FTA滤膜用于PCR检测肉中的金黄色葡萄球菌

利用FTA滤膜,采用PCR技术可直接检测肉及肉制品中的金黄色葡萄球菌,无需增菌,灵敏度高。在采用浮选与溶剂萃取相结合方法的基础上,使用FTA膜可高效地从鲜肉中提取金黄色葡萄球菌的DNA,消除PCR反应的抑制因子。以金黄色葡萄球菌耐热核酸酶基因（nuc）为靶基因,经过PCR扩增得到279bp的产物。经过DNA测序证实该产物为目的扩增产物。使用FTA滤膜处理样品,再通过PCR方法检测金黄色葡萄球菌,猪肉、牛肉、羊肉、鸡肉匀浆液的检出限均为10cfu/mL,可在6h内完成对肉中金黄色葡萄球菌的检测,比目前普遍采用的先增菌再进行PCR检测的方法缩短了12～24h 。实际检测了72份样品,同时与GB 4789.10-94方法及两种快速检测致病性金黄色葡萄球菌检测方法做比较,PCR方法的检出率为72.2％,检出时间为6h,GB 4789.10-94方法检出率为70.8%,检出时间为5d,Prfilm RSA方法的检出率为61.1％, 检出时间为18h,BairdParker R.P.F方法的检出率为69.4％,检出时间为18h,结果表明FTA滤膜用于PCR检测肉中金黄色葡萄球菌检出率高,耗时短。使用FTA滤膜法制备模板DNA,为食品中的致病菌快速检测构建了一个技术平台。     

种特异性PCR快速检测奶粉中阪崎肠杆菌研究

阪崎肠杆菌是一种目前认为以奶粉为传播媒介的食源性条件致病菌,通过α-1,4-葡萄糖苷酶基因和ompA基因分别设计引物ESF-ESR和ESSF-ESSR,进行单重和双重PCR方法研究,结果显示所有阪崎肠杆菌菌株PCR扩增均为阳性,阴性对照均未扩增出目的片段;纯菌单重PCR灵敏度分别为102cfu/mL和101cfu/mL,双重PCR灵敏度为103cfu/mL;在有或无其他细菌存在时,人工污染阪崎肠杆菌模拟样品单重PCR检测灵敏度分别为103cfu/mL和102cfu/mL,双重PCR检测灵敏度为104cfu/mL;实际样品检测显示PCR方法与传统方法具有很好的一致性。结果表明:该PCR方法具有很好的种特异性和灵敏度,能够克服奶粉中杂菌对快速检测阪崎肠杆菌造成的干扰,减少以保守序列来设计引物导致假阳性结果的出现,可以较好地应用于奶粉中阪崎肠杆菌的检测与鉴定。     

煮沸裂解法和试剂盒法提取浸矿菌基因组DNA的比较

目的:在生物浸出中,微生物群落结构分析有着重要意义,而群落分析的基础是提取纯度高、损失少的基因组DNA。为了解决这一问题,本实验通过比较两种较常用的DNA提取方法,煮沸裂解法和试剂盒法,寻找一种灵敏、快速、经济实用的制备浸矿细菌基因组DNA的方法。方法:分别用煮沸裂解法和试剂盒法提取6种浸矿菌的基因组DNA,从所提取的基因组DNA浓度、纯度、回收率和对PCR扩增反应的影响方面比较了两种方法的提取效果;用两种方法来处理不同浓度梯度的一种菌,通过实时定量PCR来比较两种方法的灵敏性。结果:相同处理量(108个)的革兰氏阳性菌(1株)、革兰氏阴性菌(4株)、古菌(1株)经两种方法提取的基因组DNA差异较大,煮沸裂解法所得的6组基因组DNA更纯,其OD260/OD280的值更接近1.8-2.0(纯DNA的OD260/OD280在1.8-2.0之间),前者所提DNA回收率最大可达后者的16.7倍;煮沸裂解法只需较少菌(102个)便能让实时定量PCR检测到所提DNA模板浓度,比试剂盒法灵敏。结论:两种方法提取的基因组DNA均可用于后续的PCR扩增,此外,前者提取的DNA浓度随细菌浓度增加而呈线性增大,而后者随菌浓度增大,所提DNA量增加有限,因此,在生物浸出中微生物基因组DNA的提取可直接采用简单快速的煮沸提取法,为实验节约成本和时间。     

乳制品中四种病原菌多重荧光PCR检测方法的建立与应用

本研究建立了可快速特异检测乳制品中阪崎肠杆菌、沙门氏菌、金黄色葡萄球菌和单核细胞增生李斯特氏菌的多重荧光PCR方法。通过设计阪崎肠杆菌、沙门氏菌、金黄色葡萄球菌和单核细胞增生李斯特氏菌的特异性引物和探针,建立了多重PCR反应体系,评价了该方法的特异性、灵敏度和精密度,并用该方法和国标法分别检测了150份乳制品中的4种致病菌,对检测结果进行对比。结果显示,检测目标菌为相应阳性,检测其他12种非目标供试菌为阴性;检测灵敏度分别为1 cfu/μL;其精密度的变异系数均小于5%;与国标检测方法相比其检测结果具有良好的一致性。     

基因标记枯草芽孢杆菌BS-68A在黄瓜上定殖

目的:野生型枯草芽孢杆菌Bacillus subtilisBS-68能有效地防治由Pythium spp.和Fusarium oxporum引起的黄瓜立枯病和枯萎病。为了探究该菌株的生防机制,利用该菌株的黄绿荧光蛋白基因和氯霉素抗性基因标记菌株BS-68A研究其在黄瓜植株各个部位的定殖能力、种群动态和在根围的分布。方法和结果:用基因标记菌株发酵液分别对黄瓜种子进行浸种和浇穴处理,播种后30d,该菌能在黄瓜根部和茎基部定殖,不能在茎部和叶部定殖。浸种处理,该菌在茎基部的种群数量为3.1×104cfu/株,大于根部的种群数量4.1×102cfu/株;浇穴处理,该菌在茎基部的种群数量8.0×103cfu/株,低于根部的种群数量2.5×104cfu/株。     

沙门菌、志贺菌、副溶血性弧菌多重PCR检测方法的研究

建立快速检测沙门菌、志贺菌和副溶血性弧菌的多重PCR方法[1-4].根据沙门菌hilA基因、志贺菌ipaH基因及副溶血性弧菌TDH基因设计特异性PCR引物[5-6],被检样品经4 h振荡培养后金属浴裂解制备DNA模板,使用全自动毛细管电泳核酸检测系统分析PCR扩增产物.在580、423和245 bp处分别出现预期的特异性DNA条带,且无非特异扩增条带出现.敏感性试验显示沙门菌在模拟标本中的检测灵敏度为101-2cfu/mL、志贺菌为101cfu/mL、副溶血性弧菌为102cfu/mL.该方法操作方便、分析时间短、特异性和灵敏度高,可用于公共卫生突发事件食源性病原菌的快速检测.     

人母乳细菌Streptococcus salivarius F286和S. parasanguinis F278对HT29细胞的细胞毒性

为了筛选到具有抗炎特性的有益菌,研究者通常将待测细菌的发酵液上清和热致死菌体与TNF-α刺激下的人类结肠腺癌细胞HT29共孵育,并测量细菌是否能够减少HT29细胞分泌的炎症因子。该测试的前提之一是待测细菌的发酵液上清或菌体不杀死或杀死<10%的HT29细胞。在前期的工作中,我们从人母乳中分离得到Streptococcus salivarius F286和S.parasanguinis F278两株菌。在研究这两株菌的抗炎能力之前,我们利用MTT法摸索不同浓度的S.salivarius F286和S.parasanguinis F278的发酵液上清和热致死菌体对HT29细胞的细胞毒性。实验表明,两株菌的发酵液上清的原液和稀释液对HT29细胞均没有细胞毒性;浓度5×10^5~7.5×10^6cfu/mL的F286热致死菌体、浓度5×10^5~2.5×10^6cfu/mL的F278热致死菌体对HT29细胞的细胞毒性低于10%,而浓度1×10^8cfu/mL的热致死F286和F278菌体分别杀死(23±5.3)%和(22±5.3)%的HT29细胞。因此,S.salivarius F286和S.parasanguinis F278的发酵液上清原液、以及浓度5×10^5~7.5×10^6cfu/mL的F286热致死菌体和5×10^5-2.5×10^6cfu/mL的F278热致死菌体可在HT29细胞模型中进行抗炎能力测试。本研究的方法可用于确定其他细菌在HT29细胞模型中进行抗炎能力测试的合理浓度范围。     

两种方法制备模板DNA在PCR-SSCP中的应用研究

目的:比较两种方法(DNA试剂盒提取法和FTA卡法)提取的DNA在PCR-SSCP反中的可靠性.方法:用DNA试剂盒从全血中提取DNA和用NaOH方法从FTA卡中提取DNA后,用分光光度计检测两种方法提取DNA的浓度及纯度,进行PCR反应之后,用2％的琼脂糖凝胶电泳检测其质量,接着进行SSCP检测,观测其效果.两种方法提取的样品相同,进行PCR反应和SSCP检测的条件完全一致.结果:用DNA试剂盒提取法和FTA卡法提取的DNA纯度分别为OD260/280=1.817,OD260/280=1.806.48份贵州荷斯坦奶牛DNA后续PCR反应和SSCP的检测结果表明,两种方法提取的DNA用于PCR反应和SSCP检测其效果没有明显差别,成功率为100％.结论:FTA卡结合DNA较稳定,用NaOH法提取DNA效果可靠且比试剂盒方法简便、快捷、经济,值得推广.     

奶粉伴侣中微生物污染特点分析

目的：了解婴幼儿经常食用的奶粉伴侣中微生物的污染状况。方法：2016年在我国8个省（直辖市）采集奶粉伴侣864份,按照食品安全国家标准方法检测菌落总数、大肠菌群、阪崎肠杆菌、单核细胞增生李斯特菌、金黄色葡萄球菌和沙门氏菌。结果：104%（9/864）的奶粉伴侣菌落总数＞104 cfu/g,012%（1/864）大肠菌群＞102 cfu/g,未检出单核细胞增生李斯特氏菌、金黄色葡萄球菌和沙门氏菌,但1份样品检出阪崎肠杆菌。结论：奶粉伴侣微生物污染水平较低。     

滁菊连作土壤中尖孢镰刀菌的分离、鉴定及变化特征

采用传统平板培养法及尖孢镰刀菌专性培养基对滁菊连作土壤的真菌和尖孢镰刀菌进行分析测定,结果表明,在滁菊连作条件下,土壤中真菌和尖孢镰刀菌的数量显著增加,平均分别达到8.71×105 cfu/g干土和3.12×104cfu/g干土,各自是对照土壤的2.24倍和5.57倍。土壤中尖孢镰刀菌数量显著增加可能是导致滁菊连作障碍的重要因素。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.