VIHOLA H,et al. Failure of MTT as a toxicity testing agent for mesoporous silicon microparticles[J].Chemical Research in Toxicology,2007,20(12):1913-1918.
[15]TSUKATANI T,SUENAGA H,HIGUCHI T,et al. Colorimetric cell proliferation assay for microorganisms in microtiter plate using water-soluble tetrazolium salts[J]. Journal of Microbiological Methods,2008,75(1):109-116.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |