The full potential of diagnostic PCR is limited, in part, by the presence of inhibitors in complex biological samples that reduce the amplification efficiency. Therefore, different pre-PCR treatments are being used to reduce the effects of PCR inhibitors. The aim of the present study was to investigate the effects of 16 amplification facilitators to enhance DNA amplification in the presence of blood, feces, or meat. Different concentrations of amplification facilitators and inhibitory samples were added to PCR mixtures containing rTth or Taq DNA polymerase. The addition of 0.6% (wt/vol) bovine serum albumin to reaction mixtures containing Taq DNA polymerase reduced the inhibitory effect of blood and allowed DNA amplification in the presence of 2% instead of 0.2% (vol/vol) blood. Furthermore, the addition of bovine serum albumin (BSA) to reaction mixtures containing feces or meat enhanced the amplification capacities of both polymerases. Taq DNA polymerase was able to amplify DNA in the presence of 4% instead of 0.4% (vol/vol) feces and 4% instead of 0.2% (vol/vol) meat, and rTth was able to amplify DNA in the presence of 4% instead of 0.4% (vol/vol) feces and 20% instead of 2% (vol/vol) meat. The single-stranded DNA binding T4 gene 32 protein (gp32) had a relieving effect similar to that of BSA, except when it was added to PCR mixtures of rTth containing meat and of Taq DNA polymerase containing feces. The relieving effects of betaine and a cocktail of proteinase inhibitors were more sample specific. The addition of 11.7% (wt/vol) betaine allowed Taq DNA polymerase to amplify DNA in the presence of 2% (vol/vol) blood, while the addition of proteinase inhibitors allowed DNA amplification by both polymerases in the presence of 4% (vol/vol) feces. When various combinations of betaine, BSA, gp32, and proteinase inhibitors were tested, no synergistic or additive effects were observed. The effects of facilitators on real-time DNA synthesis instead of conventional PCR were also studied.

The PCR is an extremely powerful method for detecting microorganisms. However, its full potential as a rapid detection method is limited by the inhibition of the thermostable DNA polymerase from Thermus aquaticus by many components found in complex biological samples. In this study, we have compared the effects of known PCR-inhibiting samples on nine thermostable DNA polymerases. Samples of blood, cheese, feces, and meat, as well as various ions, were added to PCR mixtures containing various thermostable DNA polymerases. The nucleic acid amplification capacity of the nine polymerases, under buffer conditions recommended by the manufacturers, was evaluated by using a PCR-based detection method for Listeria monocytogenes in the presence of purified template DNA and different concentrations of PCR inhibitors. The Ampli Taq Gold and the Taq DNA polymerases from Thermus aquaticus were totally inhibited in the presence of 0.004% (vol/vol) blood in the PCR mixture, while the Hot Tub , Pwo , rTth , and Tfl DNA polymerases were able to amplify DNA in the presence of 20% (vol/vol) blood without reduced amplification sensitivity. The DNA polymerase from Thermotoga maritima ( Ultma ) was found to be the most susceptible to PCR inhibitors present in cheese, feces, and meat samples. When the inhibitory effect of K and Na ions was tested on the nine polymerases, Hot Tub from Thermus flavus and rTth from Thermus thermophilus were the most resistant. Thus, the PCR-inhibiting effect of various components in biological samples can, to some extent, be eliminated by the use of the appropriate thermostable DNA polymerase.

A major inhibitor of diagnostic PCR in human plasma was identified and the mechanism of inhibition was characterized. Human blood was divided by centrifugation into buffy coat, plasma, platelets, and erythrocytes. All these blood fractions were found to be highly inhibitory to a standardized PCR mixture containing the thermostable DNA polymerase Ampli Taq Gold. PCR inhibitors in human plasma were purified by chromatographic procedures and were characterized by a process of elimination, so that the PCR-inhibitory effects of plasma fractions were tested after each purification step. The major inhibitor in human plasma, as determined by size-exclusion chromatography, anion-exchange chromatography, and chromatofocusing, was found to be immunoglobulin G (IgG) on the basis of N-terminal amino acid sequencing and electrophoretic analysis of the purified polypeptide. When different concentrations of purified plasma IgG (PIgG) were added to PCR mixtures containing 11 different thermostable DNA polymerases and 1 ng of Listeria monocytogenes DNA as template DNA, the only polymerase that resisted inhibition was rTth . The inhibitory effect was reduced when PIgG was heated at 95°C before it was added to PCR or after the addition of excess nontarget DNA to the PCR mixture. However, heating of PIgG together with target DNA at 95°C was found to block the amplification. Inhibition by PIgG may be due to an interaction with single-stranded DNA, which makes the target DNA unavailable for 10 of the DNA polymerases tested. The results show the danger of using boiling as a method of sample pretreatment or using a hot start prior to PCR. The effect of plasma PCR inhibition could be removed by mixing plasma with DNA-agarose beads prior to amplification, while plasma PCR inhibitors were found to bind to the DNA-agarose beads.

Background: Baker's yeast (Saccharomyces cerevisiae) has been engineered for xylose utilization to enable production of fuel ethanol from lignocellulose raw material. One unresolved challenge is that S. cerevisiae lacks a dedicated transport system for pentose sugars, which means that xylose is transported by non-specific Hxt transporters with comparatively low transport rate and affinity for xylose. Results: In this study, we compared three heterologous xylose transporters that have recently been shown to improve xylose uptake under different experimental conditions. The transporters Gxf1, Sut1 and At5g59250 from Candida intermedia, Pichia stipitis and Arabidopsis thaliana, respectively, were expressed in isogenic strains of S. cerevisiae and the transport kinetics and utilization of xylose was evaluated. Expression of the Gxf1 and Sut1 transporters led to significantly increased affinity and transport rates of xylose. In batch cultivation at 4 g/L xylose concentration, improved transport kinetics led to a corresponding increase in xylose utilization, whereas no correlation could be demonstrated at xylose concentrations greater than 15 g/L. The relative contribution of native sugar transporters to the overall xylose transport capacity was also estimated during growth on glucose and xylose. Conclusions: Kinetic characterization and aerobic batch cultivation of strains expressing the Gxf1, Sut1 and At5g59250 transporters showed a direct relationship between transport kinetics and xylose growth. The Gxf1 transporter had the highest transport capacity and the highest xylose growth rate, followed by the Sut1 transporter. The range in which transport controlled the growth rate was determined to between 0 and 15 g/L xylose. The role of catabolite repression in regulation of native transporters was also confirmed by the observation that xylose transport by native S. cerevisiae transporters increased significantly during cultivation in xylose and at low glucose concentration.

Two distinct forms of phosphoglucomutase were found in Lactococcus lactis subsp. lactis, strains 19435 and 65.1, growing on maltose: beta-phosphoglucomutase (beta-PGM), which catalyzes the reversible conversion of beta-glucose 1-phosphate to glucose 6-phosphate in the maltose catabolism, and alpha-phosphoglucomutase (alpha-PGM). beta-PGM was purified to more than 90% homogeneity in crude cell extract from maltose-grown lactococci, and polyclonal antisera to the enzyme were prepared. The molecular mass of beta-PGM was estimated by gel filtration to be 28 kDa; its isoelectric point was 4.8. The corresponding values for alpha-PGM were 65 kDa and 4.4, respectively. The expression of both PGM enzymes was investigated under different growth conditions. The specific activity and amount of beta-PGM per milliliter of cell extract increased with time in lactococci grown on maltose, but the enzyme was absent in lactococci grown on glucose, indicating enzyme synthesis to be induced by maltose in the growth medium. When glucose was added to maltose-grown lactococci, both the specific activity and amount of beta-PGM per milliliter of cell extract decreased rapidly. This suggests that synthesis of beta-PGM is repressed by glucose in the medium. Although the specific activity of alpha-PGM did not change during growth on maltose or glucose, lactococcal strain 19435 showed a much higher specific activity of both alpha- and beta-PGM than strain 65.1 when grown on maltose.