高温环境样品总DNA直接和间接提取方法的比较

分别采用两种环境总DNA直接提取法和一种间接提取法从6种温泉菌席样品中提取总DNA,以DNA粗产物的纯度、能否用于后续PCR扩增及PCR-DGGE(变性梯度凝胶电泳)所反映的微生物多样性为评价指标对两类方法进行比较和评价。研究发现,虽然间接提取法效率低下,但对于高温极端环境中生物量较小的样品,间接法能得到有研究价值的、纯度较高的环境样品总DNA,而直接法得到的DNA量小且不适于PCR扩增操作。在使用这2类方法都能得到可用于研究操作的DNA的情况下,间接提取法能更好的体现环境样品中微生物的多样性。     

Estimation of deciduous forest leaf area index using direct and indirect methods

This study evaluated one semi-direct and three indirect methods for estimating leaf area index (LAI) by comparing these estimates with direct estimates derived from litter collection. The semi-direct method uses a thin metallic needle to count a number of contacts across fresh litter layers. One indirect method is based on the penetration of diffuse global radiation measured over the course of a day. The second indirect method uses the LAI-2000 plant canopy analyser (PCA) which measures diffuse light penetration from five different sky sectors simultaneously. The third indirect method uses the Demon portable light sensor to measure the penetration of direct beam sunlight at different zenith angles over the course of half a day. The Poisson model of gap frequency was applied to estimate plant area index (PAI) from observed transmittances using the second and third methods. Litter collection from 11 temperate decidous forests gave values of LAI ranging from 1.7 to 7.5. Estimates based on the needle method showed a significant linear relationship with LAI values obtained from litter collections but were systematically lower (by 6–37%). PAI estimates using all three indirect techniques (fixed light sensor system, LAI-2000 and Demon) showed a strong linear relationship with LAI derived from litter collection. Differences, averaged over all forest stands, between PAI estimates from each of the three indirect methods and LAI from litter collections were below 2%. If we consider that LAI=PAI–WAI (wood area index) then, all three indirect methods underestimated LAI by an additional factor close to the value of WAI. One reason could be a local clumping of architectural canopy components: in particular, the spatial dispositions of branchlets and leaves are not independent, leading to a non-random relationship between the distributions of these two canopy components.     

Early detection and rapid identification of Streptococcus pneumoniae and Haemophilus influenzae in acute pneumonias

The comparative study of the diagnostic value of the enzyme immunoassay (EIA), indirect immunofluorescence (IF) and countercurrent immunoelectrophoresis (CIE) was made. The serological identification of the isolated and reference pneumococci (19) and H. influenzae (38) strains revealed the possibility of using all three microanalytical methods for this purpose. The study of pneumococcal and H. influenzae antigens in native sputum obtained from 74 patients with acute pneumonia showed that EIA and indirect IF were highly sensitive, their sensitivity considerably exceeding that of the bacteriological analysis. Pneumococcal antigens were detected in 66.2% of patients by EIA and in 54.0% of patients by indirect IF, while H. influenzae antigens were detected in 58.1% of patients by EIA and in 67.6% of patients by indirect IF. The sensitivity of CIE proved to be considerably lower; in the detection of pneumococcal antigens it was level with the sensitivity of the bacteriological analysis (23.0%) and H. influenzae antigens could be detected only in 27.0% of patients.     

Immunohistochemical demonstration of keratins in human ovarian neoplasms. A comparison of methods

A comparison of five immunohistochemical methods for the demonstration of keratins in human ovarian neoplasms using affinity-purified polyclonal rabbit antibody was made. The use of indirect immunofluorescence on frozen sections briefly fixed in acetone was found to be the most sensitive method and demonstrated keratin in all 14 primary and 1 metastatic ovarian epithelial neoplasms studied. Protein A-peroxidase, peroxidase--antiperoxidase (PAP), indirect peroxidase, or the avidin--biotinylated peroxidase complex (ABC) methods applied to formalin-fixed tissues were less sensitive and led to false negative results in 9 of 15, 1 of 15, 8 of 15, and 6 of 15 cases, respectively. A single case of dysgerminoma failed to reveal keratin by any method.     

Modeling Human Exposure to Phthalate Esters: A Comparison of Indirect and Biomonitoring Estimation Methods

Humans are potentially exposed to phthalate esters (PEs) through ingestion, inhalation, and dermal contact. Studies quantifying exposure to PEs include “biomarker studies” and “indirect studies.” Biomarker studies use measurements of PE metabolites in urine to back-calculate exposure to the parent diester, while indirect studies use the concentration of the PE in each medium of exposure and the rate of intake of that medium to quantify intake of the PE. In this review, exposure estimates from biomarker and indirect studies are compiled and compared for seven PEs to determine if there are regional differences and if there is a preferred approach. The indirect and biomarker methods generally agree with each other within an order of magnitude and discrepancies are explained by difficulties in accounting for use of consumer products, uncertainty concerning absorption, regional differences, and temporal changes. No single method is preferred for estimating intake of all PEs; it is suggested that biomarker estimates be used for low molecular weight PEs for which it is difficult to quantify all sources of exposure and either indirect or biomarker methods be used for higher molecular weight PEs. The indirect methods are useful in identifying sources of exposure while the biomarker methods quantify exposure.     

Methods and Errors in Measurements of Synovial Fluid Volume in Stifles with Low Volume and High Viscosity Synovial Fluid. An Experimental Study in Goats

Synovial fluid (SF) volume was calculated using various methods in the stifles of goats, in which the cranial cruciate ligament had been transected on one side. Measurements were performed prior to surgery and again 4,8, and 18 weeks following surgery, by measuring the dilution of an injected radioactive tracer diluted by the SF. Later, 7 months following surgery, SF volume measurements using simple arthrocentesis were performed on stifles in 9 of the goats, and the SF that could not be aspirated, was calculated using 2 indirect methods simultaneously on identical fluids in 3 of these goats. SF was also collected directly during staged arthrotomy of the stifles in 4 goats. There were conflicting results between methods, but the resulting calculated SF volumes seemed to be larger in the operated stifles compared to the controls for all the methods at about the same degree. The 2 indirect methods used to calculate the fluid remaining in the joints following arthrocentesis gave disparate volume calculations. The experiments revealed sources of error in all methods. Direct methods failed to acquire the total fluid volume, and indirect methods were subject to improper mixing and escape of the injected fluid or synovial fluid or both. It was concluded that none of the methods could be used to measure the “true” volume of SF, if such a concept exists and can be defined. None of the methods were considered reliable to compare volumes in different type of joints containing this type of fluid. It was, however, concluded that all the methods gave indication of increased SF volume present on a relative basis when paired joints were compared.     

Comparing direct and indirect methods to estimate detection rates and site use of a cryptic semi-aquatic carnivore

Monitoring animal populations can be challenging, particularly when working with species that are cryptic, rare, or occur at low densities. The northern river otter (Lontra canadensis) is a cryptic, semi-aquatic carnivore that has been intensively studied in recent decades, yet much of what is known about its ecology is a result of studies that have employed indirect methods of detection and monitoring. These indirect methods, such as latrine or other sign surveys, have been the primary approach used for studying distribution, abundance, and habitat use of otters, with minimal representation of direct methods. In this study, we compared direct (camera traps) and indirect (scat count surveys) methods of evaluating detection probabilities and site use patterns of otters at latrines. We found that the direct method produced a significantly greater monthly detection probability than the indirect method and that camera surveys resulted in fewer occurrences of false negatives than scat surveys. However, the number of scats deposited at a site was positively correlated with number of visits by otters at a site (Tau-b = 0.675). Thus, while cameras outperformed scat counts in terms of detection, the two methods were comparable in determining intensity of site use. We conclude that, depending on the parameter of interest, scat counts may be an acceptable surrogate for more direct methods of monitoring otters and other cryptic species. We caution, however, that in the absence of comparative methodological data, direct methods such as camera trapping should be preferred when making inferences about animal distribution, abundance, or habitat use.     

On the evaluation of productivity in hydrocarbon fermentation

Direct and indirect methods of the assessment of growth rate (productivity) in hydrocarbon fermentation are briefly reviewed. The application of an indirect method, based on alkali consumption at constant pH, is discussed from the viewpoint of varying protein content in the biomass dry weight. An automatic control of substrate addition based on the indirect method is suggested.     

Immunohistochemical localization of human carbonic anhydrase isoenzyme C.

Methods for immunohistochemical localization of human carbonic anhydrase isoenzyme C (HCA C) with indirect fluorescent antibody and immunoperoxidase techniques are described. Both methods revealed large amounts of this "high activity" isoenzyme in the mucosae of human stomach and appendix. With the indirect immunofluorescent method the presence of the enzyme in human erythrocyte cytoplasm was also demonstrated. Correlations of present findings with those obtained with the traditional histochemical methods for demonstration of carbonic anhydrase activity are discussed.     

Synthesis of multiple antigenic peptides (MAPs)—strategies and limitations

Dendrimeric platforms such as MAPs can be synthesized either entirely by solid‐phase methods (SPPS, direct approach) or by conjugation in solution of preformed, SPPS‐made building blocks (indirect approach). Although MAPs and MAP‐like constructs have been extensively and successfully used for various biological (mainly immunological) applications, experimental reports are most often lacking in chemical detail about their preparation and characterization. Here, we provide complete accounts of the synthesis and analytical documentation of MAPs and similar dendrimers by either all‐SPPS (direct) or chemoselective thioether ligation (indirect) methods. We have chosen as model epitopes a 24‐residue sequence of the ectodomain of protein M2 from influenza virus (M2e), which is found to be a rather challenging peptide epitope, and a far more manageable, shortened (12‐residue) version of the same peptide. The advantages and shortcomings of both direct and indirect methods are discussed. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Immunocytochemistry of cerebrospinal fluid

In order to determine how best to study cells in cerebrospinal fluid (CSF) by immunocytochemical techniques, several crucial technical variables and five immunocytochemical methods were examined. Immunocytochemical studies could be performed on either cell suspensions or smears. The method using cell suspensions was more sensitive, producing less background staining, but requiring more cells than that using smears. Among the five methods examined, indirect immunoperoxidase (IP) and indirect immunoalkaline phosphatase (IAP) were comparable in sensitivity. The peroxidase-antiperoxidase (PAP), alkaline phosphatase-antialkaline phosphatase (APAAP) and avidin-biotin complex-immunoalkaline phosphatase (ABC-AP) methods were comparable in sensitivity and were more sensitive than either the IP or IAP technique. The peroxidase methods were plagued with problems related to endogenous enzyme activity and the ABC-AP method may exhibit undesirable background staining. Therefore, the IAP method should be used for cell suspensions and the APAAP for cells on smears. In CSF specimens with a small number of cells, immunocytochemical studies should be done on smears by the APAAP method. These conclusions are supported by our experience with CSF specimens from patients with reactive and neoplastic lymphocytoses.     

The separation of enantiomeric sugars by chromatographic methods

This paper has reviewed the number of chromatographic methods by which one may determine the absolute configuration of sugars. Both indirect methods (converting the enantiomeric pair into diastereomers) and direct methods (using chiral stationary phases) have been discussed. Resolving reagents for the indirect methods include chiral hydroxy compounds, chiral amines, and chiral thiols; with subsequent separation of the diastereomers either by gas-liquid chromatography or by high pressure liquid chromatography. Direct methods discussed have exclusively utilized chiral substitution of organopolysiloxane phases for the separation of enantiomeric sugars as volatile derivatives by gas-liquid chromatography.     

Pathogenicity Confirmation of Ganoderma Disease of Coconut Using Early Diagnosis Technique

The pathogenicity and diagnostic methods were standardized for Ganoderma disease of coconut. The pathogenicity of Ganoderma lucidum isolated from coconut was tested using six types of inoculation techniques. Two diagnostic methods, viz. indirect enzyme‐linked immunosorbent assay (ELISA) and polymerase chain reactions (PCR) were applied for the confirmation of pathogenicity in coconut seedlings. Polyclonal antibodies (PAbs) were raised against mycelial, basidiocarp and specific proteins of Ganoderma and used for detection of Ganoderma in inoculated seedlings through indirect ELISA technique. All the three PAbs could detect Ganoderma in diseased coconut root tissues in early stage of the disease before symptom expression by indirect – ELISA at the antiserum dilution of 1 : 1000 for mycelial protein, 1 : 700 for Ganoderma specific protein and 1 : 3000 for basidiocarp protein. Low cross‐reactions were observed with saprophytic fungi occurring in coconut roots and also with other basidiomycetous fungi. In PCR, primers Gan1 and Gan2 generated from internal transcribed spacer region of rDNA were used the detection that produced a product of 167‐bp size in Ganoderma infected plants. In the present investigation, spawn inoculum responded earlier within 8 weeks compared with other methods of inoculation as expressed by OD value in ELISA test. This was also confirmed by PCR technique. The combination of these two diagnostic methods for detection of Ganoderma infection was highly reliable, rapid and sensitive.     

口蹄疫病毒P1基因在大肠杆菌中的高效表达及其生物活性的初步分析

将口蹄疫病毒 (FMDV)结构蛋白基因P1的完整cDNA序列插入原核表达性载体pGEX KG中 ,使P1基因与GST融合 ,获得融合表达质粒pKG P1,转化E .coliBL₂₁ (DE3) ,经IPTG诱导 ,SDS PADE结果表明GST P1融合蛋白获得高效表达 ,Western blot检测证实表达的融合蛋白具有免疫学活性 ,表达产物主要存在于细菌裂解液上清中。进一步采用GST纯化试剂盒纯化P1蛋白并作为诊断抗原 ,建立了P1 ELISA诊断方法 ,与FMD间接血凝 (IHA)检测方法平行检测 86 4份血清样品 ,总的符合率达87%。     

Direct and indirect measurement of somatic cell count as indicator of intramammary infection in dairy goats

Background  

Mastitis is the most important and costly disease in dairy goat production. Subclinical mastitis is common in goats and is mainly caused by contagious bacteria. Several methods to diagnose subclinical mastitis are available. In this study indirect measurement of somatic cell count (SCC) by California Mastitis Test (CMT) and direct measurement of SCC using a portable deLaval cell counter (DCC) are evaluated. Swedish goat farmers would primarily benefit from diagnostic methods that can be used at the farm. The purpose of the study was to evaluate SCC measured by CMT and DCC as possible markers for intramammary infection (IMI) in goats without clinical symptoms of mastitis. Moreover to see how well indirect measurement of SCC (CMT) corresponded to direct measurement of SCC (DCC).     

Estimation of pK(a) values using microchip capillary electrophoresis and indirect fluorescence detection

Microchip capillary electrophoresis (CE), coupled with indirect fluorescence detection was investigated for estimating the pK(a) values of non-fluorescent compounds. The CE method is based on the differences in electrophoretic mobility of the analyte as a function of the pH of the running buffer. Nine compounds were tested, including several of pharmaceutical importance, with pK(a) values from 10.3 to 4.6. All buffers contained 5-TAMRA as the fluorescent probe for indirect detection. Calculated pK(a) values agreed well with literature values obtained by traditional methods, differing not more than 0.2 from the literature value. The current work on single lane chips demonstrates the principle of microchip CE with indirect detection as a viable method for estimating pK(a) values. However, increased throughput will be required using a multilane chip to enable the approach to be used practically.     

猪口蹄疫病毒VP1结构蛋白抗体间接ELISA方法的建立

将口蹄疫病毒(FMDV)的VP1基因,通过pPROex-HT表达载体在大肠杆菌BL21(DE3)中成功表达,获得大小为31ku的融合蛋白,Westernblot检测证实表达的该蛋白具有良好的生物学活性。以纯化的融合蛋白为抗原建立了猪FMDVVP1蛋白间接ELISA检测方法。通过对80份田间血清样品的检测表明,该方法与FMDV液相阻断ELISA(国标试剂盒)的总符合率为96.25%,表明建立的VP1蛋白间接ELISA检测方法具有很好的特异性和敏感性。     

微血管内皮细胞的分离、纯化、鉴定及基因转移的研究

用胶原酶消化,梯度离心,LDL-FACS法分离及纯化小鼠肺组织微血管内皮细胞(MLMEC),并根据细胞形态及间接免疫荧光法加以鉴定。纯化后的MLMEC形态为多边形、短梭形,呈鹅卵石样排列;它能摄取RB-DiL-Ac-LDL,从而胞浆内出现亮红色颗粒;用抗PECAM抗体间接免疫荧光染色后细胞接触处呈亮绿色,细胞轮廓清晰可见。用感染法将HSV-TK基因转入MLMEC细胞内,用XTT法显示携带有HSV-TK基因的MLMEC/TK化及基因转移方法的建立将为研究MLMEC在各种病理状况下的作用及作为基因治疗的载体提供体外模型。     

Extensive pollen flow in two ecologically contrasting populations of Populus trichocarpa

Pollen-mediated gene flow was measured in two populations of black cottonwood using direct (paternity analysis) and indirect (correlated paternity) methods. The Marchel site was an area with an approximate radius of 250 m in a large continuous stand growing in a mesic habitat in western Oregon. In contrast, the Vinson site was an area with a radius of approximately 10 km and consisted of small, disjunct and isolated stands in the high desert of eastern Oregon. Pollen immigration was extensive in both populations, and was higher in the Marchel site (0.54 ± 0.02) than in the substantially larger and more isolated Vinson site (0.32 ± 0.02). Pollen pool differentiation among mothers was approximately five times stronger in the Vinson population (Φ_FT = 0.253, N  = 27 mothers) than in the Marchel population (Φ_FT = 0.052, N  = 5 mothers). Pollen dispersal was modelled using a mixed dispersal curve that incorporated pollen immigration. Predicted pollination frequencies generated based on this curve were substantially more accurate than those based on the widely used exponential power dispersal curve. Male neighbourhood sizes ( sensu Wright 1946 ) estimated using paternity analysis and pollen pool differentiation were remarkably similar. They were three to five times smaller in the Vinson population, which reflected the substantial ecological and demographic differences between the two populations. When the same mathematical function was used, applying direct and indirect methods resulted in similar pollen dispersal curves, thus confirming the value of indirect methods as a viable lower-cost alternative to paternity analysis.