犬细小病毒可视化LAMP检测方法的建立

目的:建立犬细小病毒(CPV)可视化环介导等温扩增(LAMP)检测方法。方法:比对77个不同犬细小病毒全基因组数据,从中找出一段433 bp的高度保守序列作为检测对象,通过基因合成得到含该序列的质粒,制备1×10~3、1×10~2、1×10~1拷贝/μL的质粒作为标准品,并设计一套能扩增这段序列的LAMP引物。结果:首先采用实时荧光定量LAMP扩增,确定了设计的引物能够扩增质粒标准品,最低检测浓度为1×10~1拷贝/μL;然后用pH敏感指示剂中性红进行可视化LAMP扩增检测,肉眼观察到扩增反应液变为紫红色即判断为阳性,最低检测浓度同样为1×10~1拷贝/μL;随后采用实时荧光定量LAMP和可视化LAMP测试50份犬细小病毒疑似临床DNA样品,检测出阳性样品35个,与国标诊断结果完全吻合;将阳性样品扩增后测序,经比对,确定为犬细小病毒序列;对金黄色葡萄球菌、宋内志贺菌、坂崎肠杆菌、单增李斯特菌、大肠杆菌DNA进行扩增,结果均为阴性,证明了该方法的特异性。结论:建立了高度特异和灵敏的犬细小病毒可视化LAMP检测方法。     

圈养小熊猫几种病毒的PCR检测

本文采用巢式PCR/ RT-PCR 方法,对我国10 个动物园中无临床症状的圈养小熊猫的71 个肛拭子和61 个唾液拭子样品,进行犬瘟热病毒(CDV)、犬细小病毒(CPV)、犬冠状病毒(CCV)、犬腺病毒(CAV)和犬疱疹病毒(CHV)的检测,以评估我国圈养小熊猫是否面临这几种病毒的威胁。对阳性PCR 结果进行测序分析,并与GenBank 上的序列进行比较。结果,在肛拭子样品中检测到3 个CPV 和6 个CCV 阳性结果,经测序后,与GenBank 上序列的同源性分别达99% 和100% 。而在唾液拭子样品中没有检测到任何阳性结果,且CDV、CAV和CCV 的检测结果均为阴性。从阳性CPV 的肛拭子样品中分离到一株细小病毒毒株,表明圈养小熊猫已受到细小病毒和犬冠状病毒的感染,今后应加强这两种病毒的预防工作。本文所采用的PCR 方法检测病毒性疾病,能检测到微量的病毒模板,可对小熊猫病毒性感染进行早期诊断。     

犬瘟热和犬冠状病毒联合RT-PCR检测方法的应用

塔里木农垦大学乔军、解放军大学夏咸柱、东北农业大学何宠彬等对此课题进行了研究。他们利用犬瘟热(CDV)和犬冠状病毒(CCV)牧异性引物对同一样品中CDV和CCV的RNA模板进行联合RT—PCR扩增,并优化其扩增条件,得到两条大小与试验设计完全相符的特异性扩增带,且不扩增犬细小病毒、犬腺病毒、犬副流感病毒、轮状病毒的核酸。其敏感性试验表明：该联合PCR能检测出10^-4倍稀释的CDV模板(10^6.6TCID50／0．1mL)。对7份临床发病犬病科检测,其阳性检出率可高于电镜负染检查。这表明此法有高度敏感、特异、准确、快速等特点,适用于当前民用和军用兽医临床对CDV、CCV的检测和对犬的其他疾病的鉴别诊断。     

EBOV-Z和MARV的NP基因在杆状病毒表达系统的表达及反应原性鉴定

利用昆虫杆状病毒表达系统,成功地对埃博拉(EBOV)扎伊尔型病毒和马尔堡病毒(MARV)的NP基因进行表达。Western blot结果显示,重组EBOV蛋白和重组MARV-NP与各自阳性血清有特异的反应原性,在血清学上无交叉反应。IFA检测感染重组昆虫杆状病毒的Sf9细胞表明,EBOV和MARV NP获得大量表达,呈现强烈的荧光,对照组细胞无特异荧光。该研究为EBOV和MARV流行病学调查和研制诊断试剂盒奠定了基础。     

猪细小病毒LDR-PCR检测方法的建立和应用

为准确特异灵敏地检测猪细小病毒(PPV),建立一种新的LDR-PCR方法。首先在病毒的保守区内设计一对LDR探针,LDR探针两端各连有一段引物对应序列,以连接产物为模板进行PCR,琼脂糖凝胶电泳检测结果。以标准质粒为模板,通过对LDR反应的退火温度、连接酶浓度及探针浓度等反应条件进行优化,确定了LDR最佳的反应体系,并建立了LDR-PCR方法。结果表明,可以特异地检测PPV,与猪繁殖与呼吸障碍综合征病毒(PRRSV)、猪瘟病毒(CSFV)、伪狂犬病毒(PRV)、猪圆环病毒(PCV)、猪传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)无交叉反应;最低检测限为102个拷贝。利用建立的方法对41例临床样本进行检测,14份样品PPV阳性,与普通PCR检测结果符合率为97.6%。     

多重PCR检测PRV、PPV、PCV方法的建立及其应用

根据GenBank上已发表的猪伪狂犬病病毒(PRV)的gE、gI基因序列、猪细小病毒(PPV)的E蛋白的基因序列、II型猪圆环病毒(PCV-2)的ORF2基因序列,分别设计并合成3对能特异性扩增PRV、PPV、PCV-2的引物,通过DNAstar软件分析这3对引物不存在cross d im er。建立了PCR方法分别检测PRV、PPV、PCV-2,然后通过条件的优化,建立了PCR同时检测PRV、PPV、PCV-2的方法并研制出试剂盒。对试剂盒的特异性和有效期进行了研究。结果表明,该试剂盒具有很好的特异性,有效期在-20℃至少可以保存1年。     

一种快速提取植物病毒DNA的方法

介绍在PCR检测体系中一种快速提取病毒DNA的方法。利用高盐缓冲液溶液释放病毒DNA,同时利用葡聚糖凝胶微柱纯化提取液,有效消除样品中PCR抑制物质并直接作为PCR反应模板扩增检测病毒。该法无需任何特殊设备,适合对大量植株进行大通量的检测分析。     

痘病毒科不同病毒属特异的PCR检测方法的建立

目的:建立可准确、快速地鉴别诊断可感染人的不同属痘病毒的特异PCR方法。方法:设计针对正痘病毒属、副痘病毒属和传染性软疣病毒属的多对特异引物,并制备相应的DNA模板,针对不同的模板优化引物与反应条件,分别进行检测筛选,建立病毒属特异的单独与多重PCR方法。结果:单一模板的PCR扩增反应中,正痘病毒的检测敏感性可达101拷贝/μL(引物为OPEaL-F1880/OPEaL-R2057),副痘病毒的检测敏感性可达101拷贝/μL(引物为PP2/PP3),传染性软疣病毒的检测敏感性为100 pg/μL体系(引物为MCV1/MCV2);混合模板的PCR扩增反应中,各属特异的引物均可获得预期大小的特异片段。结论:我们建立的PCR诊断方法,可用于痘病毒科不同病毒属感染的实验室特异快速鉴别诊断。     

国内信息

犬瘟热和犬冠状病毒联合RT PCR检测方法的应用塔里木农垦大学乔军、解放军大学夏咸柱、东北农业大学何宠彬等对此课题进行了研究。他们利用犬瘟热(CDV)和犬冠状病毒(CCV)牧异性引物对同一样品中CDV和CCV的RNA模板进行联合RT PCR扩增,并优化其扩增条件,得到两条大小与试验设计完全相符的特异性扩增带,且不扩增犬细小病毒、犬腺病毒、犬副流感病毒、轮状病毒的核酸。其敏感性试验表明:该联合PCR能检测出104倍稀释的CDV模板(106.6TCID50/0.1mL)。对7份临床发病犬病科检测,其阳性检出率可高于电镜负染检查。这表明此法有高度敏感…     

猫肾F_(81)传代细胞中犬细小病毒原位PCR检测方法

目的　建立在猫肾F81 细胞中犬细小病毒原位PCR的检测方法。方法　在猫肾F81 细胞上感染犬细小病毒 ,设计特异性引物 ,用直接原位PCR法在染毒 12h ,2 4h ,48h细胞片上检测出犬细小病毒 ,并与常规免疫组化的方法进行了比较。结果　在染毒 48h的细胞片上 ,用阳性记分法将两种检测方法得到的阳性细胞进行统计比较 ,差异极显著 (P <0 .0 0 1) ,用原位PCR法所得出的阳性率高。结论　原位PCR法检测犬细小病毒具有敏感性高和组织定位的优点     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.     

真菌类遗传学分析的知识结构教学

本文以认知结构理论为指导,讨论了真菌类遗传分析与高等动植物遗传分析的内在联系,认为利用这种内在联系进行教学可收到好的效果并说明了作者的具体教学过程。 Abstract:In the paper, the relationship between genetic analysis of Fungi and genetic analysis of high animal and plant was discussed.A good results were obtained when we adopted this method in the teaching.     

龙胆科药用植物化学成分的研究现状

龙胆科植物在我国的分布范围很广,且多数为药用植物,其多数种属的药用植物,至今其化学成分尚未被系统研究。综述了目前龙胆科药用植物的化学成分的研究现状及一般提取方法,对近年来发现的环烯醚萜及裂环烯醚萜类化合物进行了总结,为本科药用植物的更深入研究提供了参考。     

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Summary Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l⁻¹ glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l⁻¹) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.     

Specificity of action of piperidylcholine derivatives as substrates of cholinesterases of various origin

The review deals with study of enzymologic properties of a novel highly specific acetylcholinesterase substrate, N-(β-acetoxyethyl) piperidinium iodomethylate (“piperidylcholine”), and its 30 derivatives that were tested as effectors of cholinesterases of mammals and various species of Pacific squids. It was proven for the first time that responsible for specificity of action was structure of cyclic ammonium grouping of the alcohol part of molecule of the ester substrate. Analysis of specificity is performed based on enzymatic hydrolysis parameters—activity of catalytic center of cholinesterases and bimolecular constant of the reaction rate that are determined at optimal and low substrate concentrations. Among the specially synthesized group of thioester compounds there is revealed one more highly specific acetylcholinesterase substrate—N-(β-acetoxyethyl) piperidinium.     

Interconnection of parameters of regulation system of lipid peroxidation and of morphophysiological parameters of mouse liver

A complex analysis of seasonal fluctuations of the mean group parameters of the system of regulation of lipid peroxidation has been performed in liver of Balb/c mice. Association of lipid characteristics and morphophysiological parameters is studied in the Balb/c mouse liver. An inter-connection is revealed between the liver index and the amount of lysoforms of phospholipids, the scale and character of the interconnection differing essentially depending on proportion of phos-phatidylcholine in mouse liver phospholipids.