不同诱导条件下的VBNC状态副溶血弧菌的PMA-qPCR定量检测及其呼吸活性分析

定量检测不同诱导条件下的活的非可培养(viable but non-culturable,VBNC)状态副溶血弧菌的变化情况,并对VBNC菌的呼吸活性进行分析。采用叠氮溴化丙锭(propidium monoazide,PMA)与荧光定量聚合酶链式反应(fluorescence quantitative polymerase chain reaction,q PCR)结合的技术(PMA-q PCR)定量检测副溶血弧菌活菌数。结合激光扫描共聚焦显微镜和流式细胞仪优化传统CTC(5-cyano-2,3-ditolyl tetrazolium chloride)呼吸检测法对VBNC状态的副溶血弧菌进行分析。结果表明PMA-q PCR检测技术结合平板计数的方法,可用于定量检测副溶血弧菌诱导过程中活菌数和可培养菌数的变化情况,并利用差值测定出VBNC细胞的浓度。在不同的温度、Na Cl含量和氯霉素含量的诱导条件下,副溶血弧菌在其中的7种条件下在60 d内进入了VBNC状态。VBNC细胞终浓度最低为5.75×10~2 CFU/m L,最高为1.70×10~5 CFU/m L。在细胞呼吸实验中,采用CTC与4’,6-二脒基-2-苯基吲哚(4’,6-diamidino-2-phenylindole,DAPI)双染法能在激光扫描共聚焦显微镜下清晰地观察到VBNC细胞表面的红色荧光,有效区分死活细胞。同时利用流式细胞仪能够根据荧光强度快速区分呼吸活性呈阴性和阳性的细胞。本研究为VBNC细菌的检测和生理特性的研究提供了新的思路和依据。     

PMA-qPCR定量检测VBNC副溶血弧菌方法的建立与优化

副溶血弧菌（Vibrio parahaemolyticus）在某些不利于生长的自然和食品加工条件下能够进入活的非可培养（viable but non-culturable, VBNC）状态,普通检测方式极易漏检,因此VBNC细菌检测技术的开发与优化对副溶血弧菌感染防控具有重要意义。基于叠氮溴化丙锭结合荧光定量PCR技术（propidium monoazide quantitative PCR,PMA-qPCR）能够利用活细胞的膜完整性区分活菌和死菌的原理,以副溶血弧菌ATCC 17802为对象,通过探讨PMA-qPCR方法中各项因素对活菌计数结果的影响,优化并建立了VBNC副溶血弧菌的定量检测方法。实验结果表明,当添加PMA质量浓度为15 mg/L,黑暗孵育10 min,再进行强光照射15 min后,死细胞的DNA扩增得到有效抑制,此时提取DNA模板进行qPCR扩增准确性更高,活菌数与qPCR扩增循环数呈现显著性线性相关（R²=0.99）。PMA-qPCR法测定副溶血弧菌活菌数的标准曲线显示该方法检测范围为2.90×10¹～2.90×10     

叠氮溴化丙锭(PMA)在食源性致病菌检测中的应用

食源性致病菌是危害食品安全和人类健康的主要因素,对食源性致病菌进行检测是控制食源性疾病的关键环节。传统检测技术无法检测活的非可培养状态(VBNC)的细菌。基于核酸的检测方法克服了这一缺陷,然而难以区分"死菌"与"活菌"。叠氮溴化丙锭(PMA)为一种对死菌核酸具有高度结合能力的光敏染料,近年来,将其与核酸检测技术相结合,广泛应于多种食源性致病菌的活菌检测。本文综述了PMA的作用机理,影响PMA活菌检测的因素以及PMA在常见食源性致病菌检测中的应用,并预期PMA的进一步应用与后续研究情况。     

SD-PMA-ddPCR检测食品中沙门氏菌的研究

研究将叠氮溴化丙锭(PMA)与微滴数字PCR(dd PCR)技术相结合,建立一种沙门氏菌活菌的检测方法。结果表明,PMA不能完全有效抑制108CFU/m L的沙门氏菌死菌DNA的PCR扩增,0.1%脱氧胆酸钠(SD)和40.0μg/m L PMA协同作用,可以有效抑制108CFU/m L的沙门氏菌死菌DNA的PCR扩增,不抑制沙门氏菌活菌DNA扩增的PMA最高浓度是40.0μg/m L。经过SD和PMA对样品预处理,在不同死、活菌比例下,PMA-dd PCR可以定量检测活菌,避免了死菌DNA的干扰。本方法的检出限为8.0 copy/20μL。利用PMA-dd PCR检测人工污染鸡肉样品,最低可检出102CFU/m L的沙门氏菌。实验结果证明PMA-dd PCR方法的特异性、精确度、稳定性良好。     

表面活性剂脱氧胆酸钠在单增李斯特菌死活细胞EMA-PCR鉴别中的应用

通过脱氧胆酸钠(sodium deoxycholate,SD)结合染料叠氮溴乙锭(ethidium bromide monoazide,EMA)与PCR反应体系相结合,建立纯培养条件下区分单增李斯特菌死活细胞的方法,即SD-EMA-PCR鉴别单增李斯特菌死活细胞检测法。结果表明:向浓度为2.0×108CFU/mL的单增李斯特死菌悬液中加入终浓度为1.5 mg/mL的表面活性剂脱氧胆酸钠后,EMA激活光解最佳曝光时间为25 min。SD-EMA-PCR检测体系中,对死菌细胞DNA的PCR扩增抑制的EMA最小浓度为1.6μg/mL;而对活菌细胞DNA的PCR扩增不抑制的EMA最大浓度为9μg/mL,活菌细胞的最低检出限为20 CFU/mL。SD-EMA-PCR检测法能够减小EMA-PCR漏检死菌细胞给检测结果带来假阳性的可能,降低了检测的成本,为单增李斯特菌的快速检测提供了一种新的有效途径。     

EMA-PCR法快速检测啤酒中腐败短乳杆菌

将叠氮溴乙锭(ethidium bromide monoazide,EMA)与聚合酶链式反应(polymerase chain reaction,PCR)技术相结合,以酒花耐受基因hor C为靶基因,用啤酒腐败短乳杆菌基因组DNA作为模板进行扩增。结果发现,在前处理过程中加入EMA,当终质量浓度小于20μg/m L时,对活的短乳杆菌中靶基因的扩增没有明显抑制作用;而当EMA终质量浓度为1.0μg/m L时可有效抑制10~5 CFU/m L短乳杆菌死细胞的扩增。本实验建立的EMA-PCR检测方法的灵敏度为10~4活细胞/m L酒液样品。验证实验结果表明,在13株乳酸菌中,建立的hor C特异性EMA-PCR能有效检测到其中的全部5株啤酒污染菌,同时可区分这5株菌的活/死细胞混合体系,降低检测过程中的假阳性。     

PMA-qPCR技术在发酵食品活菌计数中的应用

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。     

PMA结合ddPCR检测食品中沙门氏菌

建立一种快速、灵敏的微滴式数字PCR方法,用于检测食品中沙门氏菌活菌。利用叠氮溴化丙锭(PMA)对死菌预处理,PMA可以选择性穿过死菌细胞膜,在光照条件下与死菌DNA发生共价交联反应,进而阻止其DNA进行PCR扩增,提取细菌基因组DNA进行微滴式数字PCR(dd PCR)检测。结果表明,强烈光照15.0 min,可以使PMA与死菌DNA共价交联,同时钝化游离的PMA;PMA终质量浓度为40.0μg/m L可以有效抑制105CFU/m L的沙门氏菌死菌DNA的PCR扩增;不抑制活菌DNA扩增的PMA最高浓度是50.0μg/m L。利用PMA处理死活菌混合液时,PMA-dd PCR可以在死菌存在的条件下,定量检测活菌,降低了"假阳性"结果的出现。灵敏度检测结果显示:PMA-dd PCR灵敏度是0.4 copy/μL。利用PMA-dd PCR检测人工污染的鳕鱼样品,最低可检出102CFU/m L的沙门氏菌,精密度和稳定性良好。     

基于叠氮溴化丙锭与恒温核酸扩增技术快速检测亚致死状态食源金黄色葡萄球菌

建立一种将荧光染料叠氮溴化丙锭（propidium monoazide,PMA）与环介导等温扩增（loop-mediatedisothermal amplification,LAMP）技术相结合的方法,用于快速高效检测金黄色葡萄球菌（Staphylococcusaureus）。同时,采用人工污染金黄色葡萄球菌的速冻水饺和奶粉作为食品样品,研究PMA-LAMP方法的检测灵敏度。结果表明,PMA溶液质量浓度3 μg/mL,650 W卤素灯下曝光5 min,PMA能够完全抑制1.2×107 copies/mL金黄色葡萄球菌死菌核酸扩增。PMA-LAMP方法能够在恒温65 ℃、60 min内完成对亚致死型金黄色葡萄球菌特异性nuc基因的特异性检测,其对亚致死状态金黄色葡萄球菌的检出限为34 CFU/mL,对食物样品速冻水饺和奶粉的检出限分别为17 CFU/mL和1.70×102 CFU/mL。建立的PMA-LAMP方法可以有效检测亚致死态金黄色葡萄球菌,提供了一种新的检测技术和解决方案。     

肉及肉制品中沙门氏菌活细胞的PCR检测研究

将荧光染料叠氮溴化丙锭(propidium monoazide,PMA)与普通聚合酶链式反应(polymerase chain reaction,PCR)结合,通过对PMA的曝光时间、浓度进行优化,确定PMA-PCR区别死活细胞的最佳条件,并制作活细胞定量标准曲线,建立肉及肉制品中沙门氏菌活细胞的PMA-PCR检测方法。结果表明：使插入死细胞DNA中的PMA活化并且光解溶液中游离PMA的最佳曝光时间为15min;不抑制沙门氏菌活细胞DNA扩增的最大PMA质量浓度为10μg/mL;完全抑制热致死细胞DNA扩增的最小PMA质量浓度为4μg/mL。经PMA处理,含有不同比例的沙门氏菌热致死细胞和活细胞的混合液中活的沙门氏菌能够通过PCR被选择性的检测,最小检测限为20CFU/PCR。而且,经研究发现在20～2×105CFU/PCR范围内,电泳条带相对荧光强度与活细胞数的对数具有线性关系。采集30份肉及肉制品样品,利用PMA-PCR方法检测出两份生肉样品中存在沙门氏菌,经过6h的富集培养后的活菌浓度分别为2.5×103CFU/mL和3.4×103CFU/mL。     

金黄色葡萄球菌活的非可培养状态复苏及PMA-qPCR检测

为研究金黄色葡萄球菌活的非可培养（VBNC）状态的复苏问题,采取温度、盐度和酸度3个因素复合诱导制备VBNC菌,尝试四种复苏方法,并以荧光定量PCR和PMA-qPCR技术结合的方法进行检测。结果证明：8%无菌Tween80可以使VBNC状态金黄色葡萄球菌48 h后复苏,同时PMA-qPCR能够有效检出VBNC状态金黄色葡萄球菌,克服传统平板计数法对于VBNC菌的漏检。     

Induction,detection, formation,and resuscitation of viable but non‐culturable state microorganisms

The viable but nonculturable (VBNC) state has been recognized as a strategy for bacteria to cope with stressful environments; in this state, bacteria fail to grow on routine culture medium but are actually alive and can resuscitate into a culturable state under favorable conditions. The VBNC state may pose a great threat to food safety and public health. To date, more than 100 VBNC microorganism species have been proven to exist in fields of food safety, environmental application, and agricultural diseases. Most harsh conditions can induce these microorganisms into the VBNC state, including food processing and preservation methods, adverse environmental conditions, and plant‐disease controlling means. The characteristics of VBNC state cells differ from those of normally growing cells and dead cells, based on which of the various detection methods are developed, and they are of great significance for potential risk assessment. To provide molecular level insights into this state, many studies on induction and resuscitation mechanisms have emerged over the past three decades, including research on omics, specific genes, or proteins involved in VBNC state formation and the roles of promoters in resuscitation from the VBNC state. In this review, microorganism species, induction and resuscitation factors, detection methods, and formation and resuscitation mechanisms of the VBNC state are comprehensively and systematically summarized.     

肠炎沙门氏菌EMA-PCR检测方法的建立

将荧光染料叠氮溴化乙锭（ethidium monoazide,EMA）与聚合酶链式反应（polymerase chain reaction,PCR）检测技术相结合,用于肠炎沙门氏菌活菌的检测。实验参数优化结果表明,当EMA终质量浓度50 μg/mL、曝光时间10 min时,可以抑制约107 CFU/mL肠炎沙门氏菌死菌DNA的扩增;活菌灵敏度检测结果显示,EMA-PCR方法检测限与单一PCR方法一样均为27.5 CFU/mL,说明EMA处理既不会影响活菌DNA的扩增也不会影响PCR方法的灵敏度。利用EMA-PCR方法检测死活混合菌液时发现,添加EMA的实验组DNA条带亮度会随着活菌比例的降低而变暗,且当样品中全是死菌时,没有目标条带出现;而不添加EMA的对照组DNA条带亮度没有变化,当样品中全是死菌时,目标条带依然清晰可见。说明添加EMA可以达到区分死活菌的目的,EMA-PCR方法只检测样品中的活菌,避免了死菌DNA造成假阳性的可能性。     

EMA-LAMP方法快速检测死/活的食源性沙门氏菌

建立应用叠氮溴化乙锭(ethidium monoazide,EMA)与环介导等温扩增(loop-mediated isothermal amplification,LAMP)相结合检测沙门氏死/ 活菌的方法。该方法具有快速、方便和灵敏度高等优点,以SYBR Green I 为显色剂、恒温65℃、反应1h 便可得出检测结果。该检测系统中,得到241bp 的目标片段和类似于梯子状的系列条带。该方法的检测限为10-10g DNA/ 管,是EMA-PCR 方法的1%。本方法实用性强,具有普遍的应用意义。     

Determination of viable wine yeast using DNA binding dyes and quantitative PCR

The detection and quantification of wine yeast can be misleading due to under or overestimation of these microorganisms. Underestimation may be caused by variable growing rates of different microorganisms in culture media or the presence of viable but non-cultivable microorganisms. Overestimation may be caused by the lack of discrimination between live and dead microorganisms if quantitative PCR is used to quantify with DNA as the template. However, culture-independent methods that use dyes have been described to remove the DNA from dead cells and then quantify the live microorganisms. Two dyes have been studied in this paper: ethidium monoazide bromide (EMA) and propidium monoazide bromide (PMA). The technique was applied to grape must fermentation and ageing wines. Both dyes presented similar results on yeast monitoring. Membrane cell recovery was necessary when yeasts were originated from ethanol-containing media. When applied to grape must fermentation, differences of up to 1 log unit were seen between the QPCR estimation with or without the dye during the stationary phase. In ageing wines, good agreement was found between plating techniques and QPCR. Most of the viable cells were also culturable and no differences were observed with the methods, except for Zygosaccharomyces bailii and Dekkera bruxellensis where much higher counts were occasionally detected by QPCR. The presence of excess dead cells did not interfere with the quantification of live cells with either of the dyes.     

Detection of viable Salmonella in lettuce by propidium monoazide real-time PCR

Contamination of lettuce by Salmonella has caused serious public health problems. Polymerase chain reaction (PCR) allows rapid detection of pathogenic bacteria in food, but it is inaccurate as it might amplify DNA from dead target cells as well. This study aimed to investigate the stability of DNA of dead Salmonella cells in lettuce and to develop an approach to detecting viable Salmonella in lettuce. Salmonella-free lettuce was inoculated with heat-killed Salmonella Typhimurium cells and stored at 4 °C. Bacterial DNA extracted from the sample was amplified by real-time PCR targeting the invA gene. Our results indicate that DNA from the dead cells remained stable in lettuce for at least 8 d. To overcome this limitation, propidium monoazide (PMA), a dye that can selectively penetrate dead bacterial cells and cross-link their DNA upon light exposure, was combined with real-time PCR. Lettuce samples inoculated with different levels of dead or viable S. Typhimurium cells were treated or untreated with PMA before DNA extraction. Real-time PCR suggests that PMA treatment effectively prevented PCR amplification from as high as 10(8) CFU/g dead S. Typhimurium cells in lettuce. The PMA real-time PCR assay could detect viable Salmonella at as low as 10(2) CFU/mL in pure culture and 10(3) CFU/g in lettuce. With 12-h enrichment, S. Typhimurium of 10(1) CFU/g in lettuce was detectable. In conclusion, the PMA real-time PCR assay provides an alternative to real-time PCR assay for accurate detection of Salmonella in food.     

Quantitative PCR coupled with sodium dodecyl sulfate and propidium monoazide for detection of viable Staphylococcus aureus in milk

Conventional quantitative PCR (qPCR) are unable to differentiate DNA of viable Staphylococcus aureus cells from dead ones. The aim of this study was to use sodium dodecyl sulfate (SDS) and propidium monoazide (PMA) coupled with lysostaphin to detect viable Staph. aureus. The cell suspensions were treated with SDS and PMA before DNA extraction. The SDS is an anionic surfactant, which can increase the permeability of dead cells to PMA without compromising the viability of live cells. The lysostaphin was applied to improve the effectiveness of DNA extraction. The reliability and specificity of this method were further determined by the detection of Staph. aureus in spiked milk. The results showed that there were significant differences between the SDS-PMA-qPCR and qPCR when a final concentration of 200 μg/mL of lysostaphin was added in DNA extraction. The viable Staph. aureus could be effectively detected when SDS and PMA concentrations were 100 µg/mL and 40 μM, respectively. Compared with conventional qPCR, the SDS-PMA-qPCR assay coupled with lysostaphin was more specific and sensitive. Therefore, this method could accurately detect the number of viable Staph. aureus cells.     

核酸交联剂在食源致病菌活菌检测中应用的 研究进展

近年来,由食源致病菌引起的食品安全问题越来越受到人们的重视,如何快速准确检测食品中是否存在食源致病菌是食品安全研究的热点问题。基于PCR检测食源致病菌的方法因快速且特异性强而被广泛应用,然而普通的PCR检测方法难以消除死菌残留DNA导致的假阳性结果,因而无法对致病菌进行准确检测。核酸交联剂是一种含有两个或两个以上烷基化官能团的烷基化试剂,目前,在食源致病菌检测中应用的核酸交联剂主要为EMA和PMA。核酸交联剂通过一定方式的诱导可选择性的透过死菌的细胞膜并与DNA产生共价交联,从而强有力的抑制死菌DNA的PCR扩增,达到鉴别死菌和活菌的效果。本文就核酸交联剂在食源致病菌活菌检测中应用的研究进展进行了综述,以期能为相关研究者开发食源致病菌活菌检测方法提供参考。