番茄褪绿病毒TaqMan荧光定量PCR快速检测方法的建立与应用

【目的】本研究旨在建立TaqMan实时荧光定量PCR(TaqMan RT-qPCR)技术,快速检测单头烟粉虱Bemisia tabaci体内的番茄褪绿病毒(tomato chlorosis virus,ToCV)。【方法】根据ToCV外壳蛋白保守序列设计了1对特异性引物和1条TaqMan探针,建立了TaqMan RT-qPCR方法;与常规PCR检测进行比较,检测该方法的灵敏度与特异性;并应用该方法对单头烟粉虱成虫体内ToCV进行了快速检测。【结果】本研究构建的TaqMan RT-qPCR检测ToCV的标准曲线,其循环阈值(Ct值)与模板浓度具有良好的线性关系,扩增效率为98%。该方法对ToCV的最低检测浓度为8.3×10 copies/μL,灵敏度是常规RT-PCR的1000倍。该方法与田间番茄两种重要病毒番茄黄化曲叶病毒(tomato yellow leaf curl virus,TYLCV)和番茄斑萎病毒(tomato spotted wilt virus,TSWV)检测无交叉反应。单头烟粉虱成虫ToCV检测结果表明,温室内ToCV侵染植株上烟粉虱携毒率为100%,田间烟粉虱的携毒率为30%。【结论】本研究建立的TaqMan RT-qPCR检测方法,可快速有效检测单头烟粉虱体内ToCV携毒情况,为该病毒病的防控提供了技术支撑。     

大鼠疑似泰勒氏病毒荧光定量检测方法的建立

目的建立一种能在临床上快速、准确地检测大鼠疑似泰勒氏病毒(Theiler’s-like virus of rats,TLV)的方法,采用TaqMan探针荧光定量聚合酶链式反应(qPCR)技术,特异性针对TLV病毒核酸进行检测。方法通过基因合成序列作为质粒标准品的模板,同时选择特异性的序列在3622~3729 nt处,设计一对引物和TaqMan性探针,优化反应体系及条件,进行qPCR扩增,从而建立TLV TaqMan探针qPCR方法,并对其灵敏度、稳定性和特异性进行评价。结果建立的TLV qPCR检测方法,标准曲线线性关系良好,R~2值可达到0.99,灵敏性最低能够检测到10个拷贝数/μL,对比普通PCR方法,高出其100倍;对其他常见大鼠病毒均无非特异反应;重复性良好,批内和批间变异系数均小于1%。结论利用TaqMan探针建立快速检测TLV的荧光定量PCR方法,该方法具有操作简便、灵敏度高、特异性好等特点。     

蜜蜂残翼病毒TaqMan-MGB探针荧光定量RT-PCR检测方法的建立

蜜蜂残翅病毒(Deform wing virus,DWV)已成为世界上最著名、分布最广、研究最为深入的昆虫病原.虽然DWV以前存在于蜜蜂种群中,但一种新的媒介一外寄生螨Varroa破坏体的到来和全球传播,极大地促进了 DWV的流行病学.为了建立快速、准确且能定量分析的蜜蜂残翅病毒,DWV检测方法,本研究参照GenBank中已登录的DWV高度保守的3C-RdRp基因区域,设计了 1对特异性引物和带有羧基荧光素(Fast Auxiliary Memory,FAM)与淬灭基团(Eclipse)标记的TaqMan探针,并对反应条件进行优化,建立了检测DWV的TaqMan-MGB探针荧光定量RT-PCR检测方法(TaqMan qRT-PCR),同时对该检测方法的特异性、敏感性、重复性进行了试验,并对临床样品进行检测.结果显示:该方法最佳上下游引物和探针浓度分别为12.5μmol/L和10μmol/L,最佳退火温度为59℃;敏感性试验中,对DWV质粒标准品检测下限为2.58×101拷贝/μL;本方法特异性较好,对蜜蜂常见病毒-蜜蜂慢性麻痹病毒(Chronic bee paralysis virus,CBPV)、蜜蜂急性麻痹病毒(Acute paralysis virus,ABPV)、黑蜂王台病毒(Black queen cell virus,BQCV)、囊状幼虫病毒(Sacbrood virus,SBV)、以色列急性麻痹病毒(Israeli acute paralysis virus,IAPV)和中蜂囊状幼虫病毒(Chinese sacbrood virus,CSBV)无交叉反应;且批内变异系数和批间变异系数均小于2％.利用该方法对49份临床疑似样本进行检测,其中DWV阳性样本为35份,检出率高于常规RT-PCR方法,且病毒载量大于1×107拷贝/只样本数共24份,表明辽宁和河北部分地区感染DWV蜂群中病毒载量较高,流行情况比较严重.因此,本研究建立的TaqMan探针荧光定量RT-PCR方法具有灵敏度高、特异性强、重复性好等特点,能够用于DWV病原监测、流行病学调查和病毒定量分析等相关研究.     

基于双探针杂交的AIV、NDV和FAV-4三重环介导PCR鉴别检测方法的建立及应用

本研究目的是建立禽流感病毒(AIV)、新城疫病毒(NDV)和禽腺病毒4型(FAV-4)三重环介导PCR检测方法,用于3种病毒混合感染的鉴别诊断。根据GenBank数据库中AIV的HA基因、NDV的F基因和FAV-4的Hexon基因序列保守性,分别设计两条序列相邻的特异性探针,标记于不同大小的拟南芥TOC1基因片段两端,作为报告基因,用于环介导PCR检测,成功建立了AIV、NDV和FAV-4三重环介导PCR鉴别诊断方法,扩增片段大小分别为200bp、270bp和360bp。特异性试验结果表明,环介导PCR对含有AIV、NDV和FAV-4核酸的样品可扩增出特异性目的片段,而含有IBDV、IVB、FPV和MDV核酸的样品则未见明显扩增,特异性良好;灵敏性试验结果表明,环介导PCR对AIV、NDV和FAV-4的最低极限浓度分别为25pg/μL、12.5pg/μL和12.5pg/μL的核酸样品,检测灵敏度高;多病原同时检测不影响环介导PCR检测特异性和灵敏性。利用该方法对117份临床样本进行检测,结果显示,环介导PCR对AIV、NDV和FAV-4阳性检出率分别为15.4%,27.4%和34.2%,分别高于常规PCR 12.8%,23.9%和32.5%,接近于SybrGreen实时PCR 15.4%,28.2%和35%;kappa一致性检验显示,环介导PCR、SybrGreen实时PCR两种方法对AIV、NDV和FAV-4检测结果高度符合,Kappa值分别为κ=0.93、κ=0.89和κ=0.94。本研究成功建立了AIV、NDV和FAV-4三重环介导PCR检测方法,适用于临床上3种病原的快速鉴别诊断。     

K亚群禽白血病病毒实时荧光RT-PCR检测方法的建立及初步应用

根据GenBank数据库K亚群禽白血病病毒(Subgroup K avian leukosis virus,ALV-K)特异性抗原gp85的基因序列,设计一组ALV-K特异性的引物和探针,并构建阳性重组质粒。在对反应体系进行优化的基础上,建立了ALV-K的实时荧光RT-PCR检测方法并进行了特异性试验、敏感性试验和重复性试验。结果显示,本方法能对ALV-K进行特异性扩增,而对A亚群、B亚群、J亚群、E亚群禽白血病毒和新城疫病毒(NDV)、鸡传染性支气管炎病毒(IBV)、禽网状内皮组织增生病病毒(REV)、禽流感病毒(AIV)等禽病病原未见扩增。该方法最低能够检测到3.4×10~1拷贝数/μL的阳性质粒,灵敏度是RT-PCR的100倍。应用本方法对人工攻毒ALV-K的SPF鸡各脏器进行病毒定量分析,结果显示,在攻毒鸡体内心、肝、脾、肺、肾等脏器中均能检测到ALV-K,且肝、肾和脾中的病毒含量显著高于心和肺(P0.01)。     

非洲猪瘟病毒B646L和F778R双基因PCR检测方法的构建

为建立一种能够快速检测非洲猪瘟病毒(ASFV)的普通PCR方法,本研究分别以ASFV B646L和F778R基因为靶序列设计了两对特异性引物,建立ASFV B646L和F778R双基因普通PCR方法,并对其特异性、灵敏性及重复性进行检测。结果显示：该PCR体系(25.0μL体系)的最优组合为Pfu酶0.2μL、10×Reaction Buffer 2.5μL、2.5 mmol/L d NTP Mixture 2.5μL、B646L上下游引物各1.5μL、F778R上下游引物各0.5μL、DNA模板1.0μL;退火温度为57℃、退火时间为30 s、72℃延伸2.5 min,30个循环。该方法仅针对ASFV B646L和F778R基因进行特异性扩增,无交叉反应。同时对B646L和F778R阳性质粒的最低检测下限分别为7.2×10⁷ copies/μL、1.5×10⁶ copies/μL,试验重复性良好。本研究对于提高ASFV临床诊断的准确性、特异性和高效性具有重要价值,为其快速检测提供了可靠的技术支撑。     

蜜蜂克什米尔病毒实时荧光RT-PCR检测方法的建立和评价

蜜蜂克什米尔病毒(Kashmir bee virus,KBV)是一种高毒力的急性蜜蜂病毒,可以引起蜜蜂的死亡和蜂群崩溃.本研究旨在建立一种灵敏、快速检测KBV的TaqMan实时荧光RT-PCR检测方法.参照GenBank中polymerase polyprotein有关基因序列,设计一组特异性引物和探针,并通过体外转录法制备RNA标准品作为阳性模板.在对反应体系进行优化的基础上,建立了 KBV的实时荧光RT-PCR检测方法并进行了特异性试验、敏感性试验、重复性试验和临床样本验证.结果显示:该方法能有效扩增8×100拷贝/μL～8×107拷贝/μL 的KBV标准品,建立的标准曲线呈现良好的线性关系.该方法的检测灵敏度为8拷贝/μL,对其他蜜蜂病毒不发生交叉反应,具有很好的特异性;重复性试验结果显示组内和组间的变异系数分别低于1％和2％,重复性良好.应用本研究建立方法与常规RT-PCR方法对样品进行检测,TaqMan实时荧光RT-PCR检测方法的特异性优于常规RT-PCR.本研究建立的实时荧光RT-PCR检测具有良好的敏感性、特异性和重复性,为KBV的检测和流行病学调查提供技术支持.     

9种呼吸道病毒微流体芯片检测体系构建与应用

近年来,越来越多新的致病性呼吸道病毒被发现,给口岸检验检疫机构的预防和控制造了成极大困扰,建立甲型流感病毒(Influenza A virus,FluA)、乙型流感病毒(Influenza B virus,FluB)、副流感病毒(Human Parainfluenza virus,HPIV)、冠状病毒(Coronavirus,Cov)、呼吸道合胞病毒(Respiratory syncytial virus,RSV)、人偏肺病毒(Humanmetapneumovirus,HMPV)、人博卡病毒(Human bocavirus,Hbov)、腺病毒(Adenovirus,Adv)和鼻病毒(Rhinovirus,Rhv)9种呼吸道病原体微流体芯片检测体系。选择人β-actin基因作为靶基因设计内标引物探针,根据9种呼吸道病原体基因组的保守区设计相应的引物探针;在Ultra Fast Labchip Real-time PCR V280平台上,筛选合适的RT-PCR Master Mix;对扩增体系中引物探针浓度进行优化,用优化后的扩增体系进行灵敏度和特异性验证;用100例临床标本进行回顾性检测。选出具有快速扩增性能的RT-PCR Master Mix,优化后病原体上游引物、下游引物和探针终浓度分别为500nmol/L、500nmol/L和250nmol/L,内标基因上游引物、下游引物和探针终浓度均为300nmol/L、300nmol/L和150nmol/L;在此扩增体系下,9种呼吸道病毒的最低检测限均为1.0×10~3拷贝/mL,不同病毒无交叉反应,对100例临床样本检测,准确率100%。因此,本研究建立了一种可同时用于9种呼吸道病毒快速检测的微流体芯片扩增体系,采用本检测体系可在25min内实现上述9种病毒核酸定性检测。     

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.