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.     

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。     

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。     

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.     

两种DNA探针杂交检测结核分支杆菌方法的研究

为改进结核杆菌DNA探针的特异性与实用性,研制了以生物素标记的两种对结核分支杆菌特异的DNA探针:一个5’端标记的20bp的寡核苷酸探针和一个采用PCR方法合成的188bp长链探针。两种探针分别与结核分支杆菌的全染色体DNA,以及基因组上IS6110序列的一段317bp的PCR扩增产物进行斑点杂交,以碱性磷酸酶(AP)催化的染色反应检测,测试了两个探针的敏感性和特异性。系统地比较研究了两种探针杂交检测条件:探针的浓度选择,杂交温度与洗膜温度的选择,以及杂交与洗膜温度对检测的敏感性与特异性的影响。寡核苷酸探针和188bp探针杂交检测纯化结核分支杆菌基因组DNA的敏感性分别为100ng与6ng,杂交检测PCR产物的敏感性分别是400pg与50pg。两探针的最佳杂交浓度均为40～160ng/ml,最佳杂交温度分别是42℃与68℃,最佳洗膜温度分别是60℃与60～68℃之间。两种探针均仅与结核分支杆菌及BCG有杂交信号,而与其它受试分支杆菌及非分支杆菌杂交结果都呈阴性。它们的特异性都很强,但188bp探针的敏感性约是寡核苷酸探针的7～16倍,而且188bp探针检测本底较低,是检测结核分支杆菌的较佳选择     

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。     

双加氧酶α亚基保守序列克隆及探针制备

制备地高辛标记的环羟基化双加氧酶的α亚基保守序列探针。利用PCR法成功地克隆了环羟基化双加氧酶的α亚基保守序列310 bp片段,并对其进行测序。利用PCR法制备地高辛标记探针,对新标记的探针进行检测,结果显示其标记效率在0.1 pg/μL;敏感性检测表明,对同源DNA的检出限量为100 pg;对提取的副溶血性孤菌质粒DNA、短小芽胞杆菌总DNA杂交均呈阴性,说明该探针具有较强的特异性。     

橡胶树转基因植株Southern杂交体系的优化

以地高辛标记的Southern杂交技术已广泛应用于多个物种中。选择橡胶树为材料,就基因组DNA的提取、探针标记方法的选择以及具体的杂交操作过程等方面对该体系进行了优化。结果表明,至少40μg高质量的DNA在300μL的大酶切体系中,酶切12 h可获得良好效果;PCR法标记的探针杂交条带清晰、背景浅,其效率明显强于随机引物法标记的探针。本研究优化的体系信号强、背景浅和灵敏度高,为橡胶树Southern杂交鉴定分析提供参考。     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

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.     

Composition and diversity of bird communities in Swedish farmland–forest mosaic landscapes

CapsuleThe amount of forest (at local and landscape scales) and occurrence of residual habitats at the local scale are shown to be the major factors influencing bird community composition in farmland–forest landscapes in central Sweden.

Aims To investigate the importance of local habitat and landscape structure for breeding birds in farmland–forest landscapes in central Sweden.

Methods Breeding birds were censused at 292 points. A detailed habitat mapping was made within 300 m of the points. Within a 300–600 m radius only two major habitats (forests and arable fields) were identified.

Results Cluster analyses of bird communities identified three site types that also differed in habitat composition: (i) partially forested sites in forested landscapes; (ii) heterogeneous sites with residual habitats in mosaic landscapes; and (iii) field-dominated farmland sites in open landscapes. A total of 19 of 25 farmland bird species (restricted to farmland or using both farmland and forest) had the highest abundance in farmland sites with mosaics of forest and farmland, while only six farmland species had the highest abundance in field-dominated sites. The bird community changed from being dominated by farmland species to being dominated by forest species (common in forest landscapes without farmland) at small proportions (10–20%) of forest at the local scale. A major difference in habitat composition between heterogeneous and field-dominated sites was the occurrence of different residual habitats (e.g. shrubby areas and seminatural grasslands). These habitats seemed to influence bird community composition more than land-use, despite covering <10% of the area. Seminatural grasslands were important for bird community composition and species-richness, but grazing seemed to be less important. Among different land-use types, cereal crops were the least preferred fields. Set-asides with tall vegetation and short rotation coppices were positively associated with species-richness of farmland birds.

Conclusion In general, the composition of the landscape was important for bird community composition, although amount and distribution of forests, occurrence of residual habitats and land-use of fields at the local scale had the strongest influence on bird community composition. The possible implications of these patterns for managing farmland–forest landscapes are discussed.     

Chemical constituents from the fruits of Rhus typhina L. and their chemotaxonomic significance

This work describes the isolation and characterization of twenty-nine compounds from the fruits of Rhus typhina L., including eleven flavonoids (1–11), eleven phenols (12–22), two pentacyclic triterpenes (23–24), two organic acids (25–26), one lumichrome (27), one courmarin (28) and one pyrimidine (29) on the basis of their spectroscopic data. Compounds apigenin (1), daidzein (4), orobol (5), 3′, 5, 5′, 7-tetrahydroxyflavanone (6), naringenin (7), butein (8), (-)-catechin (9), quercetin-3-O-α-L-(3″-O-galloyl)-rhamnoside (11), 2-hydroxybenzoic acid (13), 4-hydroxybenzaldehyde (14), vanillin (15), methyl 3,4-dihydroxybenzoate (16), 3,5-dihydroxybenzamide (18), tyrosol (19), caffeic acid (20), 3-(2,4,6-trihydroxyphenyl)-1-(4-hydroxyphenyl)-propan-1-one (21), phlorizin (22), friedelin (23), oleanolic acid (24), 4,4-dimethyl-heptanedioic acid (25), anthranilic acid (26), lumichrome (27), scoparone (28) and uracil (29) have not been recorded before in this plant. This is the first report on the occurrence of compounds 4–7, 9, 11, 13–14, 16, 18–21, 25–29 from the genus Rhus. Moreover, the chemotaxonomic significance of these isolated compounds was also summarized.