副溶血弧菌EMA-PCR检测技术的建立

PCR技术被广泛应用于副溶血弧菌的检测中, 但传统的PCR技术无法区分样品中的死细菌与活细菌, 往往使检测结果出现较高的假阳性。因此, 将叠氮溴乙锭(Ethidium monoazide bromide, EMA)与PCR技术结合, 建立一种快速、准确的副溶血弧菌检测方法。以dnaJ基因为检测副溶血弧菌的靶基因, 分别用副溶血弧菌的纯培养细胞及其基因组DNA作模板进行PCR检测, 灵敏度分别为2.5×104 CFU/mL和6×102 fg/μL。在检测样品前处理过程中加入EMA, 当EMA的浓度小于5 mg/L时, EMA对活菌靶基因的扩增没有明显的抑制; 而终浓度为2 mg/L的EMA, 能有效抑制1×108 CFU/mL副溶血弧菌死菌的扩增。活菌和死菌混合体系的PCR结果表明, EMA-PCR能有效降低副溶血弧菌检测过程中的假阳性。     

SD-PMA-ddPCR检测食品中单核细胞增生李斯特氏菌

【目的】检测食品中单核细胞增生李斯特氏菌活菌。【方法】利用脱氧胆酸钠(SD)对受损细胞预处理,然后使叠氮溴化丙锭(PMA)进入受损细胞与DNA发生共价交联,提取细菌基因组DNA进行微滴式数字PCR(dd PCR)检测。【结果】0.1%SD和5.0 mg/L PMA协同作用,可以有效抑制108 CFU/m L的单核细胞增生李斯特氏菌死菌DNA的PCR扩增。经过SD和PMA对样品预处理,dd PCR可以在死菌存在条件下,定量检测鸡肉中单核细胞增生李斯特氏菌活菌,消除了"假阳性"结果的出现。活菌灵敏度检测结果显示:SD-PMA-dd PCR的灵敏度为2.0 copies/20μL。SD-PMA-dd PCR方法精密度和稳定性良好。【结论】SD-PMA-dd PCR在检测食源性致病菌方面有巨大的发展空间。     

脱氧胆酸钠在副溶血性弧菌死活细胞叠氮溴乙锭PCR鉴别中的应用

[目的]通过将表面活性剂脱氧胆酸钠(Sodium deoxycholate,SD)与叠氮溴乙锭(Ethidium bromide monoazide,EMA)-PCR反应体系相结合,建立SD-EMA-PCR鉴别副溶血性弧菌死活细胞的检测方法.[方法]依次对加入检测体系中的脱氧胆酸钠最适浓度、EMA区分死活细胞DNA的浓度范围、EMA激活光解最佳曝光时间进行优化;确定SD-EMA-PCR方法检测副溶血性弧菌死活细胞混合体系中活细胞的最低检出限.[结果]当脱氧胆酸钠浓度≤0.5 g/L,EMA的浓度为3.2-34.0 mg/L,曝光时间为25 min时,SD-EMA-PCR检测体系仅对死细胞DNA扩增产生抑制作用.SD-EMA-PCR检测活菌细胞的最低检出限为10 CFU/mL.[结论]死活细胞混合体系的SD-EMA-PCR检测证明该方法能够明显降低EMA-PCR漏检的死菌对检测结果造成的影响,为完善食源性致病菌检测中死活菌细胞鉴别方法提供了一种有效途径.     

EMA-LAMP方法快速检测鉴别副溶血性弧菌

建立将DNA染料EMA(ethidium bromide monoazide)结合环介导等温扩增技术(loop-mediated isother-mal amplification,LAMP)的方法(EMA-LAMP),用于检测鉴别副溶血性弧菌(Vibrio parahaemolyticus)死/活菌细胞。针对副溶血性弧菌不耐热溶血素基因tlh(thermolabile hemolysin)特异性序列的6个位点设计4条引物及2条环引物,进行检测。结果表明,浓度为8.0μg/mL或更高浓度的EMA,至少经25 min的曝光处理,能够有效抑制浓度为1×108cfu/mL的副溶血性弧菌死细胞的扩增,而对用相同浓度EMA处理的副溶血性弧菌活细胞扩增没有影响。经EMA处理,含有不同比例的副溶血弧菌死细胞和活细胞的混合液中,活菌的最小检测限为1.0×102cfu/mL。EMA-LAMP方法比EMA-PCR方法区分死活细胞中的活细胞更为有效,是一种能够快速、灵敏且更为有效鉴别副溶血性弧菌死活细胞的新方法。     

德尔卑沙门氏菌分子等温活菌检测体系的建立

【背景】德尔卑沙门氏菌(Salmonella enterica subsp.enterica Derby)是危害人类生命安全的主要致病性血清型。【目的】建立一种准确、快速检测德尔卑沙门氏菌的方法。【方法】通过建立叠氮溴化丙锭(propidium monoazide,PMA)-重组酶聚合酶扩增(recombinase polymerase amplification,RPA)的方法准确有效地检测样品中的活德尔卑沙门氏菌。【结果】使用基因RU61＿00441作为检测靶点,设计引物SD1正确地鉴定了所有被测菌株。实验结果表明,PMA处理能有效区分活细胞和死细胞。基因组DNA检测限为761.2 fg/μL,活菌检测限为45 CFU/m L。此方法检测德尔卑沙门氏菌的血清型不受自然背景(猪肉、鸡肉和牛肉)菌群基因组DNA的影响。此外,该方法还可以检测出动物性食品中富集6 h后浓度低至3.9 CFU/m L的德尔卑沙门氏菌。【结论】这种PMA-RPA检测方法耗时短并具有更好的灵敏度和特异性,能为沙门氏菌的检测提供更有效的指导。     

叠氮溴化丙锭-荧光定量PCR法实时快速检测5种乳杆菌活菌数方法的建立与应用

【背景】乳杆菌属是发酵食品中最常见的微生物之一,与食品的品质和安全密切相关,定量检测乳杆菌活菌数、解析乳杆菌群落组成对发酵乃至肠道微生物等具有重要意义。【目的】建立一种在种水平上定量检测5种乳杆菌活菌数的叠氮溴化丙锭-荧光定量PCR (propidium monoazide-quantitative PCR,PMA-qPCR)检测方法并探讨其适用性。【方法】以植物乳杆菌、发酵乳杆菌、短乳杆菌、嗜酸乳杆菌和干酪乳杆菌等发酵食品中常见的5种乳杆菌为目标菌株,查找并筛选特异性引物用于荧光定量PCR (qPCR)检测,优化叠氮溴化丙锭(PMA)处理条件,测定PMA-qPCR检测法的特异性、灵敏度及可靠性。最后利用PMA-qPCR法检测黄酒酿造过程中5种乳杆菌的活菌数。【结果】PMA最佳处理条件为：浓度20 μmol/L下暗处理15 min后曝光15 min,此时可抑制样品中99.89%的死菌DNA扩增。该方法特异性高,能够准确识别5种乳杆菌;线性关系强,R2>0.98;灵敏度高,检测限为101.8?103.2 CFU/mL;重复性好,Cq值变异系数小于1%;与平板计数相比差异不显著(统计学上),p>0.05。利用该方法检测黄酒中5种乳杆菌的活菌数,发现发酵乳杆菌、干酪乳杆菌和短乳杆菌是主要的乳杆菌(总计占比59%?89%),与已知黄酒酿造中乳杆菌群落组成相符。【结论】建立的PMA-qPCR法能够快速、准确地检测5种乳杆菌的活菌数,为解析样品中乳杆菌的实时组成及检测具有活性但不可培养(viable but nonculturable,VBNC)状态的乳杆菌提供了可靠的手段。     

携带TGEV DNA疫苗减毒沙门氏菌的构建及安全性与免疫性分析

【目的】探讨以减毒沙门氏菌为载体,进行TGEV DNA疫苗口服免疫可行性。【方法】通过RT-PCR扩增TGEV四川株（SC-H）S基因5’端约2.1 kb的主要抗原位点片段,将其插入真核表达载体pVAX1,构建重组质粒pVAX-S,体外转染COS7细胞,间接免疫荧光检测S基因表达。通过电转化将pVAX-S转入减毒鼠伤寒沙门氏菌SL7207,构建SL7207（pVAX-S）重组菌,并在体外感染小鼠腹腔巨噬细胞,以RT-PCR、间接免疫荧光检测细胞内S基因的转录与表达情况。将SL7207（pVAX-S）重组菌以5×108、1×109、2×109CFU剂量口服接种BALB/c小鼠,分析其安全性,并以1×109CFU剂量的重组菌3次免疫BALB/c小鼠,通过间接ELISA检测免疫小鼠的血清IgG与肠道粘膜IgA抗体。【结果】成功构建重组质粒pVAX-S,且重组质粒能在COS7细胞中表达。重组菌SL7207（pVAX-S）感染巨噬细胞后检测到目的基因的转录、表达。小鼠口服接种不同剂量重组菌,具有良好的安全性。免疫小鼠于二免后两周可检测到针对TGEV S蛋白的特异性血清IgG与肠道粘膜IgA抗体,且三免后两周与SL7207（pVAX1）空载体免疫组间分别存在显著性差异（P<0.05）和极显著性差异（P<0.01）。【结论】携带TGEV DNA疫苗的减毒沙门氏菌小鼠试验显示了良好的免疫原性与安全性。     

添加有扩增内标的副溶血弧菌PCR检测方法的建立

摘要：【目的】发掘副溶血弧菌特异性更强的检测靶点,并人工构建扩增内标,建立可以有效避免假阴性的新PCR检测体系。【方法】利用生物信息学方法,从副溶血弧菌（Vibrio parahaemolyticus）基因组DNA中发掘特异性很高的序列,并设计相应的特异性引物,人工构建扩增内标,建立PCR检测体系。【结果】本研究发掘得到的序列vp1332特异性很强,经检索,该序列是编码ABC转运子接合蛋白组分的基因片段,根据此序列设计一对特异检测引物（vp1332L/vp1332R）,同时,构建了扩增内标,并建立了PCR检测体系。利用该体系对296株副溶血弧菌和33株非副溶血弧菌进行检测,结果显示,所有以副溶血弧菌为模板的PCR反应均可扩增到一条343 bp的特异片段,而模板来源于非副溶血弧菌的则只能扩增到一条499 bp的扩增内标片段。灵敏度实验表明,该PCR反应体系的检测灵敏度为1.6×102 cfu/mL。人工污染实验表明,起始染菌量为1.24 cfu/25 g样品时经8 h增菌,即可检测到副溶血弧菌。实际样品检测结果也证实该方法的有效性。【结论】本研究建立的PCR反应体系能特异地检测副溶血弧菌,并可有效地排除假阴性,提高检测准确率。     

氧化压力下沙门氏菌可培养性检测及其活的非可培养状态形成情况

【背景】氧化压力会导致细菌进入活的非可培养(viable but non-culturable,VBNC)状态,菌落形成能力可能受到亚致死损伤的影响。目前对于VBNC态细菌的定量检测是基于活菌数与可培养数的差值,因此可培养数的检测对于VBNC态定量研究很关键,培养基组成不合适可能会造成漏检。【目的】分析培养基组成对氧化压力下亚致死损伤细菌检测的重要影响;探究常见食源性致病菌肠炎沙门氏菌在氧化压力下形成VBNC态的情况。【方法】分别采用Luria-Bertani (LB)、beef peptone yeast (BPY)和Salmonella Shigella (SS)培养基检测并比较肠炎沙门氏菌的可培养数;采用RT-qPCR、荧光染色-激光共聚焦显微镜观测氧化压力下肠炎沙门氏菌形成VBNC态的情况。【结果】非选择性培养基LB和BPY能检出亚致死细菌,SS培养基中牛胆盐导致可培养数减少;肠炎沙门氏菌经53°C过氧化氢处理1.5 h后进入VBNC态的比例显著高于53°C过氧化氢+亚铁离子和过氧化氢+柠檬酸处理(P<0.05)。【结论】在对VBNC态的检测中应选择合适的固体培养基检测可...     

PMA-qPCR法检测冷冻基质中非可培养状态(VBNC)副溶血性弧菌

【背景】副溶血性弧菌为冰鲜产品及肉制品中常见的污染微生物,致病性强,危害严重,出入境运输及加工的肉食品常采取冷冻冷藏的处理手段来防止微生物污染及生长,以保持食物新鲜。而残留的部分副溶血性弧菌会进入活的非可培养状态(Viable but non-culturable state,VBNC),从而构成潜在的风险隐患。【目的】建立可用于冷冻食品中VBNC副溶血性弧菌的快速检测方法,并探讨其适用性。【方法】将大西洋鲑鱼1:10匀浆,加入终浓度为6.6×10~5 CFU/mL的副溶血性弧菌,-20°C分别诱导10、20、30和50 d。建立实时荧光PCR技术(qPCR)方法,测定其特异性、灵敏度及稳定性。利用PMA-qPCR法对不同冷冻时期样品中的副溶血性弧菌进行检测,同时与qPCR、平板培养法进行比较。【结果】建立的qPCR方法特异性好,与其他阴性参考株无交叉反应;灵敏度高,检测限为19.8 CFU/mL;重复性好,C_q值的变异系数(CV)均在1.5%以下;标准曲线为y=-3.272x+45.310,线性回归系数R~2为0.996,定量范围为1×10~2–1×10~9 CFU/mL。在低温诱导10-50 d后,qPCR法的C_q值在26.32-27.34之间,与诱导前相比几乎没有变化;叠氮溴化丙锭(PMA)-qPCR法的C_q值则从诱导前的26.43逐步上升到38.84,呈现明显的上升趋势,表明死菌的数量在显著上升。经过比较及统计,PMA-qPCR检测的活菌数均高于平板培养法测出的数量,差异显著(P0.05)。【结论】PMA-qPCR特异性及灵敏度高,能有效抑制对死菌的扩增,同时能克服传统平板培养法对VBNC的漏检缺陷,可方便、快捷地用于冷冻食品中受损致病微生物,尤其是进入VBNC状态的细菌检测。     

Use of ethidium bromide monoazide for quantification of viable and dead mixed bacterial flora from fish fillets by polymerase chain reaction

Ethidium bromide monoazide (EMA) was utilized to selectively allow conventional PCR amplification of target DNA from viable but not dead cells from a broth culture of bacterial mixed flora derived from cod fillets. The universal primers designated DG74 and RW01 that amplify a 370-bp sequence of a highly conserved region of all eubacterial 16S rDNA were used for the PCR. The use of 10 μg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The minimum amount of EMA to completely inhibit the PCR amplification of DNA derived from dead bacterial cells was 0.8 μg/ml. Amplification of target DNA from only viable cells in a suspension with dead cells was selectively accomplished by first treating the cells with 1 μg/ml of EMA. A standard curve was generated relating the intensity of fluorescence of DNA bands to the log of CFU of mixed bacterial cultures for rapidly assessing the number of genomic targets per PCR derived from the number of CFU. A linear range of DNA amplification was exhibited from 1 × 10² to 1 × 10⁵ genomic targets per PCR. The viable/dead cell discrimination with the EMA-PCR method was evaluated by comparison with plate counts following freezing and thawing. Thawing frozen cell suspensions initially containing 1 × 10⁵ CFU/ml at 4, 20, and 37 °C yielded a 0.8 log reduction in the number of viable cells determined by both plate counts and EMA-PCR. In contrast, thawing for 5 min at 70 °C resulted in a 5 log reduction in CFU derived from plate counts (no CFU detected) whereas the EMA-PCR procedure resulted in only a 2.8 log reduction in genomic targets, possibly reflecting greater damage to enzymes or ribosomes at 70 °C to a minority of the mixed population compared to membrane damage.     

Use of ethidium bromide monoazide for quantification of viable and dead mixed bacterial flora from fish fillets by polymerase chain reaction

Ethidium bromide monoazide (EMA) was utilized to selectively allow conventional PCR amplification of target DNA from viable but not dead cells from a broth culture of bacterial mixed flora derived from cod fillets. The universal primers designated DG74 and RW01 that amplify a 370-bp sequence of a highly conserved region of all eubacterial 16S rDNA were used for the PCR. The use of 10 microg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The minimum amount of EMA to completely inhibit the PCR amplification of DNA derived from dead bacterial cells was 0.8 microg/ml. Amplification of target DNA from only viable cells in a suspension with dead cells was selectively accomplished by first treating the cells with 1 microg/ml of EMA. A standard curve was generated relating the intensity of fluorescence of DNA bands to the log of CFU of mixed bacterial cultures for rapidly assessing the number of genomic targets per PCR derived from the number of CFU. A linear range of DNA amplification was exhibited from 1 x 10(2) to 1 x 10(5) genomic targets per PCR. The viable/dead cell discrimination with the EMA-PCR method was evaluated by comparison with plate counts following freezing and thawing. Thawing frozen cell suspensions initially containing 1 x 10(5) CFU/ml at 4, 20, and 37 degrees C yielded a 0.8 log reduction in the number of viable cells determined by both plate counts and EMA-PCR. In contrast, thawing for 5 min at 70 degrees C resulted in a 5 log reduction in CFU derived from plate counts (no CFU detected) whereas the EMA-PCR procedure resulted in only a 2.8 log reduction in genomic targets, possibly reflecting greater damage to enzymes or ribosomes at 70 degrees C to a minority of the mixed population compared to membrane damage.     

Detection of viable Escherichia coli O157:H7 by ethidium monoazide real-time PCR

Aims: The aim of this study was to develop and optimize a novel method that combines ethidium bromide monoazide (EMA) staining with real‐time PCR for the detection of viable Escherichia  coli O157:H7 in ground beef. EMA can penetrate dead cells and bind to intracellular DNA, preventing its amplification via PCR. Methods and Results: Samples were stained with EMA for 5 min, iced for 1 min and exposed to bright visible light for 10 min prior to DNA extraction, to allow EMA binding of the DNA from dead cells. DNA was then extracted and amplified by TaqMan^® real‐time PCR to detect only viable E. coli O157:H7 cells. The primers and TaqMan^® probe used in this study target the uidA gene in E. coli O157:H7. An internal amplification control (IAC), consisting of 0·25 pg of plasmid pUC19, was added in each reaction to prevent the occurrence of false‐negative results. Results showed a reproducible application of this technique to detect viable cells in both broth culture and ground beef. EMA, at a final concentration of 10 μg ml^?1, was demonstrated to effectively bind DNA from 10⁸ CFU ml^?1 dead cells, and the optimized method could detect as low as 10⁴ CFU g^?1 of viable E. coli O157:H7 cells in ground beef without interference from 10⁸ CFU g^?1 of dead cells. Conclusions: EMA real‐time PCR with IAC can effectively separate dead cells from viable E. coli O157:H7 and prevent amplification of DNA in the dead cells. Significance and Impact of the Study: The EMA real‐time PCR has the potential to be a highly sensitive quantitative detection technique to assess the contamination of viable E. coli O157:H7 in ground beef and other meat or food products.     

Discrimination of viable Vibrio vulnificus cells from dead cells in real-time PCR

Ethidium bromide monoazide (EMA) was utilized to selectively allow the real-time PCR (RT-PCR) amplification of a targeted DNA sequence in viable but not dead cells of Vibrio vulnificus. The optimized light exposure time to achieve cross-linking of DNA by the EMA in dead cells and to photolyse the free EMA in solution was at least 15 min. The use of 3.0 microg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable cells of V. vulnificus. The minimum amount of EMA to completely inhibit the RT-PCR amplification of DNA derived from heat-killed cells was 2.5 microg/ml. Amplification of DNA from dead cells in a mixture with viable cells was successfully inhibited by 2.5 microg/ml of EMA, whereas the DNA from viable cells present was successfully amplified by RT-PCR.     

Selective detection of viable Helicobacter pylori using ethidium monoazide or propidium monoazide in combination with real-time polymerase chain reaction

Because Helicobacter pylori has a role in the pathogenesis of gastric cancer, chronic gastritis and peptic ulcer disease, detection of its viable form is very important. The objective of this study was to optimize a PCR method using ethidium monoazide (EMA) or propidium monoazide (PMA) for selective detection of viable H. pylori cells in mixed samples of viable and dead bacteria. Before conducting the real-time PCR using SodB primers of H. pylori, EMA or PMA was added to suspensions of viable and/or dead H. pylori cells at concentrations between 1 and 100 μM. PMA at a concentration of 50 μM induced the highest DNA loss in dead cells with little loss of genomic DNA in viable cells. In addition, selective detection of viable cells in the mixtures of viable and dead cells at various ratios was possible with the combined use of PMA and real-time PCR. In contrast, EMA penetrated the membranes of both viable and dead cells and induced degradation of their genomic DNA. The findings of this study suggest that PMA, but not EMA, can be used effectively to differentiate viable H. pylori from its dead form.     

Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide

The distinction between viable and dead bacterial cells poses a major challenge in microbial diagnostics. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based quantification methods overestimate the number of viable cells in mixed populations or even lead to false-positive results in the absence of viable cells. On the other hand, RNA-based diagnostic methods, which circumvent this problem, are technically demanding and suffer from some drawbacks. A promising and easy-to-use alternative utilizing the DNA-intercalating dye ethidium monoazide bromide (EMA) was published recently. This chemical is known to penetrate only into "dead" cells with compromised cell membrane integrity. Subsequent photoinduced cross-linking was reported to inhibit PCR amplification of DNA from dead cells. We provide evidence here that in addition to inhibition of amplification, most of the DNA from dead cells is actually lost during the DNA extraction procedure, probably together with cell debris which goes into the pellet fraction. Exposure of bacteria to increasing stress and higher proportions of dead cells in defined populations led to increasing loss of genomic DNA. Experiments were performed using Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium as model pathogens and using real-time PCR for their quantification. Results showed that EMA treatment of mixed populations of these two species provides a valuable tool for selective removal of DNA of nonviable cells by using conventional extraction protocols. Furthermore, we provide evidence that prior to denaturing gradient gel electrophoresis, EMA treatment of a mature mixed-population drinking-water biofilm containing a substantial proportion of dead cells can result in community fingerprints dramatically different from those for an untreated biofilm. The interpretation of such fingerprints can have important implications in the field of microbial ecology.     

Unsuitable distinction between viable and dead Staphylococcus aureus and Staphylococcus epidermidis by ethidium bromide monoazide

Aims:  The DNA-intercalating dye ethidium bromide monoazide (EMA) has recently been used as a DNA binding agent to differentiate viable and dead bacterial cells by selectively penetrating through the damaged membrane of dead cells and blocking the DNA amplification during the polymerase chain reaction (PCR). We optimized and tested the assay in vitro using Staphylococcus aureus and Staphylococcus epidermidis cultures to distinguish viable from dead bacteria, with the goal of reducing false positive PCR results.
Methods and Results:  Viable and heat-inactivated bacteria were treated with EMA or left untreated before DNA extraction. A real-time PCR assay for the detection of the tuf gene in each DNA extract was used. Our results indicated that EMA influenced viable bacteria as well as dead bacteria, and the effect of EMA depended on the EMA concentration and bacterial number.
Conclusions:  EMA is not a suitable indicator of bacterial viability, at least with respect to Staphylococcus species.
Significance and Impact of the Study:  Determining the viability of pathogens has a major impact on interpreting the results of molecular tests for bacteria and subsequent clinical management of patients. To this end, several methods are being evaluated. One of these methods – intercalating DNA of dead bacteria by EMA – looked very promising, but our study found it unsatisfactory for S. aureus and coagulase-negative Staphylococci.     

Selective PCR detection of viable Enterobacter sakazakii cells utilizing propidium monoazide or ethidium bromide monoazide

Aims: The detection of viable Enterobacter sakazakii cells is important due to the association of this pathogen with outbreaks of life-threatening neonatal infections. The aim of this study was to optimize a PCR-based method for selective detection of only viable Ent. sakazakii cells in the presence of dead cells, utilizing propidium monoazide (PMA) or ethidium bromide monoazide (EMA). Methods and Results: PMA or EMA was added to suspensions of viable and/or dead Ent. sakazakii cells at varying concentrations (10, 50 or 100 μg ml⁻¹) prior to DNA isolation and PCR with Ent. sakazakii-specific primers. At concentrations of 50 and 100 μg ml⁻¹, PMA completely inhibited PCR amplification from dead cells, while causing no significant inhibition of the amplification from viable cells. PMA was also effective in allowing selective PCR detection of only viable cells in mixtures of varying ratios of viable and dead cells. EMA was equally effective in preventing amplification from dead cells, however, it also inhibited DNA amplification from viable cells. Conclusions: This study demonstrated the efficiency of PMA for viable and dead differentiation of Ent. sakazakii, as well as the lack of selectivity of EMA for this purpose. Significance and Impact of the Study: PMA-PCR, in particular, will be useful for monitoring the resistance, survival strategies and stress responses of Ent. sakazakii in foods and the environment.     

Polymerase chain reaction amplification length-dependent ethidium monoazide suppression power for heat-killed cells of Enterobacteriaceae

The polymerase chain reaction (PCR) can confirm the presence of bacteria, but it is unable to differentiate between live and dead bacteria. Although ethidium monoazide (EMA)- and propidium monoazide (PMA)-based PCR have been evaluated, a quantity of ≥ 10(3)cells/ml dead cells produces a false-positive reading at 40 to 50 cycles (K. Rudi et al., Appl. Environ. Microbiol. 71 (2005) 1018-1024). After confirming the precision of real-time PCR of a long DNA target (16S or 23S ribosomal RNA [rRNA] gene, 1490 or 2840 bp), we evaluated the degree of suppression of an EMA treatment on the 16S/23S PCR using various amplification lengths (110-2840 bp) with heat-killed cells of Enterobacteriaceae (e.g., Salmonella enteritidis). We found that the inhibition rate was proportional to the PCR amplification length; short DNA (110 bp) amplification slightly delayed the threshold cycle (C(T)) of heat-killed cells of Enterobacteriaceae when compared with no EMA treatment. Regardless of the amplification length, the C(T) delay using live cells of Enterobacteriaceae with EMA was negligible. Thus, our real-time PCR of a long DNA (16S or 23S) template following EMA treatment is a rapid viable bacterial assay, which can potentially target all genera, for testing pasteurized milk that may have originally been contaminated with high levels of dead bacteria.