DNase处理法结合数字PCR与qPCR对活的非可培养状态副溶血性弧菌检测比较研究
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摘要
目的探讨DNase处理法结合微滴化数字PCR(droplet digital PCR,ddPCR)在检测冷冻食品中活的非可培养状态(viable but non-culturable state, VBNC)副溶血性弧菌的适用性,并与实时荧光定量PCR(qPCR)进行比较。方法用无菌磷酸盐缓冲液对解冻的大西洋鲑鱼样品进行10倍梯度稀释,再加入副溶血性弧菌,终浓度为6.6×10~5 CFU/mL。-20℃分别诱导10、20和30 d。将DNase试剂与叠氮溴化丙锭(propidium monoazide,PMA)染料分别作用于不同时期的冷冻基质,结合qPCR和ddPCR进行比较检测,对比其作用效果。结果DNase-qPCR和PMA-qPCR检测3个冷冻阶段活性副溶血性弧菌的Cq值分别为31.41±0.06、32.40±0.04、34.59±0.15和31.24±0.06、32.32±0.03、34.25±0.12, 2种方法Cq值均呈现上升的趋势且数值接近。采用DNase-ddPCR和PMA-ddPCR检测各阶段活性副溶血性弧菌,可直接读出绝对拷贝数,分别为233±6.43、108±5.57、28±3.21和256±6.56、126±3.06、35±2.52。2种方法检测的拷贝数差异不大,重复性好。相对标准偏差均在可接受范围内,符合欧盟定量检测要求。结论DNase处理试剂与PMA对有活性的副溶血性弧菌检测效果相当,与PMA相比,DNase处理法无需强光照射,操作简便快捷,无试剂毒性。ddPCR不需要标准品即能实现对基质中微量活性副溶血弧菌精准定量检测。
Objective To explore the applicability of DNase treatment combined with droplet digital PCR(ddPCR) in the detection of viable but non-culturable state(VBNC) Vibrio parahaemolyticus, and compare with real-time fluorescence quantitative PCR(qPCR). Methods The thawed Atlantic salmon samples were diluted by 10 times gradient with sterile phosphate buffered saline, and V. parahaemolyticus was added. The final concentration was 6.6×10~5 CFU/mL induced of 10, 20 and 30 d at-20℃. The DNase reagent and the propidium monoazide(PMA)dye were applied to the frozen substrates at different stages, and the effects were compared by qPCR and ddPCR,respectively. Results The Cq of V. parahaemolyticus in 3 freezing stages detected by DNase-qPCR and PMA-qPCR was 31.41±0.06, 32.40±0.04, 34.59±0.15 and 31.24±0.06, 32.32±0.03, 34.25±0.12, respectively. The Cq values of both methods showed an upward trend and the values were close to each other. When detected by DNase-ddPCR and PMA-ddPCR for V. parahaemolyticus, the absolute number of copies could be read directly: 233±6.43, 108±5.57,28±3.21, and 256±6.56, 126±3.06, 35±2.52. There was little difference in the number of copies detected by the two methods, and they had good repeatability. Relative standard deviation was acceptable and met the requirement of quantitative detection in EU. Conclusion The DNase treatment reagent and PMA have the similar effect on the detection of active V. parahaemolyticus. Compared with PMA, the DNase treatment method does not require strong light irradiation, and the operation is simple and rapid, and there is no reagent toxicity. ddPCR can detect trace amounts of active V. parahaemolyticus in the matrix and achieve accurate quantification without the need for standards.
Objective To explore the applicability of DNase treatment combined with droplet digital PCR(ddPCR) in the detection of viable but non-culturable state(VBNC) Vibrio parahaemolyticus, and compare with real-time fluorescence quantitative PCR(qPCR). Methods The thawed Atlantic salmon samples were diluted by 10 times gradient with sterile phosphate buffered saline, and V. parahaemolyticus was added. The final concentration was 6.6×10~5 CFU/mL induced of 10, 20 and 30 d at-20℃. The DNase reagent and the propidium monoazide(PMA)dye were applied to the frozen substrates at different stages, and the effects were compared by qPCR and ddPCR,respectively. Results The Cq of V. parahaemolyticus in 3 freezing stages detected by DNase-qPCR and PMA-qPCR was 31.41±0.06, 32.40±0.04, 34.59±0.15 and 31.24±0.06, 32.32±0.03, 34.25±0.12, respectively. The Cq values of both methods showed an upward trend and the values were close to each other. When detected by DNase-ddPCR and PMA-ddPCR for V. parahaemolyticus, the absolute number of copies could be read directly: 233±6.43, 108±5.57,28±3.21, and 256±6.56, 126±3.06, 35±2.52. There was little difference in the number of copies detected by the two methods, and they had good repeatability. Relative standard deviation was acceptable and met the requirement of quantitative detection in EU. Conclusion The DNase treatment reagent and PMA have the similar effect on the detection of active V. parahaemolyticus. Compared with PMA, the DNase treatment method does not require strong light irradiation, and the operation is simple and rapid, and there is no reagent toxicity. ddPCR can detect trace amounts of active V. parahaemolyticus in the matrix and achieve accurate quantification without the need for standards.
引文
[1] Zhong QP, Tian J,Wang B,et al. PMA based real-time fluorescent LAMP for detection of Vibrio parahaemolyticus in viable but nonculturable state[J]. Food Control, 2016,63:230-238.
[2] Pinto D, Santos MA, Chambel L. Thirty years of viable but nonculturable state research:Unsolved molecular mechanisms[J]. Crit Rev Microbiol,2015,41(1):61-76.
[3] Catherine MB, Andrea G, Nadia G, et al. The response of foodbome pathogens to osmotic and desiccation stresses in the food chain[J]. Int J Food Microbiol, 2016,221:37-53.
[4] Oliver JD. Recent findings on the viable but nonculturable state in pathogenic bacteria[J]. FEMS Microbiol Rev, 2010,34(4):415-425.
[5] Zhao XH, Zhong JL, Wei CJ, et al. Current perspectives on viable but non-culturable state in foodbome pathogens[J]. Front Microbiol, 2017,8(703813):580.
[6] Forghani F, Langaee T, Eskandari M, et al. Rapid detection of viable Bacillus cereus emetic and enterotoxic strains in food by coupling propidium monoazide and multiplex PCR(PMA-mPCR)[J]. Food Control,2015, 55:151-157.
[7] Zhang ZH, Liu W, Xu HY, et al. Propidium monoazide combined withreal-time PCR for selective detection of viable Staphylococcus aureus in milk powder and meat products[J]. J Dairy Sci, 2015, 98(3):1625-1633.
[8] Chang CW, Lin MH. Optimization of PMA-qPCR for Staphylococcus aureus and determination of viable bacteria in indoor air[J]. Indoor Air,2017, 28(1):64-72.
[9] Villarreal JV, Jungfer C, Obst U, et al. DNase I and proteinase K eliminate DNA from injured or dead bacteria but not from living bacteria in microbial reference systems and natural drinking water biofilms for subsequent molecular biology analyses[J]. J Microbiol Methods, 2013, 94:161-169.
[10] Reyneke B, Ndlovu T, Khan S, et al. Comparison of EMA-, PMA-and DNase qPCR for the determination of microbial cell viability[J]. Appl Microbiol Biotechnol, 2017, 101(19):7371-7383.
[11] Massanella M, Singhania A, Bwliakova BN, et al. Differential gene expression in HIV-infected individuals following ART[J]. Antiv Res, 2013,100(2):420-428.
[12] GB4789.1-2010食品安全国家标准食品微生物学检验总则[S].GB 4789.1-2010 National food safety standard-General principles for food microbiological testing[S].
[13] Huang XX, Yang J, Zheng J, et al. Propidium monoazide based real-time PCR to detect Vibrio parahaemolyticus in a viable but nonculturable state[J]. Microbiol China, 2018,45(4):934-940.
[14] European Network of GMO Laboratories(ENGL). Definition of minimum performance requirements for analytical methods of GMO testing[Z].
[15] Nebe-von CG, Badley RA. Viability assessment of bacteria in mixed populations using flow cytometry[J]. J Microscopy, 1995, 179:55-66.
[16] Pan Y, Breidt JF. Enumeration of viable Listeria monocytogenes cells by realtime PCR with propidium monoazide and ethidium monoazide in the presence of dead cells[J]. Appl Environ Microbiol, 2007, 73:8028-8031.
[17] Duarte A, Botteldoorn N, Coucke W, et al. Effect of exposure to stress conditions on propidium monoazide(PMA)-qPCR based Campylobacter enumeration in broiler carcass rinses[J]. Food Microbiol, 2015, 48:182-190.
[18] Pacholewicz E, Swart A, Lipman LJ, et al. Propidium monoazide does not fully inhibit the detection of dead Campylobacter on broiler chicken carcasses by Qpcr[J]. J Microbiol Methods, 2013, 95(1):32-38.
[19] Zhong JL, Zhao XH. Detection of viable but non culturable Escherichia coli 0157:H7 by PCR in combination with propidium monoazide[J].Biotech, 2018,8:28.
[20] Lovdal T, Befring HM, Bjorkblom B, et al. Propidium monoazide combined with real-time quantitative PCR underestimates heat-killed Listeria innocua[J]. J Microbiol Methods, 2011,85:164-169.
[2] Pinto D, Santos MA, Chambel L. Thirty years of viable but nonculturable state research:Unsolved molecular mechanisms[J]. Crit Rev Microbiol,2015,41(1):61-76.
[3] Catherine MB, Andrea G, Nadia G, et al. The response of foodbome pathogens to osmotic and desiccation stresses in the food chain[J]. Int J Food Microbiol, 2016,221:37-53.
[4] Oliver JD. Recent findings on the viable but nonculturable state in pathogenic bacteria[J]. FEMS Microbiol Rev, 2010,34(4):415-425.
[5] Zhao XH, Zhong JL, Wei CJ, et al. Current perspectives on viable but non-culturable state in foodbome pathogens[J]. Front Microbiol, 2017,8(703813):580.
[6] Forghani F, Langaee T, Eskandari M, et al. Rapid detection of viable Bacillus cereus emetic and enterotoxic strains in food by coupling propidium monoazide and multiplex PCR(PMA-mPCR)[J]. Food Control,2015, 55:151-157.
[7] Zhang ZH, Liu W, Xu HY, et al. Propidium monoazide combined withreal-time PCR for selective detection of viable Staphylococcus aureus in milk powder and meat products[J]. J Dairy Sci, 2015, 98(3):1625-1633.
[8] Chang CW, Lin MH. Optimization of PMA-qPCR for Staphylococcus aureus and determination of viable bacteria in indoor air[J]. Indoor Air,2017, 28(1):64-72.
[9] Villarreal JV, Jungfer C, Obst U, et al. DNase I and proteinase K eliminate DNA from injured or dead bacteria but not from living bacteria in microbial reference systems and natural drinking water biofilms for subsequent molecular biology analyses[J]. J Microbiol Methods, 2013, 94:161-169.
[10] Reyneke B, Ndlovu T, Khan S, et al. Comparison of EMA-, PMA-and DNase qPCR for the determination of microbial cell viability[J]. Appl Microbiol Biotechnol, 2017, 101(19):7371-7383.
[11] Massanella M, Singhania A, Bwliakova BN, et al. Differential gene expression in HIV-infected individuals following ART[J]. Antiv Res, 2013,100(2):420-428.
[12] GB4789.1-2010食品安全国家标准食品微生物学检验总则[S].GB 4789.1-2010 National food safety standard-General principles for food microbiological testing[S].
[13] Huang XX, Yang J, Zheng J, et al. Propidium monoazide based real-time PCR to detect Vibrio parahaemolyticus in a viable but nonculturable state[J]. Microbiol China, 2018,45(4):934-940.
[14] European Network of GMO Laboratories(ENGL). Definition of minimum performance requirements for analytical methods of GMO testing[Z].
[15] Nebe-von CG, Badley RA. Viability assessment of bacteria in mixed populations using flow cytometry[J]. J Microscopy, 1995, 179:55-66.
[16] Pan Y, Breidt JF. Enumeration of viable Listeria monocytogenes cells by realtime PCR with propidium monoazide and ethidium monoazide in the presence of dead cells[J]. Appl Environ Microbiol, 2007, 73:8028-8031.
[17] Duarte A, Botteldoorn N, Coucke W, et al. Effect of exposure to stress conditions on propidium monoazide(PMA)-qPCR based Campylobacter enumeration in broiler carcass rinses[J]. Food Microbiol, 2015, 48:182-190.
[18] Pacholewicz E, Swart A, Lipman LJ, et al. Propidium monoazide does not fully inhibit the detection of dead Campylobacter on broiler chicken carcasses by Qpcr[J]. J Microbiol Methods, 2013, 95(1):32-38.
[19] Zhong JL, Zhao XH. Detection of viable but non culturable Escherichia coli 0157:H7 by PCR in combination with propidium monoazide[J].Biotech, 2018,8:28.
[20] Lovdal T, Befring HM, Bjorkblom B, et al. Propidium monoazide combined with real-time quantitative PCR underestimates heat-killed Listeria innocua[J]. J Microbiol Methods, 2011,85:164-169.