荧光定量PCR技术检测样品中SeMNPV有效含量

采用NCBI提供的BLAST软件在线检索SeMNPV特异性基因ORF22、ORF40,并将其构建到pMD18-T载体上。以纯化的pMD18-T-ORF22、pMD18-T-ORF40质粒为标准样品,已计数野生型SeMNPV的基因组作对照,用荧光定量PCR检测样品中SeMNPV多角体含量。荧光定量PCR检测得到标准曲线方程为con=10(-0.282CT 9.965)(相关系数:R2=0.9997)和con=10(-0.296CT 9.945)(相关系数:R2=0.9995)。测得一个SeMNPV多角体包含102核酸分子,检测到SeMNPV复合剂包含633PIBs/mg、691PIBs/mgSeMNPV多角体,两者结果一致。与复合剂中SeMNPV实际含量相符。     

家蚕质型多角体病毒荧光定量PCR检测方法的建立

根据GenBank报道的家蚕质型多角体蛋白基因的保守序列设计特异性引物扩增118bp核苷酸片段,使用含有目的基因片段的重组质粒标准品绘制标准曲线,建立家蚕质型多角体病毒的实时荧光定量PCR检测方法。结果表明,标准曲线中模板拷贝数(X)与Ct值(Y)关系为Y=-3.582lgX+38.748,相关系数R2=0.999,构建的实时荧光定量PCR检测方法具有良好的敏感性、特异性和重复性,可用于家蚕质型多角体病毒病的快速检验及该病的流行性调查研究。     

美国白蛾核型多角体病毒实时荧光定量PCR检测方法的建立及应用

为了建立对昆虫核型多角体病毒（nucleopolyhedrovirus）进行定量检测方法,本研究选用美国白蛾Hyphantria cunea核型多角体病毒的polyhedrin基因为目的基因,经引物设计、PCR 扩增、目的片段与载体连接转化以及重组质粒的鉴定,并对重组质粒标准品和样品DNA进行检测,构建出美国白蛾核型多角体病毒SYBR GreenⅠ荧光定量标准曲线。统计学分析显示标准品浓度的对数值与Ct 值之间存在良好的线性关系（R=0.998）。该方法的检测灵敏度为10¹～10²拷贝/μL,线性范围达5个数量级,扩增产物形成单一的特异性熔解峰,组内组间的变异系数均小于3%。根据已建立的方法对不同感染时间段的感病幼虫进行检测,每毫克幼虫样本内病毒polyhedrin基因拷贝数增殖倍数对数值与感染时间（d）呈正相关（R=0.987）。这些结果表明,建立的美国白蛾核型多角体病毒荧光定量 PCR检测方法是可靠的,具有灵敏度高、重复性好等特点,可为昆虫核型多角体病毒体内增殖动态研究及昆虫病毒的标准化生产提供参考。     

EvaGreen实时荧光定量PCR检测猪圆环病毒2型方法的建立

根据GenBank中的猪圆环病毒2型(PCV2)ORF4基因序列设计一对特异性引物,通过常规PCR扩增PCV2的ORF4基因,并将其纯化的PCR产物克隆入pMD18-T载体中,构建重组质粒。应用该重组质粒进行EvaGreen实时荧光定量PCR,建立了PCV2 DNA的标准曲线,并进行熔解曲线分析。结果显示:建立的EvaGreen实时荧光定量PCR特异性强,与其他非靶标病毒基因不发生交叉反应;灵敏度高,最高可检测到10拷贝/μL的病毒量;重复性好,3个浓度的批间和批内的变异系数均小于2.5%;对118份临床样品进行检测,阳性样品25份,阳性率为21.2%,检测结果与普通PCR相符率为99.2%,其中一份样品经荧光PCR检测为PCV2阳性,普通PCR检测为阴性。结果表明,建立的EvaGreen实时荧光定量PCR方法具有特异性强、敏感度高、重复性好、简便快捷等特点,可用于临床PCV2感染的早期诊断以及分子流行病学调查。     

甘蔗杆状病毒荧光定量PCR检测方法的建立及初步应用

为建立一种能够快速、灵敏、特异的检测甘蔗杆状病毒(sugarcane bacilliform virus, SCBV)的SYBR GreenⅠ荧光定量PCR方法,针对SCBV的基因序列,设计了特异性扩增引物,利用构建的标准品建立和优化针对SCBV的荧光定量PCR检测方法,并对该方法进行了特异性、稳定性、灵敏性等的测试,随后用于田间样品的检测。结果表明:将含有SCBV基因组序列的重组质粒进行梯度稀释制成标准品,利用标准品进行荧光定量PCR,获得标准曲线y=-3.482 1x+37.264,相关系数r~2=0.999 9,说明CT值与反应起始模板数量呈线性关系,可进行准确定量;组内和组间变异系数在0.19%～1.68%之间,表明检测方法重复性良好;建立的荧光定量PCR方法最低可检测到10个拷贝重组质粒/μL,是常规PCR检测灵敏度的100倍。使用建立的荧光定量PCR方法和常规PCR方法对采集的90份甘蔗叶片样品进行检测,常规PCR检出53份阳性样品,荧光定量PCR检出56份阳性样品,表明所建立的荧光定量PCR方法较常规PCR敏感性高,且准确性高。本研究建立的SCBV荧光定量PCR检测方法重复性好,灵敏度高,为构建甘蔗健康种苗体系提供了一种高效检测方法。     

杀鲑气单胞菌SYBR Green Ⅰ实时荧光定量PCR检测方法的建立与应用

根据GenBank中杀鲑气单胞菌铁载受体(ferric siderophore receptor,fst A)基因序列设计并合成一对特异性引物,经过反应体系优化后建立了检测杀鲑气单胞菌的SYBR Green I实时荧光定量PCR检测方法。将fst A基因克隆到p MD18T载体上构建重组质粒pMD18T-fst A并制备标准曲线,结果显示该标准曲线的线性关系良好,扩增所得标准曲线为y=-4.255 2x+39.644,相关系数R2为0.988;熔解曲线分析显示产物为单一的特异峰。特异性检测结果表明,该方法对杀鲑气单胞菌及其亚种具有良好的特异性,与其他细菌不发生交叉反应;最低检测限为35拷贝/μL,较常规PCR的灵敏度高出约100倍。应用建立的方法对人工感染的虹鳟病样进行了检测,待检样品均呈阳性反应,表明该方法具有很好的适用性。研究表明,所建立的实时荧光定量PCR方法具有特异、灵敏、快速、定量的优点,可用于杀鲑气单胞菌的快速诊断及定量检测。     

甜菜夜蛾核多角体病毒在离体细胞中连续传代时重复DNA片段的产生和分析

野生型甜菜夜蛾核多角体病毒 (SeMNPV)US1分离株 (SeUS1 )通过空斑法纯化 ,PCR、长片段PCR和限制性内切酶分析筛选和鉴定 ,获得一株基因型较为均一且具完整基因组的克隆株 ,命名为Se 4。Se 4在其宿主细胞系Se3 0 1中无稀释连续传代至 1 0代 ,各代被感染细胞中的病毒DNA经限制性内切酶分析 ,发现在第 7代时病毒基因组中出现了一条新增的 3 .5kb片段 ,随着代数的增加 ,该片段的摩尔量逐渐增加 ,在第 1 0代时已成为超摩尔带 ,推测该片段为SeMNPVDNA复制的顺式作用元件。序列分析表明 ,该片段覆盖了SeMNPV 81 0 1 4～ 845 3 8nt共 3 5 2 5bp的序列 ,包含被预测为杆状病毒的DNA复制原点的non hr区域以及一些SeMNPV特有的ORF。研究结果为核多角体病毒II组的non hr在病毒复制过程中具有重要作用的观点提供了体外实验的证据     

棉铃虫单核衣壳核多角体病毒(HaSNPV)Hind Ⅲ-L片段的序列分析

本文报道了棉铃虫单核衣壳核多角体病毒(Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus,HaSNPV)基因组的HindⅢ-L片段的全序列.该片段全长2 635bp,包括5个有意义的开放阅读框HaSNPV ORF227,晚期表达因子10基因(lef10),vp1054基因,Ac55(AcMNPV ORF55的同源基因),Ac56(AcMNPV ORF56的同源基因).与其它6种杆状病毒的氨基酸序列比较表明,HaSNPV的lef10基因与甜菜夜蛾核型多角体病毒(SeMNPV)的同源性最高,为64%,与冷杉毒蛾核型多角体病毒(OpMNPV)的同源性最低,为43%;HaSNPV的vp1054基因与SeMNPV的同源性最高,为65%,与OpMNPV的同源性最低,为49%.序列比较表明,HaSNPV的LEF10与VP1054蛋白与其它6种杆状病毒具有相同的保守区和亮氨酸拉链(1eucine zipper)     

TaqMan荧光定量RT-PCR检测水稻RAc1基因mRNA方法的建立

构建包含RAc1基因cDNA片段的质粒,作为水稻肌动蛋白基因RAc1之mRNA定量检测的标准品,建立检测方法,为水稻其他基因的定量建立内参.从水稻叶总RNA中逆转录扩增总cDNA,PCR扩增RAc1基因中设计的目的片段,将纯化的目的片段与pMD19-T Simple载体进行连接,转化宿主菌JM-109,提取重组质粒DNA,PCR鉴定并测序分析.纯化质粒并检测260nm吸光值,确定重组质粒原液的拷贝浓度并以此制备荧光定量PCR梯度浓度标准品,进行实时荧光定量PCR实验.建立了RAc1基因mRNA表达实时荧光定量PCR检测方法,特异性好,检测灵敏度达102拷贝,线性范围为102～107拷贝,阈值循环数(Ct)与PCR体系中起始模板量的对数值之间有着良好的线性关系(r=1.000),扩增效率高(E=98.2%).建立了基因RAc1实时定量PCR的质粒标准品.     

一种检测马尔尼菲青霉菌实时荧光定量PCR方法的建立和应用

目的:建立一种检测马尔尼菲青霉菌的实时荧光定量PCR的方法。方法:针对马尔尼菲青霉菌5.8S rRNA设计特异性PCR引物,采用核酸荧光染料SYBR GreenⅠ进行实时荧光定量PCR检测,探讨该方法的灵敏度和特异性,并进行临床样品检测验证。结果:该方法的特异性较好,与该菌属内的其他细菌间无交叉反应;灵敏度可检测出10个细胞/mL全血,在检测范围内线性良好,相关系数R2=0.981。临床样品检测和传统的培养方法结果完全相符。结论:该方法特异性好,灵敏度高,操作简单,检测时间短;临床样品检测具有很好的准确性,从本研究的结果显示实时荧光定量PCR方法在检测马尔尼菲青霉菌中的应用可以大大缩短临床的诊断时间,提高临床诊断的准确度和效率。     

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.