Spatial Peculiarities in the Colonization of the Plant Rhizoplane by Microscopic Fungi

Spatial peculiarities in the colonization of the tomato, cucumber, and barley rhizoplanes by microscopic fungi were studied. The apical zone of roots was colonized with a limited number of Rstrategists (the order Mucorales, Fusariumsp., Aspergillus niger, and Mycelia sterilia). The fungal population of the root hairs and the basal zone of roots was 2- to 3-fold denser due to the prevalence of Kstrategists. Fusaria, Fusarium oxysporumin particular, colonized roots in earlier terms than the genera Trichoderma, Penicillium, Gliocladium, and others. The F. oxysporumpopulation was at a maximum in the rhizoplane zone nearest the root tip.     

曼陀罗植物内生真菌产生物碱菌株的筛选研究

本文从植物曼陀（Datura arborea）中分离得到77株内生真菌。对它们分别进行菌株发酵,发酵产物经乙醇浸提,采用TLC和生物碱显色方法,观察到菌株Ym311977、Ym312023呈生物碱阳性反应。结合HPLC分析结果,表明该生物碱并非曼陀罗植物中的主要生物碱成份。经鉴定,Ym311977属镰孢菌属（Fusarium sp．）,Ym312023属毛壳菌属（Chaetomium sp．）。     

The Distribution of Microscopic Fungi along Moscow Roads

The distribution of microscopic fungi in soils and surface air along some Moscow roads was studied from 1994 through 2000 at several distances (0, 5, 10, and 20 m) from the roads. Soil fungal complexes in roadside zones with different levels of pollution from automobile engines were found to differ in their composition, species structure, and biomorphology. Relatively far from the roads, the content of fungal mycelium diminished, while that of fungal spores increased. Close to the roads, fungal complexes in soil and surface air were dominated by dark melanin-containing fungi. The microscopic fungal complexes of soil and surface air along the roadsides were similar but differed in the relative abundance of some species.     

Screening for Ligninolytic Enzyme Production by Diverse Fungi from Tunisia

Summary This work represents the first report on the ability of autochthonous fungi from Tunisia to produce ligninolytic enzymes. Three hundred and fifteen fungal strains were isolated from different Tunisian biotopes. These fungal strains were firstly screened on solid media containing Poly R-478 or ABTS as indicator compounds that enabled the detection of lignin-modifying enzymes as specific color reactions. Of the 315 tested strains, 49 exhibited significant ABTS-oxidation activity expressed within the first week of incubation and only 18 strains decolorized the Poly R-478. Liquid cultivations and laccase, manganese peroxidase and lignin peroxidase activity assays of positive strains confirmed that eight efficient enzyme producers were found in the screening. These strains were attributed to the most closely related species using PCR amplification and sequencing of the internal transcribed spacer ‘ITS’ regions of the ribosomal DNA. The identification results showed fungal genera such as Oxyporus, Stereum and Trichoderma which have been only rarely reported as ligninolytic enzyme producers in the literature. Culture conditions and medium composition were optimized for the laccase producer Trametes trogii CTM 10156. This optimization resulted in high laccase production, 367 times more than in non-optimized conditions and which reached 110 U ml^-1 within 15 days of incubation.     

用PCR法直接快速筛查重组阳性克隆

本研究目的是应用PCR法快速筛查插入有苯丙氨酸脱氨酶（PAL）cDNA重组阳性克隆,用于PCR扩增的引物是位于载体pET23b启动子处的T7启动子引物和位于目的基因PALc DNA3’端终止密码TAA处的引物,以灭菌吸头挑一单菌落加PCR体系扩增。结果：在筛查的3个克隆中,有2个阳性克隆,并且插入方向正确,经DNA序列测定得到进一步证实,结果表明,以PCR方法筛查重组阳性克隆,可以简便快速鉴定插入片段的大小和方向,不需提取质粒。     

实验犬布氏杆菌的多重PCR检测与分型鉴定

目的建立实验犬及相关生物制品布氏杆菌的多重PCR检测与分型鉴定方法。方法选择布氏杆菌Omp2基因同源性较高的区域设计引物对布氏杆菌进行多重PCR扩增,扩增结果一致的样本进行酶切以区分不同型,同时进行序列测定,以确定该方法的准确性;然后验证该方法的特异性和敏感性。结果成功扩增得到目的条带,并通过酶切区分五种布氏杆菌;PCR产物与布氏杆菌DNA序列同源性达到99%,并验证了该方法的检测结果。实验结果证明该方法特异性较好,灵敏性为1.8×10^-7μg/mL。结论成功建立布氏杆菌多重PCR检测与分型鉴定方法,所建立的方法特异性好,灵敏度高。本研究对保证实验犬群的质量,保护饲养人员、实验人员的身体健康具有重要意义。     

聚合酶链反应技术检测禽网状内皮组织增殖病病毒

目的建立聚合酶链反应(PCR)技术检测禽网状内皮组织增殖病病毒(REV)的方法。方法提取感染REV-T和脾坏死病毒(SNV)的SPF鸡胚成纤维细胞DNA为模板,利用前病毒长末端重复序列(LTR)区引物进行扩增。采集肿瘤病鸡,以及人工感染REV 28 d后鸡肝脏、脾脏、肾脏、心脏、胸腺、法氏囊等器官,进行扩增。同时将采集的脏器组织,进行HE染色和免疫组化试验(IHC)。结果REV-T感染的组织未检测出电泳条带,而SNV感染的细胞中检测到了一条300bp特异而清晰的电泳条带,而且SNV感染的鸡组织中,PCR方法检测到了特异的条带。通过HE染色和免疫组化技术观察到了肿瘤组织,肿瘤细胞的形态、分布。结论PCR检测REV更快捷,特异更好。     

The Effect of Ecological Factors on Spore Germination and the Viability of the Mycelial Fragments of Microscopic Fungi

Spore germination and the viability of the mycelial fragments of the microscopic fungi Alternaria alternata, Penicillium spinulosum, and Mucor hiemaliswere studied with respect to the action of certain ecological factors: sucrose concentration (0, 0.2, 2, 10, and 100 g/l), temperature (4, 20, 25, and 30°C), pH (3.5, 4.0, 5.0, 6.2, and 7.0), and cadmium concentration (0, 2, 10, and 100 mg/l). The spore germination and the viabilities of different mycelial fragments were found to reach their maxima at different values of the ecological factors studied. This finding suggests that the vegetative and asexual types of reproduction of microscopic fungi may have different ecological optima.     

利用Taq DNA聚合酶对PCR产物直接进行顺序分析

Taq DNA聚合酶具有反应速度快、温度作用范围广及良好的续进性等特点,可视为一种理想的DNA顺序分析酶。本文首先对非对称性PCR扩增过程中单、双链DNA产物的积累情况进行了分析,然后采用标记延伸二步法,对Taq DNA聚合酶的性质及影响因素进行分析。为进一步改进Taq DNA聚合酶测序的方法,本反应建立了“Klenow-型”的直接掺入标记同位素测序法,即在反应液中加入与标记核苷酸相应的一定浓度的冷dNTP。此法不但解决了二步法中引物后部分DNA顺序无法读出的缺点,而且简化了反应步骤,亦能得到令人满意的顺序分析结果,每次可读出至少400碱基的序列。     

用PCR方法定量检测多药抗性基因的表达

肿瘤细胞对化疗药物的抗性是癌症有效化疗的主要障碍。人类细胞中多药抗性基因(MDR1)编码一种p-糖蛋白,后者功能是能量依赖的跨膜药物外输泵,可降低细胞毒药物在胞内的积累。定量分析MDR1表达水平可说明病人抗药能力的高低。本文应用PCR技术建立了灵敏度高、专一性好、可定量检测临床标本中MDR1表达水平的方法。对周血标本的初步检测表明:白血病化疗效果与MDR1表达水平的高低有关。     

The Effect of Hydrogen Peroxide on the Growth of Microscopic Mycelial Fungi Isolated from Habitats with Different Levels of Radioactive Contamination

The effect of hydrogen peroxide (10^?9–10^?1 M) on the mycelial growth of the fungi Alternaria alternata, Cladosporium cladosporioides, Mucor hiemalis, and Paecilomyces lilacinus has been studied. The growth of fungi isolated from habitats with a background level of radioactive contamination was stopped by H₂O₂ concentrations equal to 10^?3 and 10^?2 M, whereas the growth of fungi that were isolated from habitats with high levels of radioactive contamination was only arrested by 10^?1 M H₂O₂. The response of the different fungi to hydrogen peroxide was of three types: (1) a constant growth rate of fungal hyphae at H₂O₂ concentrations between 10^?9 and 10^?4 M and a decrease in this rate at 10^?3 M H₂O₂, (2) a gradual decrease in the growth rate as the H₂O₂ concentration was increased, and (3) an increase in the growth rate as the H₂O₂ concentration was increased from 10^?6 to 10^?5 M. The melanin-containing species A. alternata and C. cladosporioides exhibited all three types of growth response to hydrogen peroxide, whereas the light-pigmented species M. hiemalis and P. lilacinus showed only the first type of growth response. A concentration of hydrogen peroxide equal to 10^?1 M was found to be lethal to all of the fungi studied. The most resistant to hydrogen peroxide was found to be the strain A. alternata 56, isolated from the exclusion zone of the Chernobyl Nuclear Power Plant.     

Polymerase Chain Reaction for the Detection of Chlamydia psittaci in the Feces of Budgerigars

The polymerase chain reaction (PCR) targeting the ompA gene of Chlamydia psittaci was evaluated for its ability to detect chlamydiae in fecal specimens of budgerigars as compared with isolation procedures using cell culture and embryonated egg inoculations. Several procedures for PCR template DNA preparation were compared so as to determine their detection levels for chlamydiae propagated in cell culture in the presence of fecal materials. Tween-20 and proteinase K treatments followed by centrifugation of the template DNA were found to be an appropriate procedure for DNA preparation for primary PCR. Subsequent nested PCR was shown to detect 4.8 IFU/ml or 84 particles/ml of chlamydiae. Chlamydiae in 50 fecal specimens from apparently healthy budgerigars were examined by nested PCR and several other methods. Nested PCR detected chlamydiae at a higher rate (12/50, 24%) than the isolation procedure in embryonated eggs (6/50, 12%). Primary PCR combined with the isolation procedure in cell culture gave a detection rate (5/50, 10%) similar to that of isolation from embryonated eggs. Detection rates by primary PCR (1/50, 2%) and in cell culture (0%) were inferior to the other procedures.     

半嵌套式PCR技术区别检测犬细小病毒疫苗株和强毒株的研究

Two pairs of PCR primers were designed according to the sequances of the vaccine strain and virulent strain of CPV. Heminested PCR method was established. Result of the first PCR amplification showed the same amplified products of 574bp length, after the second PCR amplification, the virulent strain produced the length 364bp fragment, but the vaccine strain couldn' t produce that. The products of PCR were examined by electrophoresis and restriction enzyme digestion. The result showed the length of the fragment and enzyme sites were as the same as those designed. The PCR assay of CPV was proved to be specific and sensitive. It shows that this method may be used in discriminating the vaccine strain and virulent strain of CPV or monitoring the vaccinated canine in order to aviod disease and financial losing.     

Detection of Human Cytomegalovirus DNA in Breast Milk by Means of Polymerase Chain Reaction

Three hundred and twenty-five breast milk samples were examined for the occurrence of human cytomegalovirus (HCMV) by cell culture method. Virus was isolated from the milk in 1 of 177 samples collected within 6 days after delivery, 2 of 115 samples collected during the period of 7 days to 1 month after delivery, 10 of 33 samples collected over 1 month after delivery. Next, we tried to amplify HCMV DNA from the breast milk samples from HCMV seropositive mothers and seronegative mothers at 1 month after delivery by polymerase chain reaction. HCMV DNA was detected in 12 of 13 samples from seropositive mothers and in none of 7 samples from seronegative mothers. It was thought that all women seropositive for HCMV principally shed the virus into their breast milk at 1 month after delivery.     

应用差异PCR技术研究MTX耐药细胞的遗传学改变

基因扩增是介导细胞产生耐药性的一种普遍性机制。为探讨MTX耐药的小鼠细胞中与耐药机制形成有关的分子遗传学背景,应用差异PCR技术,检测了MTX耐药细胞中DHFR基因的扩增和过表达,并对c-myc及p53基因状态与dhfr扩增之间的相关性进行了探讨。结果表明：dhfr的扩增和过表达直接介导细胞耐药。未检测到c-myc的扩增和p53拷贝数的变化;也未检测到c-myc mRNA水平的改变,但p53基因的mRNA水平明显升高,显示p53可正常表达。这些结果表明dhfr的扩增与这两个侯选基因的状态无关,提示该耐药细胞中可能存在其他的分子遗传学改变允许基因大量扩增。     

Detection of Various Variant Verotoxin Genes in Escherichia coli by Polymerase Chain Reaction

We constructed common primers for the polymerase chain reaction to detect the genes for various Verotoxins reported, that is, VT1 (or SLT-I), VT2 (or SLT-II), VT2vha, VT2vhb, SLT-IIv (or VT2vp1, VTe) and SLT-IIva (or VT2vp2). A total of 80 Verocytotoxin-producing Escherichia coli strains isolated from humans, domestic animals and meats gave a positive result by PCR with the designed common primers. Digestion by restriction endonucleases BglII and EcoT14I of the amplicon of the VT2vp2 gene gave specific bands of the expected sizes, but not of the amplicons of other VT genes, suggesting a possible method for identification of the VT2vp2 gene. Application of the PCR with the designed primers in diagnostic and epidemiological studies on VTEC infection is also discussed.     

Sperm Mediated Transfer of Genes into Goldfish and Detection by Polymerase Chain Reaction

Goldfish sperms were mixed with eggs for fertilization after incubation with antifreeze protein gene(AFP)from ocean pout for 30 min.A number of embryos and 145 adult goldfish were obtained.DNA from adult goldfish and embryos was extracted separately.Results of the amplification by PCRand Southern blot molecular hybridization indicate the integration of exogenous antifreeze gene intothe genome of a part of the recipient goldfish.Of the 45 samples detected by PCR,twelve showedpositive reaction with distinct hybridization band.The positive rate was 26%.     

用于聚合酶链式反应(PCR)引物设计的计算机程序

 本文报道了两个用于PCR引物设计的计算机程序PCRDESN和PCRDESNA。PCRDESN程序主要从以下4个方面评价用户自己设计的一对引物的质量:(1)引物内的碱基反向重复或发夹结构,(2)两个引物之间的碱基互补配对,(3)两个引物之间的同源性,(4)引物的碱基组成及特点和T_m值计算。通过用多例文献发表的及本院有关实验室提供的引物对序列的验证,确定了程序的运算参数,证明该程序能较好地检验引物对的质量和解释某些PCR实验失败的原因。PCRDESNA程序采用逐级优化的方法和比PCRDESN所选用的更严紧的引物选择参数对用户提供的核酸序列进行快速检索,以确定所有可能的和合适的引物对。     

New Buffers to Improve the Quantitative Real-Time Polymerase Chain Reaction

Real-time PCR is a potent technique for nucleic acid quantification for research and diagnostic purposes, the wide dynamic range being one of the advantages over other techniques like the microarray. Several additives and enhancers have been studied to expand the PCR dynamic range in order to be more efficient in quantifying low quantities of nucleic acids, increase the yield and improve reaction efficiency. Shown here is that a combination of new buffers with the regularly used Tris buffer makes it possible to expand the real-time PCR dynamic range and to improve the efficiency and correlation coefficient. Mixing HEPES, TEA or MOPS with Tris was more efficient than Tris alone. It was also found that, if the pH value of the Tris buffer was calibrated with phosphoric acid instead of hydrochloric acid, then the dynamic range was significantly improved and low quantities could be detected and quantified more efficiently. Mixing more than one compound with the Tris buffer was also effective for expanding the dynamic range and increasing the efficiency and correlation coefficient in quantitative real-time PCR.