Pooling hair samples to increase DNA yield for PCR

Hairs are useful non-invasive sources of DNA, but the DNA yield can be very small, thus promoting genotyping errors. Using multiple hairs can counter this problem, but may introduce multiple contributors to a sample if collected remotely. With microsatellite genotyping, samples representing multiple animals are obvious if three or more alleles are detected at any locus: these samples can then be removed from any analyses. However, some multiple-individual samples may have only one or two alleles at each of the loci examined. We investigated the probability of failing to identify mixed pooled samples by simulating pooled samples (10 000 replicates) from microsatellite data from the northern and southern hairy-nosed wombats (NHNW, Lasiorhinus krefftii; SHNW, L. latifrons), species with low and high genetic diversity respectively. The majority (81.7%) of the 40 000 simulated samples had three or four alleles, so were readily identified as mixed. In the remaining 1-or-2-allele SHNW samples, forensic science software (DNAMIX) correctly identified mixed versus single-individual samples for all cases when the probability of locus failure was low (P _(LF) = 0.1), and 99% of samples when locus failure was high (P _(LF) = 0.5). For NHNW however, the probability of failing to identify a mixed sample was too high for population size estimation (0.05), even when the probability of locus failure was low. In cases such as this, pooled samples may be adequate for less demanding tasks, such as estimation of allele proportions. However, for animal populations with at least average levels of genetic variation, pooling of samples could safely be utilised for most applications. This revised version was published online in July 2006 with corrections to the Cover Date.     

PCR test systems for genetic identification of enterohemorrhagic Escherichia coli

The analysis of modern data on the development of amplification test systems for the gene indication of enterohemorrhagic E. coli (EHEC) is presented. In this work the emphasis is laid on the importance of using specific primers whose nucleotide sequence is closely linked with genes controlling the key factors of EHEC pathogenicity; these factors include the determinants of the synthesis of adhesins and invasins (bfp, eae, tir), shiga-like toxins (stx1, stx2), enterohemolysin (ehx), serine protease (epsA) and specific LPS of O-antigen (rfb). The problem of using primers whose sequence is not linked with virulence genes, but which may also be used for the gene indication of E. coli O157:H7 (uid, fliC) is discussed.     

Sensor systems for medical application based on hemoproteins and nanocomposite materials

Recent advances in nanotechnologies resulted in significant progress in the development of sensor systems based on nanocomposite materials. This review summarizes own and literature data on the sensor system based on hemoproteins which are functionally important for medicine. Special attention is paid to the electrochemical nanobiosensors and principles of functioning of the nanobiosensor medical systems.     

Functional unit for systems using natural raw materials

The necessity of a functional unit, which considers the equality of all benefits, is underlined especially for systems using such natural raw materials as wood. The example of identifying the ecological optimal extent of paper recycling is therefore examined by using the data of 11ASA [1]. It can be shown that the calculated quantity of the ecological optima particularly depend on the selected model of the comparison. In general, a functional unit of LCA should be based on a model which considers all benefits of the compared systems. The additional benefits of forests have to be taken into account as well. Otherwise, no statement concerning the ecological optima is possible.     

日光温室水肥耦合对甜瓜产量影响研究初探

在日光温室滴灌条件下,采用四因素二次回归通用旋转组合设计,对影响甜瓜的灌水上限、钾、磷、氮的耦合效应进行了研究.得到了总产量 y 与灌水上限 x1 、施钾量 x2 、施磷量 x3 、施氮量 x4 的水肥耦合回归模型.分析结果表明:1在本试验条件下水、钾、氮、磷各因素影响甜瓜产量的顺序为K>P>H2O>N;2各因素间存在交互作用,但交互作用未达显著水平.通过计算机模拟,得出了结果期各因素的最佳组合.     

Effect of cellular physiology on PCR amplification efficiency

Culture conditions, and other variables that modulate a cell's physiology, can bias a polymerase chain reaction (PCR) amplification against generating a representative population profile. Two Pseudomonas putida nahR alleles were constructed in pUC19 that differ solely in a 31-bp internal segment whose sequence has been inverted. After PCR amplification, the products could be distinguished on the basis of a change in a unique restriction site. When an Escherichia coli strain carrying one nahR allele is submitted to different growth conditions, the consequences of such variations on the relative PCR amplification of whole cells can be ascertained through coamplification with a strain carrying the other allele and subsequent restriction analysis. Cells in stationary phase displayed improved amplifiability while cells grown at 42°C were equally amplifiable as compared to cells grown at 37°C. However, sublethal levels of tetracycline or growth in minimal medium made the PCR target in these cells relatively less amplifiable. When cells are completely lysed and the plasmid DNA is purified beforehand, the coamplification bias is eliminated. These results suggest that mixed populations containing cells in different physiological states may not be representatively amplified by PCR unless a DNA extraction step is included.     

Effect of highly fragmented DNA on PCR.

We characterized the behavior of polymerase chain reactions (PCR) using degraded DNA as a template. We first demonstrated that fragments larger than the initial template fragments can be amplified if overlapping fragments are allowed to anneal and extend prior to routine PCR. Amplification products increase when degraded genomic DNA is pretreated by polymerization in the absence of specific primers. Secondly, we measured nucleotide uptake as a function of template DNA degradation. dNTP incorporation initially increases with increasing DNA fragmentation and then declines when the DNA becomes highly degraded. We demonstrated that dNTP uptake continues for >10 polymerization cycles and is affected by the quality and quantity of template DNA and by the amount of substrate dNTP. These results suggest that although reconstruction of degraded DNA may allow amplification of large fragments, reconstructive polymerization and amplification polymerization may compete. This was confirmed in PCR where the addition of degraded DNA reduced the resultant product. Because terminal deoxynucleotidyl transferase activity of Taq polymerase may inhibit 3' annealing and restrict the length of template reconstruction, we suggest modified PCR techniques which separate reconstructive and amplification polymerization reactions.     

New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis

Background  

Johne's disease, a serious chronic form of enteritis in ruminants, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). As the organism is very slow-growing and fastidious, several PCR-based methods for detection have been developed, based mainly on the MAP-specific gene IS900. However, because this gene is similar to genes in other mycobacteria, there is a need for sensitive and reliable methods to confirm the presence of MAP. As described here, two new real-time PCR systems on the IS900 gene and one on the F57 gene were developed and carefully validated on 267 strains and 56 positive clinical faecal samples.     

Comparison of three granular support materials for anaerobic fluidized bed systems

Summary Three different materials, kaolin, pozzolana and biolite (a material used in a commercial anaerobic fluidized bed treatment process) when tested as supports for an anaerobic fluidized bed system had similar physical and fluidization properties but behaved differently towards the biomass hold-up. However, all three systems attained similar removal efficiency rates.Nomenclature U Fluidization velocity (m/s) - U₁ Terminal fluidization velocity (m/s) - g Local acceleration due to gravity (m/s²) - _s Solid density (kg/m³) - _f Fluid density (kg/m³) - P Pressure drop (Pa) - HRT Hydraulic retention time (days) - H_mf Height of bed at minimum fluidization (m) - H Height of bed (m) - C_d Drag coefficient (dimensionless) - W Mass of solids in bed (kg) - dp Particle diameter (m) - A Cross-sectional area of column (m²) - h column height (m) - Rc_t Terminal Reynolds no. - Voidagc (fractional free volume, dimensionless) - _mf Voidage (fractional free volume) at minimum of fluidization (dimensionless)     

Effect of genome combinations on stability of yield and yield components in wheats and triticales

Summary The data on number of grains/spike, 100 grain weight and grain yield/plant in eighteen genotypes of four genome combinations (AABB- 4 genotypes, AABBDD- 6 genotypes, AABBRR- 5 genotypes and AABBDDRR- 3 genotypes) were recorded for eight environments created by combining two dates of sowing, two fertilizer regimes and two spacings. Two stability parameters-regression coefficient (b) and deviation from regression (S_d ²) were computed. Joint regression analyses revealed that the genotypes differed significantly for these characters. A significant variation due to environments was also found. A comparative study of performance of genotypes belonging to four genome combinations revealed that the genes for stability are not uniformly distributed in these genome combinations. Stability may largely depend on gene combination rather than on genome combination.     

Integrated real-time PCR for detection and monitoring of Legionella pneumophila in water systems

We evaluated a ready-to-use real-time quantitative Legionella pneumophila PCR assay system by testing 136 hot-water-system samples collected from 55 sites as well as 49 cooling tower samples collected from 20 different sites, in parallel with the standard culture method. The PCR assay was reproducible and suitable for routine quantification of L. pneumophila. An acceptable correlation between PCR and culture results was obtained for sanitary hot-water samples but not for cooling tower samples. We also monitored the same L. pneumophila-contaminated cooling tower for 13 months by analyzing 104 serial samples. The culture and PCR results were extremely variable over time, but the curves were similar. The differences between the PCR and culture results did not change over time and were not affected by regular biocide treatment. This ready-to-use PCR assay for L. pneumophila quantification could permit more timely disinfection of cooling towers.     

An international trial of quantitative PCR for monitoring Legionella in artificial water systems

Aims: To perform an international trial to derive alert and action levels for the use of quantitative PCR (qPCR) in the monitoring of Legionella to determine the effectiveness of control measures against legionellae. Methods and Results: Laboratories (7) participated from six countries. Legionellae were determined by culture and qPCR methods with comparable detection limits. Systems were monitored over ≥10 weeks. For cooling towers (232 samples), there was a significant difference between the log mean difference between qPCR (GU l^?1) and culture (CFU l^?1) for Legionella pneumophila (0·71) and for Legionella spp. (2·03). In hot and cold water (506 samples), the differences were less, 0·62 for Leg. pneumophila and 1·05 for Legionella spp. Results for individual systems depended on the nature of the system and its treatment. In cooling towers, Legionella spp. GU l^?1 always exceeded CFU l^?1, and usually Legionella spp. were detected by qPCR when absent by culture. The pattern of results by qPCR for Leg. pneumophila followed the culture trend. In hot and cold water, culture and qPCR gave similar results, particularly for Leg. pneumophila. There were some marked exceptions with temperatures ≥50°C, or in the presence of supplementary biocides. Action and alert levels for qPCR were derived that gave results comparable to the application of the European Guidelines based on culture. Algorithms are proposed for the use of qPCR for routine monitoring. Conclusions: Action and alert levels for qPCR can be adjusted to ensure public health is protected with the benefit that remedial actions can be validated earlier with only a small increase in the frequency of action being required. Significance and Impact of the Study: This study confirms it is possible to derive guidelines on the use of qPCR for monitoring the control of legionellae with consequent improvement to response and public health protection.     

Novel carbon materials in biosensor systems

In this work, novel carbon materials are evaluated as transducers, stabilizers and mediators for the construction of amperometric biosensors. It is shown that materials such as fullerenes and carbon nanotubes are promising materials as electrochemical mediators and enzyme stabilizers. Additionally porous carbon and porous glassy carbon are excellent transducers for amperometric measurements, while they provide cavities adequate for enzyme immobilization. At the same time, the sensitivity to peroxide is shown to depend on the activation procedures. Treatment that introduces oxygen groups increases the sensitivity of the carbon-based sensor to hydrogen peroxide considerably. These materials are used for the construction, mediation and stabilization of glucose biosensor.