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。     

实时荧光等温扩增法检测4种常见食源性致病菌

目的建立实时荧光等温扩增法(loop-mediated isothermal amplification,LAMP)检测金黄色葡萄球菌、沙门氏菌、副溶血性弧菌、单核细胞增生李斯特氏菌4种常见食源性致病菌的分析方法。方法根据4种致病菌特异性基因合成LAMP引物,在反应前加入荧光染料,在63℃条件下反应45 min;反应结果可以根据扩增曲线变化直接判断。并通过在实际样品中添加标准菌株菌液,测定了其在增菌0、6、24 h的检测灵敏度。结果金黄色葡萄球菌、沙门氏菌、单核细胞增生李斯特氏菌等基因组DNA的LAMP检测灵敏度为1 pg,副溶血性弧菌的检测灵敏度为10 pg;在样品加标实验中,其灵敏度在6 h分别可达到101、100、100、102 CFU/mL。结论该方法用于食源性致病菌的快速检测,具有可实时监控反应过程、反应快速,灵敏度和特异性高等优点,适合于基层食品安全监管领域现场快速检测。     

3种常见食源性致病菌多重重组酶聚合酶扩增检测方法研究

目的建立了食品中沙门菌、单核细胞增生李斯特菌和蜡样芽胞杆菌多重重组酶聚合酶扩增（recombinase polymerase amplification,RPA）快速检测方法。方法选择沙门菌invA基因、单增李斯特菌hlyA基因和蜡样芽孢杆菌16S RNA序列为目标基因进行扩增,建立并优化多重RPA扩增体系和扩增条件;评价反应体系的特异性和灵敏度,并对人工污染食品样品和实际样品进行检测。结果多重RPA反应体系能够在20 min完成三种目标基因的扩增,特异性强;对沙门菌、单增李斯特菌和蜡样芽孢杆菌的灵敏度分别为2.70×105、1.30×105、1.44×104 CFU/mL;能够用于人工污染样品和实际样品的检测。结论本研究建立的多重RPA等温扩增方法特异性强,操作快速、简单,为食源性致病菌的快速检测提供新方向     

多重重组酶介导等温扩增技术检测食品中3种食源性致病菌

建立快速、灵敏的多重重组酶介导等温扩增方法（recombinase aided amplification,RAA）同时检测食品中的大肠埃希氏菌O157:H7、沙门氏菌和金黄色葡萄球菌。方法 根据大肠埃希氏菌O157:H7的rfbE基因、沙门氏菌的invA基因以及金黄色葡萄球菌的nuc基因的序列保守区域设计RAA特异性引物,筛选出最优的引物组合并优化各项实验条件;通过标准菌株验证方法特异性和灵敏度,并通过人工污染实验,验证方法在实际食品样本中的检测能力。结果 多重RAA方法最佳扩增温度为37℃,反应时间为50 min;方法特异性良好,仅对目标菌株存在特异性扩增条带,与其他常见食源性致病菌无交叉反应;方法对基因组DNA的检测灵敏度为0.10 ng/μL;方法对经过简单增菌的人工污染牛奶样本和牛肉干样本中目标菌株的检出限为100 CFU/mL。结论 本研究建立的多重RAA扩增方法具有特异性好、灵敏度高、检测通量高等优势,适用于多种场景下食品样本中3种食源性致病菌的快速检测。     

建立DARQ-LAMP方法快速检测单增李斯特菌

该研究针对单增李斯特氏菌的特异性基因hlyA设计引物，优化反应条件，建立实时荧光环介导等温扩增（realtime loop-mediated isothermal amplification,real-time LAMP）的检测方法，在此基础上，建立检测单增李斯特氏菌的基于淬灭基团释放环介导等温扩增检测方法（detection of amplification by release of quenching-LAMP,DARQ-LAMP），分析其特异性和灵敏度。结果表明，该方法的适宜反应条件为反应温度63℃、Bst DNA 3.0聚合酶0.32 U/μL、淬灭探针双链（quenching probe double chain,QPD）探针浓度10%、Mg2+浓度6 mmol/L；对单增李斯特菌的检测限为7.3×101copies/mL，灵敏度是普通聚合酶链式反应（polymerase chain reaction,PCR）的100倍。该方法效率高、特异性好、灵敏度高，为环介导等温扩增技术检测食源性致病菌的研究提供参考。     

反转录-环介导等温扩增技术快速检测志贺氏菌

建立了反转录-环介导等温扩增技术的志贺氏菌快速检测方法。针对志贺氏菌特异保守基因ipa H设计多套引物,建立优化了的反应体系,并评价其准确性、特异性、灵敏度。以人工污染脱脂乳样品比较RT-LAMP与RT-PCR的灵敏度。结果表明,在65℃等温条件下,RT-LAMP反应可在30 min内完成。在活菌/损伤菌模型中,该方法表现出比国标法更好的准确性。在23株细菌标准菌株中仅对4株志贺氏菌有特异性检出。志贺氏菌RNA模板的检测灵敏度为7 fg/μL。人工污染脱脂乳样品检测灵敏度达到40 CFU/g,比RT-PCR方法高出2个数量级。表明所建立的志贺氏菌RT-LAMP扩增方法可以准确的检测志贺氏菌,同时具有快速、特异、灵敏的优势,可用于志贺氏菌的快速筛查和现场监控。     

乳中志贺氏菌的荧光定量PCR检测方法的建立

为了快速、有效的检测乳中的志贺氏菌,建立一种特异性强、灵敏度高的荧光定量PCR方法。根据GenBank公布的志贺氏菌ipaH基因的保守序列设计特异性引物,对提取的志贺氏菌DNA模板进行PCR扩增,对目的基因进行克隆和测序,然后利用荧光定量PCR方法,对志贺氏菌进行检测,确定其扩增条件。建立的方法特异性强,检测的灵敏度可达到10-6 ng/μL。利用建立的方法可检测乳中2.84 cfu/mL的志贺氏菌。该方法为快速、准确检测乳中的志贺氏菌提供了参考。     

环介导恒温扩增方法快速检测乳中单增李斯特菌

建立了一种快速检测灭菌乳中单增李斯特菌的环介导恒温扩增(Loop-Mediated Isothermal Amplification,LAMP)方法。以hlyA基因作为靶基因,对人工污染乳中单增李斯特菌进行了LAMP方法的灵敏度试验,同时与PCR方法进行比较。并对单增李斯特菌和7种其他乳中常见致病菌进行了LAMP检测,以验证该方法的特异性。结果表明,LAMP检测单增李斯特菌的特异性强,检出限为42 mL-1,其灵敏度比普通PCR高10倍。并且检测时间比PCR更短,在1.5 h内即可完成扩增反应。此方法快速、特异、简单、灵敏,具有较高的推广价值。     

三重PCR（聚合酶链式反应）检测鸡肉中常见病原菌的研究

建立鸡肉中常见3种致病菌即小肠耶尔森氏菌、大肠杆菌O157:H7和沙门氏菌的多重PCR(聚合酶链式反应)检测方法。方法:分别选择小肠耶尔森氏菌、沙门氏菌和大肠杆菌O157:H7基因组中高特异性的ail基因、inv A基因和ECs3032,设计并筛选具有高特异性的引物用于PCR(聚合酶链式反应)扩增。建立一种此3种致病菌的三重PCR检测方法,并通过人工污染实验对该法进行验证。结果:3对引物能特异地扩增出251、347、469 bp的目的条带;灵敏度:对这3种致病菌的单菌培养物检测灵敏度均为101CFU/m L。同时检测3种菌的灵敏度为103 CFU/m L。结论:该检测方法高效、精确、特异性强、灵敏度高,6 h~8 h内可同时快速检测多种致病菌,节省了大量时间。在实际检测中具有很好的实用价值和开发前景,可在食品检测和临床检验等领域大力推广。     

葡萄酒中4种酚酸类化合物的色谱电化学分析

目的:建立测定葡萄酒中4种酚酸的色谱电化学分析方法,并用该方法测定了5种国内不同品牌的葡萄酒。方法:采用反相高效液相色谱电化学检测法,色谱柱为HypersilODS柱(250mm×4.0mm,5.0μm),流动相为甲醇-2%醋酸,梯度洗脱,流速为0.8ml/min,检测电压为0.7V,柱温为30℃。结果:4种酚酸得到很好的分离,线性关系良好,并且电化学检测器的检测灵敏度是紫外检测器的7～600倍。结论:该方法是一种快速简便、灵敏准确的分析方法,可以为葡萄酒的质量控制提供科学依据。     

凤窝酒曲中乳酸菌的分离及其作用下的米酒品质评价

通过传统可培养法对从湖北孝感市采集的凤窝酒曲中乳酸菌进行了分离,共得到12株乳酸菌。经鉴定,它们分别属于短乳杆 菌、植物乳杆菌、发酵乳杆菌及融合魏斯氏菌4个菌种,归于乳杆菌属（Lactobacillus）与魏斯氏菌属（Weissella）共2个菌属。 使用分离 到的乳酸菌制作米酒,与未接菌处理米酒相比,分离到的乳酸菌对米酒有机酸种类与含量、固形物含量与米酒的滋味产生了显著影 响（P＜0.05）,但是对米酒pH影响不显著（P＞0.05）,且同一类乳酸菌对米酒滋味具有类似的影响。 植物乳杆菌MJ1-3使米酒中乳酸含 量增加了79%,而另一株融合魏斯氏菌MJ2-1,能显著降低米酒中草酸、琥珀酸、乳酸及乙酸的含量,可用于调节米酒有机酸含量。 因 此,分离到的乳酸菌MJ1-3与MJ2-1是用于米酒复合酒曲开发的优良候选菌株。     

Application of molecular methods to demonstrate species and strain evolution of acetic acid bacteria population during wine production

The growth of acetic acid bacteria on grapes or throughout the winemaking process influences the quality of wine, mainly because it increases the volatile acidity. The objective of this study was to analyse how the acetic acid bacteria population evolves in the changing environment of the grape surface and during wine fermentation. We have analysed the influence of yeast inoculation and SO2 addition on acetic acid bacteria populations. These bacteria were analysed at both the species and the strain level by molecular methods such as Restriction Fragment Length Polimorfism (RFLP) of amplified 16S rDNA, and amplification by polymerase chain reaction of Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) and Repetitive Extragenic Palindromic (REP-PCR). Our results show that the increases in population size are normally accompanied by a proliferation of Acetobacter aceti, which is the main species during fermentation. The diversity of strains is considerable in natural environments such as the grape surface. Changes in the environment during alcoholic fermentation substantially reduce the survival and the diversity of acetic acid bacteria. Few strains are able to survive these conditions and they seem to originate from both the grapes and the winery. To the best of our knowledge this is the first time that acetic acid bacteria are analysed at the strain level in grape surfaces and during winemaking.     

红茶菌酒饮料的研究

本文研究了红茶菌酒饮料的生产方法:以酵母菌、醋酸菌和乳酸菌为工作菌种,用糖、茶作为营养成分,在菌种互为条件,共同生存的情况下产生代谢产物;然后以发酵茶菌液为基液,加入一定数量的基酒,制成一种酒饮料。实验结果表明:红茶菌酒饮料酸甜适宜,既具茶的清香,也具有酒的清香,营养丰富、风味独特,有利于吸收,对人体有一定的营养价值和医疗保健作用。     

Fast identification of wine related lactic acid bacteria by multiplex PCR

The microflora of must and wine consists of yeasts, acetic acid bacteria and lactic acid bacteria (LAB). The latter group plays an important role for wine quality. The malolactic fermentation carried out by LAB leads to deacidification and stabilisation of wines. Nevertheless, LAB are often associated with wine spoilage. They are mainly responsible for the formation of biogenic amines. Furthermore, some strains produce exopolysaccharide slimes, acetic acid, diacetyl and other off-flavours. In this context a better monitoring of the vinification process is crucial to improve wine quality. Moreover, a lot of biodiversity studies would also profit from a fast and reliable identification method.     

PCR-核酸试纸条法检测食品中假结核耶尔森菌

目的 建立一种基于PCR-核酸试纸条技术快速检测食品中假结核耶尔森菌的方法。方法 将10株假结核耶尔森菌株和9株其他耶尔森氏菌及18株来源菌株作为实验菌株进行特异性实验; 通过纯菌液计数、干扰菌实验检测进行灵敏度验证。结果 DNA检测可达到10?3 μg/mL, 25 g样品加菌实验灵敏度可达 100 CFU/25 g, 添加10倍干扰菌不会降低检测灵敏度。利用建立方法对市场购买的食品进行筛查并与国标方法进行比较, 建立方法的灵敏度优于国标方法。结论 该方法检测结果准确, 灵敏度高, 适用于检测食品中假结核耶尔森菌。     

Antimicrobial activity of nisin against Oenococcus oeni and other wine bacteria

Nisin is a bacteriocin used against food spoilage bacteria. Sulphur dioxide is a potent antioxidant as well as an antimicrobial agent widely used in the wine industry. In this study we describe the effect of these important antibacterial agents on the growth of a collection of 64 lactic acid bacteria (23 Oenococcus, 29 Lactobacillus, 3 Leuconostoc and 9 Pediococcus), 23 acetic acid bacteria and 20 yeast isolates, most of them recovered from wine. Minimal inhibitory concentrations (MIC) and minimal bactericide concentrations of nisin, potassium metabisulphite and ethanol were determined. Nisin MIC(50) values for the tested isolates were as follows: 0.024, 12.5, 200 and > or micro for oenococci, lactobacilli-pediococci-leuconostoc, acetic acid bacteria and yeasts, respectively. Synergistic effects on bacterial growth inhibition were observed, and potassium metabisulphite MIC(50) values decreased from one to three orders of dilution when it was combined with subinhibitory concentrations of nisin in the growth media. This effect was observed in all lactic acid bacteria species of our study. Significant differences in nisin sensitivity were observed between Gram-positive and Gram-negative bacteria, and between Oenococcus oeni and other species of lactic acid bacteria. It is concluded that appropriate combinations of nisin and metabisulphite could control the growth of spoilage bacteria in wine and therefore allow a decrease in the levels of sulphur dioxide currently used by the wine industry.     

Acetic acid bacteria spoilage of bottled red wine -- a review

Acetic acid bacteria (AAB) are ubiquitous organisms that are well adapted to sugar and ethanol rich environments. This family of Gram-positive bacteria are well known for their ability to produce acetic acid, the main constituent in vinegar. The oxidation of ethanol through acetaldehyde to acetic acid is well understood and characterised. AAB form part of the complex natural microbial flora of grapes and wine, however their presence is less desirable than the lactic acid bacteria and yeast. Even though AAB were described by Pasteur in the 1850s, wine associated AAB are still difficult to cultivate on artificial laboratory media and until more recently, their taxonomy has not been well characterised. Wine is at most risk of spoilage during production and the presence of these strictly aerobic bacteria in grape must and during wine maturation can be controlled by eliminating, or at least limiting oxygen, an essential growth factor. However, a new risk, spoilage of wine by AAB after packaging, has only recently been reported. As wine is not always sterile filtered prior to bottling, especially red wine, it often has a small resident bacterial population (<10(3) cfu/mL), which under conducive conditions might proliferate. Bottled red wines, sealed with natural cork closures, and stored in a vertical upright position may develop spoilage by acetic acid bacteria. This spoilage is evident as a distinct deposit of bacterial biofilm in the neck of the bottle at the interface of the wine and the headspace of air, and is accompanied with vinegar, sherry, bruised apple, nutty, and solvent like off-aromas, depending on the degree of spoilage. This review focuses on the wine associated AAB species, the aroma and flavour changes in wine due to AAB metabolism, discusses the importance of oxygen ingress into the bottle and presents a hypothesis for the mechanism of spoilage of bottled red wine.     

五粮浓香型白酒窖房和曲房空气细菌相关性的初步研究

从宜宾华夏酒业窖房和曲房空气中分离得到44株细菌,对所得菌株进行了16S rDNA基因系统发育及部分酿造特性分析。窖房和曲房空气的细菌在属和种一级的相似性分别为40.0%和36.4%,且均以Bacillus cereus、Bacillus anthracis细菌为优势种,表明2个细菌群落具有一定的相似性。分离自窖房空气的菌株有20株（占95.2%）耐受7%的无水乙醇,2株菌耐受pH4.5,且分别有2株、9株能够产纤维素酶和淀粉酶;分离自曲房空气的菌株均不耐受7%的乙醇和pH4.5,产纤维素酶和淀粉酶的菌株分别有6株、3株。结果表明,经过长期的酿酒生产,曲房和窖房空气中均形成了较为稳定的细菌区系,其菌落组成与酿酒生产存在较为密切的相关性。在生产中,2个细菌群落之间存在广泛的交流。     

凝固型苦荞酸奶对小鼠肠道菌群的调节作用

通过动物实验,利用实时荧光定量聚合酶链式反应方法、气相色谱-质谱、Illumina高通量测序技术,对小鼠肠道菌群数量和种类,以及主要代谢产物——短链脂肪酸（乙酸、丙酸、丁酸、乳酸）的含量进行分析,探究凝固型苦荞酸奶对小鼠肠道菌群的调节作用。结果表明：与对照组相比,凝固型苦荞酸奶使肠道中3 种有益菌（乳酸杆菌、双歧杆菌、柔嫩梭菌）的数量分别增加了523.81、911.01、12.18 倍,3 种有害菌（肠杆菌、肠球菌、拟杆菌）的数量分别减少了99.89%、97.31%、99.02%。同时,凝固型苦荞酸奶组小鼠粪便中的乙酸、丙酸、丁酸、乳酸的含量分别增加了36.87%、75.59%、56.26%、92.36%,且效果优于普通酸奶组。高通量测序结果表明,凝固型苦荞酸奶较普通酸奶可以显著增加小鼠肠道菌群多样性,在门水平上显著降低厚壁菌门和疣微菌门的丰度,显著提高拟杆菌门的丰度（P＜0.05）;且在属水平上显著提高norank_f__Bacteroidales_S24-7_group、Faecalibaculum菌属的相对丰度,显著降低Ruminococcaceae_UCG-014、unclassified_f__Lachnospiraceae、Lachnospiraceae_UCG-006菌属的相对丰度（P＜0.05）,提高肠道菌群优势菌属的地位。综上所述,凝固型苦荞酸奶相较于普通酸奶,可以更好地促进小鼠肠道内有益菌的增殖并抑制有害菌的增殖,促进短链脂肪酸的生成,增加肠道菌群多样性,优化菌群结构。     

Differentiation of acetic acid bacteria based on sequence analysis of 16S-23S rRNA gene internal transcribed spacer sequences

The 16S-23S gene internal transcribed spacer sequence of sixty-four strains belonging to different acetic acid bacteria genera were analyzed, and phylogenetic trees were generated for each genera. The topologies of the different trees were in accordance with the 16S rRNA gene trees, although the similarity percentages obtained between the species was shown to be much lower. These values suggest the usefulness of including the 16S-23S gene internal transcribed spacer region as a part of the polyphasic approach required for the further classification of acetic acid bacteria. Furthermore, the region could be a good target for primer and probe design. It has also been validated for use in the identification of unknown samples of this bacterial group from wine vinegar and fruit condiments.