PCR-微孔板杂交-ELISA法检测巨细胞病毒感染

建立PCR-微孔板杂交-ELISA法检测人血清标本中巨细胞病毒DNA。将5’端标记生物素的PCR扩增产物与5’端标记地高辛的探针呈液相混合,90℃ 2min,55℃,1min杂交。杂交后产物被链霉亲和素酶标板固定,经酶标记抗地高辛标记抗体结合显色。本试剂检测灵敏度为2.5×104 copies/mL,比传统PCR和ELISA敏度性高,特异性强,与单纯疱疹病毒Ⅰ型和Ⅱ型,风疹病毒、EB病毒、腺病毒核酸无交叉反应,批内CV为8.9%,批间CV为10.5%。该法可用于定性和定量检测HCMV DNA。     

核酸诊断技术规范化的研究

针对PCR技术应用中的一些问题,从下述几方面开展了核酸诊断技术标准化的研究:①探讨了设计引物的影响因素,发现根据同一基因设计的不同引物对PCR敏感性影响较大,可相差20倍,进一步研究表明,引物的自由能,尤其是引物3'端6个碱基的自由能可能决定着PCR的敏感性;②研制成功室温稳定的PCR试剂,将所有PCR成分(加入一种酶保护剂)配制并分装于微量离心管中,并干燥,试剂室温放置8个月,37℃破坏2周对检测结果无任何影响;③因尿嘧啶糖基化酶(UDG)可特异降解DNA双链中的尿密啶核苷,PCR体系中以dUTP代替dTTP,并在PCR扩增前用UDG消化15 min,就可以特异防止PCR产物的污染.对炭疽杆菌、鼠疫杆菌、土拉杆菌和布鲁氏菌扩增结果表明,0.1 U的UDG可完全消化103～108个产物分子的污染;④建立了微孔板杂交技术代替电泳方法检测扩增产物,将PCR产物克隆作为捕获探针包被聚乙烯微孔板,PCR引物5'末端生物素标记,扩增后作为待检测探针杂交,通过比较影响微孔板杂交的包被、杂交和显色等因素,建立了PCR-EIA技术.通过比较包被缓冲液发现镁离子及其离子强度是影响DNA包被的重要因素,包被的DNA以线型质粒最佳,每孔包被300ng左右的DNA 杂交效果最好;杂交液中加和不加甲酰胺对杂交影响不大;用链霉亲和素化的辣根过氧化物酶或碱性磷酸酶酶联显色均能达到检测杂交体的目的,但前者显色较快,更为实用.确定了PCR-EIA技术的最佳的包被、杂交和显色的条件,比常规的PCR-电泳检测敏感性高10倍;⑤比较了不同标本处理方法,确定了各种临床标本和动物组织的较为理想的处理方法.通过上述不同方面的研究,确定了从引物设计、试剂配制、标本处理、UDG防污染和产物微孔板杂交-酶联显色检测等方面的优化条件,建立起敏感性高、特异性强、操作简便和检测客观的核酸检测技术,为临床实验室的常规应用和反生物战病原微生物的核酸诊断奠定了基础.     

荧光MAPREC分析Ⅰ型Sabin株脊髓灰质炎病毒突变率方法的建立和应用

目的使用CY5荧光标记引物建立定量检测I型Sabin株脊髓灰质炎病毒480-A和525-C突变率的MAPREC。方法提取I型Sabin株脊髓灰质炎病毒样品RNA,反转录合成c DNA后,进行非对称PCR扩增以生成单链DNA,之后采用CY5荧光标记引物进行一步扩增反应以合成双链产物,以DNA内切酶Dde I和Nci I酶切后电泳分离,获取荧光信号,计算样品的突变率。检测4个国际标准品(100%DNA突变标准品、IS DNA标准品、LMVR标准品和HMVR标准品)的突变率,重复5次,验证方法的准确性和精密度,并进行初步应用。结果使用CY5荧光标记引物,建立了定量检测I型Sabin株脊髓灰质炎病毒480-A和525-C突变率的MAPREC。100%DNA突变标准品、IS DNA标准品、LMVR标准品以及HMVR标准品的标示值分别为100.00%、2.00%、1.84%、2.56%,检测结果分别为95.09%、2.06%、1.45%、1.92%,各标准品的实验间CV值均<15%。I型Sabin株口服脊髓灰质炎减毒活疫苗工作种子、单次收获液和原液的突变率为1.05%~1.22%,批间一致性良好。结论初步建立了基于荧光素标记的MAPREC,可以代替同位素法进行I型Sabin株脊髓灰质炎病毒突变率的定量检测。     

地高辛标记DNA探针杂交法检测Vero细胞DNA残留量的适用性验证及应用

目的对地高辛标记DNA探针杂交法检测人用狂犬病疫苗(Vero细胞)DNA残留量进行适用性验证及应用。方法对地高辛标记DNA探针杂交法检测人用狂犬病疫苗(Vero细胞)DNA残留量进行特异性、灵敏度及稳定性验证,并应用该方法检测3批人用狂犬病疫苗(Vero细胞)的DNA残留量。结果地高辛标记探针的标记效率为0.1 pg。验证结果显示探针与非同源DNA无杂交;最低检测限度为1 pg;探针在-20℃放置7个月后,检测灵敏度仍可达到1 pg;3批人用狂犬病疫苗(Vero细胞)中残留DNA含量均符合《中国药典》规定质量控制标准。结论地高辛标记DNA探针杂交法特异性、灵敏度好,结果稳定,适用于人用狂犬病疫苗(Vero细胞)中Vero细胞DNA残留量的检测及疫苗生产过程和其成品的质量控制,对其他以Vero细胞为基质的病毒性疫苗质量控制具有借鉴意义。     

膜芯片检测A组轮状病毒的初步研究

建立表面带正电荷的尼龙膜为基片的基因芯片,采用RT-semi-nested PCR方法,对A组轮状病毒RNA实现高度扩增,并通过5'端带地高辛标记的上游引物实现扩增产物的标记.通过同膜芯片上探针的杂交和免疫显色,实现对A组轮状病毒的检测.结果表明,膜芯片对探针的固定效果、杂交吸脱和检测结果可靠,空白对照和阴性对照均为阴性,探针均显示为阳性信号,并且信号强弱与探针浓度关系不大,而主要与探针本身同互补链的结合相关.以上结果说明已经初步建立了快速检测A组轮状病毒的膜基因芯片检测技术.     

转基因甘蔗植株Southern杂交体系的优化北大核心CSCD

对甘蔗Southern杂交体系的优化,旨为转基因甘蔗Southern杂交鉴定分析提供参考。以转基因甘蔗为材料,就探针不同标记方法的比较、甘蔗基因组DNA的提取、基因组DNA酶切量、酶切时间及杂交过程等方面,对地高辛标记的Southern杂交技术进行了优化研究。结果表明,改良的CTAB法提取的甘蔗DNA能满足后期实验的要求;PCR法标记探针的效率较随机引物法标记探针的效率高,更适合用于Southern杂交;40μg的DNA在400μL酶切体系中,酶切10 h可获得良好的酶切效果;杂交温度40℃,杂交18 h,可获得清晰的杂交条带。     

转基因甘蔗植株Southern杂交体系的优化

对甘蔗Southern杂交体系的优化,旨为转基因甘蔗Southern杂交鉴定分析提供参考。以转基因甘蔗为材料,就探针不同标记方法的比较、甘蔗基因组DNA的提取、基因组DNA酶切量、酶切时间及杂交过程等方面,对地高辛标记的Southern杂交技术进行了优化研究。结果表明,改良的CTAB法提取的甘蔗DNA能满足后期实验的要求;PCR法标记探针的效率较随机引物法标记探针的效率高,更适合用于Southern杂交;40μg的DNA在400μL酶切体系中,酶切10 h可获得良好的酶切效果;杂交温度40℃,杂交18 h,可获得清晰的杂交条带。     

cDNA文库法在HCV-1b检测芯片研制中的应用

制备丙型肝炎病毒(HCV) 1b亚型诊断芯片并进行初步验证评价.采用cDNA文库法制备探针,用限制性内切酶Sau3AⅠ消化HCV 1b全长cDNA ,所得的酶切片段72℃补平加A ,AT克隆,PCR初步鉴定,并测序.将筛选出的片段打印在氨基修饰的玻片上制备成检测芯片并进行杂交验证分析.运用cDNA文库法,得到2 2个大小相对一致(2 5 0～75 0bp)的基因片段.序列分析表明,均属于HCV 1b基因,可以作为诊断芯片探针;样品标记采用限制性显示(restrictiondisplay ,RD)技术,标记后进行杂交.杂交结果显示,样品和诊断基因芯片杂交的敏感性和特异性均佳.批内和批间精密度CV值分别为5 4 %和6 8% ,表明用cDNA文库法收集片段是一种快速、简便制备芯片探针的实用方法.     

荧光定量PCR检测人巨细胞病毒的方法学建立

目的建立人巨细胞病毒（HCMV）的TaqMan MGB探针荧光定量PCR（FQ—PCR）检测方法。方法选取HCMV MIE exon4为PCR扩增靶序列,经TA克隆构建重组质粒作为定量标准品,经FQ—PCR反应条件的优化及方法学评价,再将其应用于临床检测。结果FQ—PCR最适循环参数为：95℃ 5 min;95℃ 20 s,60℃ 60 s（40 cycles）,20μl最适反应体系为：2．0mmol／L Mg^2＋、0．5μmol／L引物、1．5μmol／L探针、200μmol／L dNTP、2110×buffer、1．0 U Taq酶、2．0μl DNA模板。检测批内CV（变异系数）值为1．32％,批间CV值为1．96％;特异性较好;线性范围为10^2-10^8copies／μl。结论成功地建立了检测HCMV的FQ—PCR法,完全适用于临床检测。     

磁珠富集法分离小黄李基因组微卫星DNA片段

将微卫星探针5′端生物素化后与链亲和素磁珠特异结合,用磁珠和探针的结合物与两端连接已知序列人工接头的中国李品种小黄李(Prunus salicinacv.Xiaohuangli)基因组DNA酶切片段杂交,以此杂交片段为模板用人工接头序列为引物进行PCR扩增,根据PCR产物测序结果设计引物作为微卫星DNA的标记引物.结果在随机挑选的36个克隆进行菌落PCR检测时,从31个阳性克隆中挑选18个克隆进行测序后获得了12条特异序列,设计的8对SSR引物均在5个中国李受试品种上获得了预期的扩增产物,其中4对引物在受试品种上表现出多态性.     

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.