婴幼儿配方乳粉和发酵乳中沙门氏菌检验方法的比对研究

目的 对比美国FDA/BAM、ISO 6579-2017和GB4789.4-2016沙门氏菌检验方法的检出限、灵敏度,筛选出针对婴幼儿配方乳粉、发酵乳产品以及环境样品中沙门氏菌污染最灵敏的检测方法。方法 对20种血清型沙门氏菌进行平行检测,对比美国FDA/BAM、ISO 6579-2017和GB4789.4-2016沙门氏菌检验方法的检出限、灵敏度。使用不同基质(婴幼儿配方奶粉、酸奶样品和环境样品)人工染菌样品,对三个方法的样品适用性进行比较与评价。结果 三个标准方法的检出限为10_^-1-10_⁰CFU/样品。FDA/BAM方法、ISO 6579-2017方法、GB 4789.4-2016方法在10_¹CFU/样品染菌水平灵敏度均为100%;10_⁰CFU/样品染菌水平,灵敏度为95%、90%、90%;10_^-1CFU/样品染菌水平,灵敏度为65%、50%、45%。对三类人工污染沙门菌的样品,FDA/BAM方法的检出率显著高于ISO和GB4789方法(P＜0.05),ISO和GB4789方法检出率无显著性差异(P>0.05)。结论 三种方法检出限一致 ,均可以有效检测食品中沙门氏菌。染菌浓度低于10_⁰CFU/样品时, FDA/BAM方法的灵敏度显著高于ISO和GB4789方法。不同基质人工污染沙门氏菌样品对比,FDA/BAM方法的检出率显著高于ISO6579-2017方法和GB4789.4-2016方法。     

肉枣加工过程中细菌群落消长规律

目的：确定导致样品胀袋的关键控制点，加强肉枣加工过程中微生物污染防控。方法：采用微生物传统培养法结合高通量测序手段分析肉枣生产全过程中不同加工工序样品及胀袋样品的微生物变化情况。结果：整体来看，原料中菌落总数较低，其中鸡皮中菌落总数最高达9.10×10⁴ CFU/g，蒸煮后菌落总数下降至30 CFU/g，但真空包装后菌落总数显著上升，说明真空包装工序存在较大污染风险。胀袋样品中微生物增殖明显，菌落总数为1.03×10⁶~3.30×10⁶ CFU/g。肉枣原料中的优势菌属为不动杆菌属（Acinetobacter）、嗜冷杆菌属（Psychrobacter）和假单胞菌属（Pseudomonas）。加工过程中样品的主导菌属为葡萄球菌属（Staphylococcus），灭菌后其相对丰度降至0.02%。而胀袋样品中均以枝芽孢菌属 （Virgibacillus）为主，平均相对丰度为99.37%；该属在灌肠工序开始出现，且耐热性较强，灭菌后其相对丰度增加。结论：灌肠和包装工序可能为肉枣加工过程中的关键污染点。     

干酪乳杆菌SB27降低N-二甲基亚硝胺对大鼠肠黏膜细胞损伤作用研究

本研究选用分离自甘肃牦牛乳,且具有潜在益生功能的干酪乳杆菌SB27(Lactobacillus casei,SB27)进行研究,分析其降低N-二甲基亚硝胺(NDMA)对大鼠肠道黏膜细胞IEC-6毒性作用的能力。研究通过CCK-8实验、HE染色、TUNEL染色和透射电镜等进行全面分析。CCK-8实验结果显示,干酪乳杆菌SB27可改善NDMA致大鼠肠道黏膜细胞IEC-6毒性的影响,且该作用与干酪乳杆菌SB27的剂量有关。HE染色、TUNEL染色和透射电镜实验结果显示,NDMA(80 μg/mL)诱导了IEC-6细胞的损伤,在此过程中干酪乳杆菌SB27(10_⁵ CFU/mL、10_⁶ CFU/mL)可保护IEC-6细胞免受NDMA的毒性损伤。以上实验结果表明,干酪乳杆菌SB27降低NDMA对IEC-6细胞的毒性损伤,为该菌株降低NDMA毒性作用机理研究奠定了基础。     

脉冲强光对饮料瓶盖杀菌效果

目的：研究脉冲强光杀菌技术对饮料瓶盖上芽孢杆菌的杀灭效果,拓展其在PET无菌灌装中的应用前景。方法：以初始菌落数、脉冲照射距离、脉冲闪烁次数为影响因子,探究脉冲强光杀菌技术对38 mm及28 mm饮料瓶盖上芽孢杆菌的灭菌效果。结果：初始菌落数越高、照射距离越短、闪烁次数越多,杀菌效率越高,且对38 mm瓶盖的杀菌效率高于28 mm瓶盖的。脉冲照射距离为7 cm,闪烁次数5次时,脉冲强光对38 mm瓶盖的最高杀菌效率为4.87 log,对28 mm瓶盖的最高杀菌效率为4.71 log,最高可将初始菌落数为1×10² CFU的瓶盖上芽孢杆菌完全杀灭;照射距离为7 cm,闪烁次数为10次时,脉冲强光对38 mm瓶盖最高杀菌效率为5.34 log,对28 mm瓶盖的最高杀菌效率为5.28 log,最高可将初始菌落数1×10³ CFU的瓶盖上芽孢杆菌完全杀灭。结论：脉冲强光杀菌技术对饮料瓶盖上芽孢杆菌具有较强的杀灭作用,可用于饮料无菌灌装过程中HDPE瓶盖的杀菌。     

食品检测用产肠毒素大肠埃希氏菌标准物质的制备及评价

目的 制备并评价产肠毒素大肠埃希氏菌（ETEC）标准物质。方法 分别用基质辅助激光解吸电离（MALDI-TOF MS）、生化、16 s RNA基因序列测定三种方法确认菌株种属。采用冷冻干燥技术制备600个ETEC标准样品（10³ CFU/样品）;从中随机抽取20个进行均匀性检验;模拟25 ℃和37 ℃的运输温度测试样品的运输稳定性,同时在4 ℃和-20 ℃的条件下进行保藏稳定性检验。组织 3家实验室对样品进行协同验证。最后选择20件食品基质来验证样品的使用效果。结果 生化、MALDI-TOF MS、16 s RNA基因序列鉴定CMCC（B）43208为大肠埃希氏菌, lt、stp、sth基因确认其为ETEC。均匀性检验中,单因素方差分析得F=1.48,小于F_INV（0.05,19,20）。稳定性检验中,在25 ℃和37 ℃保藏7 d后结果为10³ CFU/样品,在-20 ℃保藏60 d和4 ℃保存28 d后结果为10³ CFU/样品。协同标定实验中,3 家实验室检测样品中菌株均为ETEC（10³ CFU/标准样品）。使用效果评价中,ETEC标准样品加入20种食品基质后均可检出,而本底对照均未检出。结论 本实验制备的ETEC标准样品均匀且稳定,协同验证、食品基质验证实验结果均与预期设置的10³ CFU/样品一致,可以作为标准物质使用。     

食品检验用解脂耶氏酵母菌标准物质制备研究

目的 为满足实验室酵母菌日常检验质量控制需求及实验室间比对,制备解脂耶氏酵母菌检验用标准物质。方法 通过生化、基质辅助激光解吸电离飞行时间质谱及ITS位点测序对研究用菌株进行菌种鉴定确认后,刮取适宜菌苔于保护剂中混匀后冻干,制备10_⁴ CFU/样品浓度的标准物质。参照CNAS-GL003等标准要求进行均匀性检验后,采用单因素方差分析对结果进行评价。将样品放于25 ℃和37 ℃及-20 ℃、4 ℃条件下,分别进行运输稳定性检验及储存稳定性检验。依据国家标准GB 4789.15,将研制的本标准物质加入7类食品基质共20件样品中进行检验,验证真实食品样品中适用性。组织3家实验室,对本标准物质进行协作标定。结果CMCC98025经生化鉴定为解脂耶氏酵母菌,准确度为93%;MALDI-TOF MS鉴定结果为解脂耶氏酵母,分数为2.012;ITS位点测序结果与NCBI Genbank中已有序列比对,匹配最优结果为解脂耶氏酵母Yarrowia lipolytica(Accession number ：CP061015.1;Query Cover：95%;Ident：100%)。均匀性测试结果符合正态分布,通过单因子方差分析计算F_0.05(19,20)=2.137,F值为1.697,F＜F_0.05,符合均匀性要求。25 ℃及37 ℃培养箱中放置7天,标准物质中菌含量仍保持在10_⁴ CFU/样品,可保持稳定。标准物质于4 ℃储存28天及-20 ℃储存90天,菌含量为10_⁴ CFU/样品,复苏率均在91%以上,说明4 ℃放置较短时间及-20 ℃放置较长时间样品稳定。7类食品样品基质中加入本标准物质后进行检验,均可检出,回收率为81.3%。经三家实验室协作标定,本标准物质平均浓度为2.0～3.0×10_⁴ CFU/样品。结论 本研究制备的解脂耶氏酵母菌标准物质均匀性、稳定性、真实食品样品中应用验证及协作标定结果均符合要求,可应用于食品中解脂耶氏酵母菌定性检验,今后可作为实验室日常检验工作的阳性对照质控样品,也可作为实验室间比对样品发放,以保障日常检验工作结果可靠性,进一步提升检验机构人员的检验水平。     

基于MTT比色和响应面法优化侧孢短芽孢杆菌最大活菌数培养条件

目的：探索获得侧孢短芽孢杆菌（Brevibacillus laterosporus）最大活菌数的最佳培养基成分及培养条件。方法：在建立MTT比色法与平板计数法的相关回归方程基础上,对获得最大活菌数的侧孢短芽孢杆菌最适培养基成分（碳源、氮源、无机盐）和培养条件（初始pH、温度、接种量、磷酸二氢钾）进行优化。结果：MTT比色法与平板计数法对活菌数测定结果表现出显著的线性关联（R²>0.999）;麦芽糖、氯化钙、初始pH、磷酸二氢钾为显著影响因子,最佳发酵条件为麦芽糖8.75 g/L,氯化钙0.17 g/L,初始pH 7.07、磷酸二氢钾3.73 g/L,此条件下活菌数为8.12×10⁸ CFU/mL,与理论活菌数（8.25×10⁸ CFU/mL）无显著差异。结论：基于MTT比色和响应面法优化侧孢短芽孢杆菌最大活菌数培养条件,优化后的活菌数较优化前提高了3.02倍。     

2021年湖北省部分地市液态乳微生物污染状况调查

目的 掌握2021年湖北省部分地市液态乳微生物污染状况。方法 采集生产、批发、零售等环节的生乳、巴氏杀菌乳、灭菌乳共101份,检测样品中菌落总数、需氧芽孢总数、嗜热需氧芽孢总数、碱性磷酸酶4项指标。结果 生乳、巴氏杀菌乳、灭菌乳总体超标率（4项指标任意一项超标）分别为52.94%、8.70%、1.64%。生乳、巴氏杀菌乳、灭菌乳的需氧芽孢总数检出率分别为52.94%、4.35%、1.64%。1份零售环节的全脂巴氏杀菌乳碱性磷酸酶检测值为409 mU/L。巴氏杀菌乳上架天数与碱性磷酸酶结果无相关性（r=0.10,P=0.65）。生产加工环节和流通环节阳性样品检出率分别为52.94%、3.57%,差异有统计意义（χ²=32.92,P<0.001）,零售环节中批发市场、便利店与商场、超市阳性样品检出率差异无统计学意义（P>0.05）。结论 湖北省生乳中需氧芽孢总数污染较为普遍,巴氏杀菌乳和灭菌乳存在微生物污染超标问题。建议进一步调查超标原因,分析污染来源,为采取针对性措施提供依据,并加大对液态乳中需氧芽孢总数、碱性磷酸酶等指标的监测,为制定相应限量标准提供基线数据。     

我国现制饮料微生物污染状况研究

目的 了解我国目前现制饮料中微生物的污染状况。方法 使用随机抽样原则在我国具有代表性的饭店、饮品店、快餐店、集体学校食堂等采集现制饮料和食用冰,共计3 583份,采用国标方法检测菌落总数、大肠菌群、金黄色葡萄球菌、沙门菌。结果 菌落总数>10⁵ CFU/mL所占比例为8.42%,大肠菌群>10² MPN/mL所占比例为19.32%,金黄色葡萄球菌检出率为2.56%,沙门菌检出率为0.09%,计数结果均<10² MPN/mL。蛋白类饮品污染状况最严重,菌落总数与大肠菌群检出比例最高,菌落总数>10⁵ CFU/mL和大肠菌群>10² MPN/mL所占比例分别为21.35%和32.97%。3—5月现制饮料的卫生状况最差,菌落总数>10⁵ CFU/mL所占比例最高,为14.80%。不同采样地点中饭店、酒店的金黄色葡萄球菌检出率最高为5.24%。结论 我国现制饮料的食品安全状况总体较好,但相关部门也应加强加工场所卫生状况的监督抽查。     

NaCl对嗜热脂肪芽孢杆菌耐热性影响的研究

为了确定在100℃以下NaCl对嗜热脂肪芽孢杆菌耐热性的影响,本研究以体积个数为107 CFU/mL的嗜热脂肪芽孢杆菌菌悬液为热处理时的初始菌液,在菌悬液的中心温度在约150 s达到70℃、80℃、90℃条件下,研究不同浓度的NaCl制备的菌悬液经热处理后,嗜热脂肪芽胞杆菌的残存体积个数和热致死率,并对嗜热脂肪芽孢杆菌的残存体积个数初步建立各温度和各NaCl浓度条件下的热失活模型。研究结果表明,嗜热脂肪芽孢杆菌菌悬液在各条件下的失活曲线均可用Origin 8.0软件中Dose Resp模型拟合,且判定系数R2均可达到0.96以上。对嗜热脂肪芽孢杆菌在不同温度、不同时间点和不同浓度NaCl下的热致死率进行单因素方差分析,空白在不同热处理温度前40 s致死率均达到95%以上,而不同浓度氯化钠在70℃下前120 s均达到95%以上,在80℃下前60 s均达到93%以上,在90℃下前60 s均达到98以上,可见不同浓度的NaCl对嗜热芽孢杆菌具有保护作用,其中2%NaCl的保护作用显著大于其它浓度的NaCl。     

A new sterility test for cow's milk using alanine racemase gene as the index

Oligonucleotide primers designed from consensus sequences of alanine racemase genes were used for a sterility test of cow's milk by polymerase chain reaction (PCR). Commercial cow's milk in two 250 ml packages was separately centrifuged at 5000 x g for 10 min, bacterial cells in each precipitate were cultivated at 30 and 55 degrees C for 5 h in Luria-Bertani medium, and the cells from each culture were mixed and used for the PCR after being treated with 0.1 N NaOH at 60 degrees C for 10 min. When we performed the PCR using DNAs from various bacteria and eukaryotes as the templates, a unique PCR product of about 390 bp was amplified only from the bacteria. The sensitivity of the PCR method was such that an initial inoculum of 1 CFU of Bacillus stearothermophilus, Escherichia coli, and Pseudomonas fluorescens per 250 ml of cow's milk could be detected. When we analyzed 14 types of commercial cow's milk, all samples which were positive by the standard sterility test at 30 or 55 degrees C were also found positive by the PCR method.     

金黄色葡萄球菌RAA-LFD快速检测方法的建立与应用

靶向金黄色葡萄球菌（Staphylococcus aureus）保守基因（nuc）序列设计特异性引物,经引物筛选和反应条件的优化,建立金黄色葡萄球菌的重组酶等温扩增（recombinase aided amplification,RAA）结合侧流层析试纸条（lateral flow dipstick,LFD）的快速检测方法。该方法在引物浓度200 nmol/L、33 ℃反应20 min即可实现靶标基因片段的有效扩增。特异性分析结果表明RAA-LFD方法检测金黄色葡萄球菌与其他常见致病菌菌株间不存在交叉反应,灵敏度分析结果表明RAA-LFD方法检测金黄色葡萄球菌的检出限为4 fg/μL（DNA）与1.83×102 CFU/mL（纯菌液）。用人工污染牛奶样品验证RAA-LFD方法的可靠性,结果显示在增菌6 h时,RAA-LFD方法可检测到1.83×101 CFU/mL金黄色葡萄球菌。分别采用微生物生化鉴定法、PCR法与RAA-LFD方法对64 份牛奶样品进行金黄色葡萄球菌检测,结果表明,RAA-LFD方法与微生物生化鉴定法总符合率为95.3%,与PCR法总符合率（98.4%）表现出较高的一致性。本研究建立的可视化检测方法具有特异性强、灵敏度高、快速高效、操作简单、检测结果直观、对仪器设备要求低等特点,为牛奶中金黄色葡萄球菌的检测提供新的发展方向。     

Inactivation of Listeria monocytogenes in Skim Milk and Liquid Egg White by Antimicrobial Bottle Coating with Polylactic Acid and Nisin

ABSTRACT: This study was to develop an antimicrobial bottle coating method to reduce the risk of outbreaks of human listeriosis caused by contaminated liquid foods. Liquid egg white and skim milk were inoculated with Listeria monocytogenes Scott A and stored in glass jars that were coated with a mixture of polylactic acid (PLA) polymer and nisin. The efficacy of PLA per nisin coating in inactivating L. monocytogenes was investigated at 10 and 4 °C. The pathogen grew well in skim milk without PLA/nisin coating treatments, reaching 8 log CFU/mL after 10 d and then remained constant up to 42 d at 10 °C. The growth of Listeria at 4 °C was slower than that at 10 °C, taking 21 d to obtain 8 log CFU/mL. At both storage temperatures, the PLA coating with 250 mg nisin completely inactivated the cells of L. monocytogenes after 3 d and throughout the 42-d storage period. In liquid egg white, Listeria cells in control and PLA coating without nisin samples declined 1 log CFU/mL during the first 6 d at 10 °C and during 28 d at 4 °C, and then increased to 8 or 5.5 log CFU/mL. The treatment of PLA coating with 250 mg nisin rapidly reduced the cell numbers of Listeria in liquid egg white to undetectable levels after 1 d, then remained undetectable throughout the 48 d storage period at 10 °C and the 70 d storage period at 4 °C. These data suggested that the PLA/nisin coating treatments effectively inactivated the cells of L. monocytogenes in liquid egg white and skim milk samples at both 10 and 4 °C. This study demonstrated the commercial potential of applying the antimicrobial bottle coating method to milk, liquid eggs, and possibly other fluid products.     

基于微滴数字PCR技术的婴幼儿配方乳粉中双歧杆菌定量检测及应用

应用微滴式数字聚合酶链式反应（droplet digital polymerase chain reaction，ddPCR）技术，建立婴幼儿配方乳粉中双歧杆菌定量检测方法。以双歧杆菌的单拷贝特异性基因rpsL为目标基因设计引物探针，对ddPCR条件进行优化，考察方法的特异性、灵敏度和重复性，并与平板计数方法进行对照验证。结果表明，建立的方法具有良好的特异性、灵敏性和重复性。细菌纯培养液的检出限为296 CFU/mL，模拟样品检出限为7 300 CFU/g，不与10 种近缘乳酸菌发生交叉反应，且重复性较好，采用已建立的ddPCR方法和平板计数方法对市售婴幼儿配方乳粉样品进行检测，2 种方法测定值结果偏差小于10%，结果一致性较好。本研究建立的ddPCR方法对婴幼儿配方乳粉中的双歧杆菌定量检测能够更快速、灵敏、准确，具有一定的应用前景。     

Evaluation of the possible use of potentially probiotic Lactobacillus strains in dairy products

In the study, the ability of two potentially probiotic strains Lactobacillus plantarum 14 and Lactobacillus fermentum 4a to milk fermentation and the possibility to use them in yogurt production were investigated. The strains did not acidify milk during 24 h and 72 h fermentation at 37C, but grew well and remained at the level of 10⁸ colony-forming units (CFU)/mL during 21 days of cold storage. Their application to yogurt production along with commercial starter culture consisted on L. delbrueckii ssp. bulgaricus and S. thermophilus allowed to obtain products with typical sensory properties, pH values and numbers of potentially probiotic bacteria at desired level 10⁷ CFU/mL.     

恒温实时荧光法快速检测奶粉中阪崎肠杆菌方法的建立

为实现奶粉中快速检测阪崎肠杆菌,本文建立了检测阪崎肠杆菌的恒温实时荧光法。针对阪崎肠杆菌16S r RNA设计三组LAMP引物,采用Deaou-308C恒温实时荧光检测平台,选取常见病原菌标准株进行引物特异性检测;选取阪崎肠杆菌标准菌株进行基因组DNA灵敏度和最低检测限测定,同时利用人工污染方式检测此方法在脱脂和全脂奶粉中的灵敏度和最低检测限,利用Real Amp法和国标法对20份市售奶粉进行对比实验。结果显示,引物组16S-11扩增效率最优,与常见病原菌无交叉反应,对阪崎肠杆菌基因组DNA、阪崎肠杆菌污染的脱脂和全脂奶粉的灵敏度分别达到102 CFU/m L、102 CFU/m L和103 CFU/m L;对阪崎肠杆菌基因组DNA和阪崎肠杆菌污染的脱脂和全脂奶粉的最低检测限分别达到103 CFU/m L、103 CFU/m L和104 CFU/m L;在20份市售奶粉样品中Real Amp检测结果与传统国标培养结果一致,表明本文建立的阪崎肠杆菌Real Amp检测方法适用于阪崎肠杆菌的快速检测。     

Survival of Listeria monocytogenes in vanilla-flavored soy and dairy products stored at 8 degrees C

The survival of Listeria monocytogenes V37 in vanilla-flavored yogurt (low-fat and nonfat) and soy milk (low-fat and Plus) stored at 8 degrees C for 31 days was investigated. Commercial samples of yogurt and soy milk were used. These samples were inoculated with either 10(4) or 10(7) CFU of L. monocytogenes per ml. Sampling was carried out every 3 to 4 days initially and was then carried out weekly, for a total storage time of 31 days. Each time a sample was collected, the pH of the sample was measured. After 31 days, low-fat plain, low-fat vanilla, and nonfat plain yogurt samples inoculated with 10(4) CFU/ml showed 2.5-log reductions in viable cell populations, and nonfat vanilla yogurt showed a 3.5-log reduction. For yogurt inoculated with 10(7) CFU/ml, reductions of 2.5 log CFU/ml were observed for plain low-fat and nonfat yogurts, and reductions of 5 log CFU/ml were observed for vanilla-flavored low-fat and nonfat yogurts. In vanilla-flavored and plain low-fat and Plus soy milk samples, cell counts increased from 10(4) and 10(7) CFU/ml to 10(9) CFU/ml at 7 and 3 days of storage, respectively, at 8 degrees C. Coagulation in soy milk samples was observed when the cell population reached 10(9) CFU/ml. In soy milk, the L. monocytogenes population did not change for up to 31 days. Vanillin had an inhibitory effect on L. monocytogenes in yogurt but not in soy milk.     

Effect of Inoculation Level of Lactobacillus rhamnosus and Yogurt Cultures on Conjugated Linoleic Acid Content and Quality Attributes of Fermented Milk Products

ABSTRACT:  The effect of inoculation concentration of Lactobacillus rhamnosus and yogurt cultures and storage time on conjugated linoleic acid (CLA) content and quality attributes of fermented milk products was determined. Yogurt culture ( Lactobacillus bulgaricus and Streptococcus thermophilus, 1:1 ratio, YC ) , L. rhamnosus (LB), and LB co-cultured with yogurt culture, were inoculated at 10⁶, 10⁷, 10⁸ CFU/mL into a milk with hydrolyzed soy oil as the lipid source. CLA content, microbial counts, acidity, texture, and volatile flavor profile of the fermented milk products were stable during storage at 4 °C for 14 d. Total CLA contents ranged from 0.51 to 1.00 mg CLA/g lipid following 14 d of storage. Inoculation level of L. rhamnosus and yogurt cultures had no significant effect on CLA content and texture, but affected acidity and the volatile flavor profile of the fermented milk products. The fermented milk products produced by L. rhamnosus co-cultured with yogurt culture with 10⁷ CFU/mL total inoculation level resulted in a high CLA content and desirable quality characteristics. This research demonstrated that the optimal inoculation concentration and the combination of L. rhamnosus and yogurt cultures were important factors to produce fermented milk products with CLA content and acceptable quality attributes.     

Application of SYBR green real-time PCR assay for specific detection of Salmonella spp. in dairy farm environmental samples

The objective of this study was to develop and evaluate a SYBR Green 1 real-time PCR method for the specific detection of Salmonella spp. in dairy farm environmental samples. Previously reported 119-bp invA gene was selected for specificity, and 124 Salmonella spp. including type strains and 116 non-Salmonella strains were evaluated. All Salmonella strains tested were invA-positive and all non-salmonella strains yielded no amplification products. The melting temperature (Tm=79 degrees C) was consistently specific for the amplicon. Correlation coefficients of standard curves constructed using the threshold cycle (C(T)) versus copy numbers of Salmonella Enteritidis showed good linearity in broth (R2=0.994; slope=3.256) and sterilized milk (R2=0.988; slope=3.247), and the minimum levels of detection were >10(2) and >10(3) colony forming units (CFU)/ml, respectively. To validate the real-time PCR assay, an experiment was conducted with both spiked and naturally contaminated samples. Lagoon water, feed/silage, bedding soil, and bulk tank milk samples obtained from dairy farms were spiked with 10(0) to 10(5) CFU/ml of Salmonella Enteritidis. Sensitivities for detecting Salmonella in these sources were 10(3) to 10(4) CFU/ml of inoculums in broth without enrichment. Detection limits were reduced to <10 CFU/ml of inoculum in broth after 18 h enrichment. Ninety-three environmental samples including fecal slurry, feed/silage, lagoon water, drinking water, bulk tank milk, farm soil, and bedding soil were analyzed for the presence of Salmonella by real-time PCR, results were compared with those obtained by conventional culture methods. All samples analyzed were negative for Salmonella by both real-time PCR and standard culture method. No false positive or false negative results were detected.     

流式分析技术快速定量检测牛乳中大肠杆菌O157:H7

建立一种基于流式分析技术的快速定量检测牛乳中大肠杆菌O157:H7的方法。用偶联有异硫氰酸荧光素的大肠杆菌O157单克隆抗体对大肠杆菌O157:H7进行特异性标记,通过优化抗体反应条件,建立流式检测方法,然后对磷酸盐缓冲溶液（phosphate buffer saline,PBS）和人工污染牛乳样品中不同浓度的大肠杆菌O157:H7进行定量检测。本研究建立的流式检测方法的在PBS中的检测范围为2.57×103～1.12×108?CFU/mL,灵敏度达到2.57×103?CFU/mL。将所建立的流式检测方法应用于牛乳样品检测,当人工污染牛乳样品中大肠杆菌O157:H7的浓度在2.31×104～1.48×108?CFU/mL之间时,流式检测方法与平板计数方法检测结果基本一致,方法的灵敏度为2.31×104?CFU/mL,检测时间为35?min。该方法能快速、定量地检测出牛乳样品中的大肠杆菌O157:H7,在食源性致病菌的快速筛查和监控中具有重要的应用价值。