基于ttr基因的mini-MPN-qLAMP法快速定量检测食品中沙门氏菌

该研究通过3管型微量MPN计数法（Mini Most Probable Number,mini-MPN）建立了一种快速定量检测食源性沙门氏菌的荧光定量环介导等温扩增（Quantitative Loop-Mediated Isothermal Amplification,qLAMP）方法。依据沙门菌属ttr基因设计了qLAMP和荧光定量PCR（Quantitative Polymerase Chain Reaction,qPCR）引物,结合5 h BPW增菌和MPN计数法建立了mini-MPN-qLAMP沙门氏菌快速定量检测方法;使用两种人工污染样品对mini-MPN-qLAMP法进行验证,使用Bland-Altman分析比较不同检测方法检测结果的一致性。结果表明,建立的qLAMP法与qPCR法反应特异性均良好,纯培养时qLAMP法检出限为500 CFU/mL。通过Bland-Altman分析表明所建立的mini-MPN-qLAMP法在速冻乌米饭中检测结果与mini-MPN-qPCR、mini-MPN计数法、平板计数法相比均具有较高的一致性,r2≥0.994,检出限为-0.44 lg MPN/mL;而在速冻鸡胸肉中该法检测结果与mini-MPN-qPCR结果一致性最佳,r2=0.990,检出限为-0.64 lg MPN/mL。肉制品中腐败杂菌会影响mini-MPN计数和平板计数结果,mini-MPN-qLAMP可排除肉制品中腐败杂菌对检测结果的影响。该研究所建立的mini-MPN-qLAMP法简单易行,准确度高,可用于食品中沙门氏菌的快速定量检测。     

涂布平板计数法与MPN法定量检测禽肉和蛋中沙门菌的比较

为选择良好的禽肉和鸡蛋中沙门菌定量检测方法 ,通过染菌实验 ,分析比较了涂布平板计数法和MPN法 ,结果表明平板计数法优于MPN法。应用平板计数法检测了 16份鸡蛋和 2 6份禽肉试样。沙门菌检出限分别为 1CFU ml和 10 0CFU g。 16份蛋类试样中均未检出沙门菌。 2 6份鸡肉试样中 ,2 0份为沙门菌阳性 ,阳性率为 77%。其中 9份试样沙门菌数低于检出限 10 0CFU g ,其余试样菌数范围为 1 5× 10 2 ～ 2 5× 10 5CFU g。对 3份 - 2 0℃保存 5d的阳性禽肉试样应用MPN法进行了冷冻前后沙门菌含量的比较分析 ,储存后的菌数比冷冻前略有增加。实验结果表明 ,在食物试样中杂菌污染严重或沙门菌水平较低的情况下 ,平板法不能准确进行沙门菌计数 ,仍需进行MPN定量检测。     

荧光定量环介导等温扩增法快速检测食品中沙门氏菌

目目的 建立一种快速简易检测食源性沙门氏菌的实时荧光定量环介导等温扩增（quantitative loop-mediated isothermal amplification, qLAMP）方法。方法 依据沙门菌属invA基因序列设计引物, 并结合短时间增菌构建沙门氏菌快速检测qLAMP法, 使用人工污染样品进行验证。结果 建立的qLAMP法最佳反应时间为40 min, 最佳反应温度是65 ℃, 最佳Mg2+浓度为6 mmol/L, Bst 2.0 WarmStart聚合酶的浓度为0.40 U/μL, 反应特异性良好, 纯培养实验表明方法检出限为100 CFU/mL。将沙门氏菌人工添加至鸡胸肉、果蔬沙拉、山泉水中, 经过7 h BPW有效增菌后所建立的qLAMP法在食品基质中的检出限达到5 CFU/25 g。结论 该方法准确、简单易行, 实验成本低, 可用于食品中沙门氏菌的快速检测。通过WarmStart Colorimetric LAMP试剂盒无需昂贵仪器即可快速检测,为沙门氏菌现场即时检测的场景提供技术支持。     

鼠李糖乳杆菌HN001菌株水平快速定量方法的建立及应用

应用荧光定量PCR技术(Quantitative Real-time PCR,qPCR),建立鼠李糖乳杆菌HN001菌株水平的快速定量检测方法。通过比对鼠李糖乳杆菌HN001的近缘菌株的基因组,筛选出鼠李糖乳杆菌HN001菌株水平的特异基因设计引物及探针,优化了反应体系和条件,建立鼠李糖乳杆菌HN001株水平的qPCR检测方法。对方法的特异性,灵敏度,检测限进行了验证,并以平板计数法进行对照验证进行了实际样品的检测。结果表明,所建立的方法特异性强与近缘菌株无交叉反应,鼠李糖乳杆菌HN001纯培养液检出限为102 CFU/mL,灵敏度可达到650copies/μL,可在3小时内完成样品检测。采用已建立的qPCR检测方法和平板技术法对市售添加鼠李糖乳杆菌HN001益生菌产品进行检测,该方法与平板计数方法检测结果一致性较好,在菌株水平上检测差异不显著(P＞0.05)。本研究建立的qPCR检测方法可有效应用于益生菌产品中鼠李糖乳杆菌HN001菌株水平定量检测,具有快速、灵敏、准确的特点。     

SD-PMA-qPCR快速检测冷冻肉制品中活性沙门氏菌的研究

为实现冷冻肉制品中活性沙门氏菌的快速检测与控制,本文利用PMA（叠氮溴化丙锭）和SD（脱氧胆酸钠）消除死菌和损伤菌的影响,建立了运用SD-PMA-qPCR法检测活性沙门氏菌的反应体系和反应条件,并进行人工染菌样品检测试验。SD的最佳浓度确定为0.1%,最佳孵育时间为20 min。对-20 ℃低温冷冻处理3~4 d的沙门氏菌处理液,使用平板计数法、qPCR、PMA-qPCR和SD-PMA-qPCR进行计数,结果发现qPCR与PMA-qPCR二者检测值接近且明显高于平板计数值,而SD-PMA-qPCR检测值与平板计数值相近,这表明SD和PMA的联合使用能有效的消除死菌和损伤菌的影响。人工染菌样品试验表明,沙门氏菌检出限在102 CFU/g,同时106 CFU/g大肠杆菌O157:H7的存在不会影响检测结果。本研究所建立的SD-PMA-qPCR检测方法特异性好、灵敏度高,有望成为快速检测冷冻肉制品中活性沙门氏菌的新方法,具有很好的研究价值和应用前景。     

实时荧光环介导等温扩增快速检测鸡肉中沙门氏菌

根据沙门氏菌invA基因序列设计特异性引物,利用Midori Green新型核酸荧光染料,建立了沙门氏菌实时荧光定量环介导等温扩增检测方法。结果表明,检测过程仅需45 min,灵敏度达6 CFU/管,而且细菌数量对数值（lg（CFU/管））与扩增荧光指数增加时间（Tp值）具有良好正相关线性关系,R2为0.985 1。通过模拟沙门氏菌人工污染25 g鸡肉样品,经37 ℃保温4 h,提取样品制备DNA模板用于实时荧光定量环介导等温扩增检测,结果表明灵敏度达450 CFU/g,共需时约7 h。随机从市场购买冷冻鸡肉样品21 份,采用国标沙门氏菌检测方法和鸡肉样品实时荧光定量环介导等温扩增检测进行对比检测,结果两种方法均检测出同一份样品为沙门氏菌阳性,其余20 份样品均为沙门氏菌阴性,表明本研究建立的鸡肉沙门氏菌实时荧光定量环介导等温扩增检测,其灵敏性和准确性与国标检测方法相当,但检测时间可缩短至7 h。     

鸡肉中弯曲菌定量检测方法的建立与初步应用

目的参照国标方法,建立鸡肉中弯曲菌定量检测的CCDA平板计数法。方法通过对选择性CCDA培养基中添加不同浓度组合的抗生素和生长促进剂,确定最佳添加浓度组合,并与普通CCDA平板计数法进行比较,建立鸡肉中弯曲菌定量检测方法,同时评价该方法的特异性和检测限。结果添加一定浓度抗生素和生长促进剂的选择性CCDA培养基和普通CCDA培养基对弯曲菌标准菌株定量计数的吻合度为(100±5)%;除弯曲菌外,其他5种肠道细菌在该CCDA培养基中均不能生长;该方法对弯曲菌菌悬液可检测至10 CFU/mL。运用该方法对50份鸡肉样品进行调查研究,共检测出22份阳性样品,弯曲菌检出率为44%,平均检测值为20CFU/100 cm~2,检测结果与国标方法阳性符合率为100%。结论该方法简便、特异性好、准确度高,为定量检测鸡肉中弯曲菌提供了新技术。     

荧光定量PCR法检测益生菌饮料中Lactobacillus casei Zhang和Bifidobacterium lactis V9

益生菌活菌数是益生菌产品最重要的指标,而检测益生菌方法的准确性和科学性则至关重要.本文采用荧光定量PCR法同时定量检测益生菌饮料中Lactobacillus casei Zhang(L.casei Zhang)和Bifidobacterium lactis V9(B.lactis V9)的活菌数,并与平板菌落计数法进行比较.结果表明,荧光定量PCR法测得L.casei Zhang活菌数与平板菌落计数法测得活菌数差异不显著;而采用荧光定量PCR法测得B.lactis V9活菌数显著高于平板菌落计数法.荧光定量PCR法灵敏、特异、简便快速,可定量检测并真实反应L.casei Zhang和B.lactis V9的活菌数.     

空肠弯曲菌定量检测方法的建立和优化

目的 建立一种空肠弯曲菌定量检测方法,为食品安全风险评估提供准确的数据支持。方法 参照ISO/TS 10272—3和GB/T 4789.9—2008,基于MPN原理建立空肠弯曲菌定量检测方法,对定量加标的生鸡肉和鸡粪样品进行不同增菌培养基、不同培养环境和不同培养方式优化比对研究,用加标回收试验对建立的方法进行验证。结果 增菌培养基的优化选择:Preston肉汤对加标菌量10cfu/g的生鸡肉和鸡粪样品进行空肠弯曲菌检测的平均值分别为12.26和10.96MPN/g,Bolton肉汤检测的平均值分别为2.38和2.32MPN/g,二者比较差异有统计学意义(P＜0.05);微需氧环境的优化选择:用三气培养箱法、产气袋法、抽气换气法和烛缸法对加标生鸡肉和鸡粪中的空肠弯曲菌检测的平均值分别为12.26和12.12MPN/g、12.26和10.96MPN/g、12.26和12.86MPN/g、10.82和12.12MPN/g,和三气培养箱法两两相比差异无统计学意义(P＞0.05);培养方式:静止培养法对加标生鸡肉和鸡粪空肠弯曲菌的检测值分别为15.8和15.2MPN/g,振荡培养法检测值分别为11.36和8.72MPN/g,二者比较差异有统计学意义(P＜0.05);用该法对低、中、高浓度加标生鸡肉样品的回收率分别为115.25%、111.5%和95.0%。结论 此方法可以对高污染样品中空肠弯曲菌进行准确定量,特异度高,值得推广应用。     

甲型副伤寒沙门菌特异性基因筛选及PCR检测体系建立

以甲型副伤寒沙门菌为检测目标,通过比较基因组和聚合酶链式反应（polymerase chain reaction,PCR）验证方法筛选到4 个该血清型的特异性基因,其中以gene_3105作为该血清型的检测靶点设计引物PA23;并结合沙门菌属特异性引物139-141,建立一种甲型副伤寒沙门菌的PCR检测方法。优化PCR反应体系,并对该检测体系的特异性、灵敏度、抗干扰能力及人工污染样品检出限等方面进行评价。结果表明,当样品中含有甲型副伤寒沙门菌时,该体系能扩增出2?条特异性条带,含有其他血清型的沙门菌仅能扩增出284?bp条带,不含沙门菌无扩增条带产生。灵敏度评价表明,基因组DNA和纯菌菌落检出限分别为32.4?pg/μL和4.3×103?CFU/mL;抗干扰能力实验显示,当鸡肉背景菌群和猪肉背景菌群浓度在106?CFU/mL和4.87×107?CFU/mL时,检出限为6.43×104?CFU/mL。当无菌的鸡肉和猪肉样品中添加N?CFU/25?g甲型副伤寒沙门菌时,经10?h增菌,检测结果为阳性（0＜N＜10）。实验建立甲型副伤寒沙门菌PCR检测方法具有较好的特异性和灵敏度,有很好的应用价值,可在食品安全领域广泛应用。     

Detection and enumeration of Salmonella enteritidis in homemade ice cream associated with an outbreak: comparison of conventional and real-time PCR methods

Salmonellosis caused by Salmonella Enteritidis (SE) is a significant cause of foodborne illnesses in the United States. Consumption of undercooked eggs and egg-containing products has been the primary risk factor for the disease. The importance of the bacterial enumeration technique has been enormously stressed because of the quantitative risk analysis of SE in shell eggs. Traditional enumeration methods mainly depend on slow and tedious most-probable-number (MPN) methods. Therefore, specific, sensitive, and rapid methods for SE quantitation are needed to collect sufficient data for risk assessment and food safety policy development. We previously developed a real-time quantitative PCR assay for the direct detection and enumeration of SE and, in this study, applied it to naturally contaminated ice cream samples with and without enrichment. The detection limit of the real-time PCR assay was determined with artificially inoculated ice cream. When applied to the direct detection and quantification of SE in ice cream, the real-time PCR assay was as sensitive as the conventional plate count method in frequency of detection. However, populations of SE derived from real-time quantitative PCR were approximately 1 log higher than provided by MPN and CFU values obtained by conventional culture methods. The detection and enumeration of SE in naturally contaminated ice cream can be completed in 3 h by this real-time PCR method, whereas the cultural enrichment method requires 5 to 7 days. A commercial immunoassay for the specific detection of SE was also included in the study. The real-time PCR assay proved to be a valuable tool that may be useful to the food industry in monitoring its processes to improve product quality and safety.     

陕西省某活鸡屠宰场肉鸡相关样品中沙门菌定量检测及污染分析

目的了解肉鸡屠宰加工中不同时间和环节沙门菌的污染情况,分析污染关键点。方法 2016年11月至2017年11月从陕西省某活鸡屠宰场不同环节定期采集活鸡肛拭子标本、整鸡胴体和鸡肉样品,使用最大可能数(MPN)法对沙门菌进行定量检测,同时分离菌株;采用聚合酶链式反应(PCR)技术对沙门菌进行鉴定,同时结合血清凝集技术对沙门菌鉴定结果进行确认。结果采集的284份样品中有67份检出沙门菌,检出率为23. 6%,平均MPN值为0. 051 6 MPN/g。2017年7月采集的样品沙门菌污染最为严重,检出率为37. 8%(14/37),平均MPN值为0. 064 7 MPN/g; 2016年11月检出率最低,为13. 9%(5/36),平均MPN值为0. 043 6 MPN/g。不同屠宰环节中,浸烫褪毛后整鸡胴体样品中沙门菌检出率最高(43. 3%,26/60),平均MPN值为0. 060 5 MPN/g;分割后冷冻前鸡胸脯肉样品中沙门菌检出率最低(18. 3%,11/60),平均MPN值为0. 036 8 MPN/g,略高于储存配送过程整鸡胴体/鸡胸脯肉样品中沙门菌的污染水平(0. 035 8 MPN/g)。结论活鸡屠宰过程沙门菌的检出率与MPN值具有较强的季节性,在不同屠宰加工环节存在纵向和交叉污染,应对活鸡屠宰加工过程沙门菌污染严重的环节进行重点控制。     

食品中绿脓杆菌检测方法的研究

目的:研究和建立食品中绿脓杆菌的检测方法。方法:通过比较不同增菌液增殖效果,分离培养基的分离效果和确定生化项目建立检验方法,并通过制备人工污染样品验证方法的可行性。结果:确定了SCDLP为增菌液,C-F-C和C-N为分离培养基,以O/F葡萄糖、氧化酶为主的生化鉴定项目,以及MPN法作为定量方法。结论:本方法适用于食品中绿脓杆菌定性和定量检测。     

测试片法在食品菌落总数检测中的应用研究

目的探究菌落总数测试片法检测食品中菌落总数的可行性。方法用菌落总数测试片法和国家标准方法同时对制备的菌悬液、自然污染食品样品和人工污染食品样品的菌落总数进行检测,采用t-检验分析2种方法检测结果的差异,采用Pearson相关分析确定2种检测方法之间的相关性。结果菌悬液和自然污染食品样品菌落总数测试片法的变异系数为0.16%~3.76%,人工污染食品样品的变异系数为0.78%~8.61%;菌落总数测试片与国家标准方法在不同类型食品样品检测结果之间没有显著性差异(P<0.05),2种方法的检测结果呈正相关(r2>0.976,P<0.001)。结论菌落总数测试片法的检测效果与国家标准方法相当,可作为替代方法,用于检测食品中的菌落总数。     

Most-probable-number determination of salmonella levels in naturally contaminated raw almonds using two sample preparation methods

Pathogens occurring in particulate foods may be unevenly distributed, which may impact interpretation of most-probable-number (MPN) values. The MPN analysis of Salmonella in naturally contaminated raw almonds was conducted using two sample preparation methods. Raw almond kernels (3,698 samples) and inshell almonds (455 samples) were collected from almond processors throughout California during the 2006 and 2007 harvests, and 100-g samples were enriched for Salmonella. The prevalence of Salmonella on kernels and inshell almonds was 1.6 and 0.9%, respectively, in 2006, and 0.83 and 2.2%, respectively, in 2007. Almond kernel samples from 2006 were further enriched for Salmonella, and levels of the organism were determined for positive samples by three-tube MPN analysis (25 g, 2.5 g, 0.25 g). Almonds were either divided into subsamples prior to blending and enrichment (method A), or samples were blended in enrichment broth prior to preparation of subsamples (method B). Salmonella was not isolated (<1.2 MPN/100 g) upon retesting of 19 of 31 (method A) or 23 of 29 (method B) positive samples. When detected, levels were 1.4 to 15.5 MPN/100 g (average 2.3 MPN/100 g) or 1.4 to 18.3 MPN/100 g (average 2.1 MPN/100 g) using methods A or B, respectively. A total of 23 different Salmonella serovars were identified from the original almond samples. Salmonella Muenchen was the most frequently isolated serovar (15%) from the 53 Salmonella-positive samples, followed by Newport (12%), Enteritidis (10%), and Typhimurium (8%). No correlation was found between presence of Salmonella and E. coli levels, aerobic plate counts, or counts of yeasts or molds.     

A rapid most-probable-number-based enzyme-linked immunosorbent assay for the detection and enumeration of Salmonella Typhimurium in poultry wastewater

The rapid and accurate detection and enumeration of low levels of Salmonella Typhimurium in food processing facilities are critical components of an effective hazard analysis critical control point program. The objective of this study was to develop a rapid (8 h) most probable number (MPN)-enzyme-linked immunosorbent assay (ELISA) for the detection and enumeration of Salmonella Typhimurium in wastewater. The specific objectives were to (i) characterize poly- and monoclonal Salmonella Typhimurium-specific antibodies in order to select the most specific and sensitive antibody for Salmonella Typhimurium detection, and (ii) validate the MPN assay through a correlation between the 8-h MPN-ELISA and the traditional 48-h Salmonella Typhimurium MPN method in poultry scald water. Poultry scald water samples were spiked with 10 and 50 CFU/ml of Salmonella Typhimurium. The traditional MPN method used a 48-h enrichment period followed by an analysis, while the MPN-ELISA used a 5-h enrichment period followed by a 3-h ELISA analysis. No differences (P < 0.05) were found between the traditional MPN and the MPN-ELISA, indicating the promise of the MPN-ELISA for the rapid detection and enumeration of Salmonella Typhimurium within an 8-h shift. This abbreviated assay will permit increased product sampling and more rapid movement of food between production and processing, resulting in reduced spoilage and quality losses.     

整鸡中弯曲菌定量检测方法的建立与优化

目的建立一种简便、灵敏、稳定的整鸡中弯曲菌定量检测方法,并用北京地区市售整鸡样品进行验证。方法分别对37株鸡肉中常污染的背景杂菌、24株弯曲菌菌株进行抗生素敏感性测试,对弯曲菌分离用选择性培养基及抗生素添加剂进行优化和改进;用77份人工定量污染弯曲菌的鸡淋洗液进行添加回收实验,以生长指数评价选择性平板对鸡肉样品中背景杂菌的抑制情况和弯曲菌生长状况影响。结果在改良后的Karmali和Preston选择性平板上,弯曲菌生长状态稳定,并可有效抑制鸡肉样品中常见的背景干扰杂菌的生长繁殖,此方法对鸡肉中弯曲菌检出的灵敏度可达2.5 CFU/g。结论所建方法灵敏度高、操作简便、计数准确,Karmali和Preston平板组合后可有效提高鸡肉中弯曲菌的检出率,适用于禽类食品中弯曲菌的定量检测。     

Quantification of Salmonella in food samples from India using the MINI-MSRV MPN and modified MINI-MSRV MPN methods

Salmonella is one of the most important food-borne pathogens. The MINI-MSRV MPN method was modified by replacing the isolation and confirmation of Salmonella on selective chromogenic agar with PCR/RT-PCR. This modification reduced the time for quantification by the MINI-MSRV MPN method by 24 h. Ninety-seven different food samples, comprising chicken, mutton, fish, and sprouts from different markets in Mumbai, India, were analyzed for quantification of Salmonella species by the MINI-MSRV MPN and modified MINI-MSRV MPN methods. Seventy-four percent of the chicken samples were found positive for Salmonella. However, Salmonella was found to be absent in fish, mutton, and sprouts samples. Salmonella load in the chicken sample was in the range of 1.30 to 120 MPN/g. This genotypic confirmation has advantage over variable phenotypic confirmation of pathogens.     

微滴式数字聚合酶链式反应定量检测食品中金黄色葡萄球菌方法的研究

目的 建立微滴数字聚合酶链式反应(ddPCR)快速定量检测食品中金黄色葡萄球菌的方法。方法 以金黄色葡萄球菌nuc基因为靶序列,筛选出同时适用于实时荧光定量PCR(qPCR)和微滴数字PCR(ddPCR)的引物探针,建立食品中金黄色葡萄球菌ddPCR快速定量检测方法,并对该方法进行特异性、灵敏度、准确性和重复性实验。结果 梯度稀释金黄色葡萄球菌纯培养液,确定ddPCR方法的检出限(LOD)和定量限(LOQ)均为110 CFU/mL;通过人工添加实验,同时进行ddPCR与平板计数检测,LOQ可达1 000 CFU/g,且4个添加水平的检测结果偏差最大为4.77%,5个平行重复的检测变异系数均小于20%,展现出良好的准确性和重复性。结论 本研究建立的金黄色葡萄球菌ddPCR定量检测方法特异性好、灵敏度高、结果准确,为快速定量检测金黄色葡萄球菌提供了参考。