BACKGROUND: Quantitative real-time PCR (qPCR) is becoming increasingly important for DNA genotyping and gene expression analysis. For continuous monitoring of the production of PCR amplicons DNA-intercalating dyes are widely used. Recently, we have introduced a new qPCR mix which showed improved amplification of medium-size genomic DNA fragments in the presence of DNA dye SYBR green I (SGI). In this study we tested whether the new PCR mix is also suitable for other DNA dyes used for qPCR and whether it can be applied for amplification of DNA fragments which are difficult to amplify. RESULTS: We found that several DNA dyes (SGI, SYTO-9, SYTO-13, SYTO-82, EvaGreen, LCGreen or ResoLight) exhibited optimum qPCR performance in buffers of different salt composition. Fidelity assays demonstrated that the observed differences were not caused by changes in Taq DNA polymerase induced mutation frequencies in PCR mixes of different salt composition or containing different DNA dyes. In search for a PCR mix compatible with all the DNA dyes, and suitable for efficient amplification of difficult-to-amplify DNA templates, such as those in whole blood, of medium size and/or GC-rich, we found excellent performance of a PCR mix supplemented with 1 M 1,2-propanediol and 0.2 M trehalose (PT enhancer). These two additives together decreased DNA melting temperature and efficiently neutralized PCR inhibitors present in blood samples. They also made possible more efficient amplification of GC-rich templates than betaine and other previously described additives. Furthermore, amplification in the presence of PT enhancer increased the robustness and performance of routinely used qPCRs with short amplicons. CONCLUSIONS: The combined data indicate that PCR mixes supplemented with PT enhancer are suitable for DNA amplification in the presence of various DNA dyes and for a variety of templates which otherwise can be amplified with difficulty.

• MeSH
• DNA chemie   genetika   MeSH
• fluorescenční barviva chemie   MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• polymerázová řetězová reakce přístrojové vybavení   MeSH
• propylenglykol chemie   MeSH
• trehalosa chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Amplification of DNA templates from whole blood with Taq DNA polymerase remains a difficult task worldwide. Using a real-time PCR setup and a buffer supplemented with 1M 1,2-propanediol, 0.2M trehalose, and SYBR green I we show a reliable technique for genotyping in mice and detection of single-nucleotide polymorphisms/mutations in humans. Elimination of DNA extraction and use of the common Taq DNA polymerase and DNA dye bring about substantial savings in labor and cost.

• MeSH
• DNA chemie   MeSH
• genotyp MeSH
• jednonukleotidový polymorfismus genetika   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• lidé MeSH
• myši MeSH
• organické látky chemie   MeSH
• propylenglykol chemie   MeSH
• pufry MeSH
• Taq-polymerasa chemie   MeSH
• trehalosa chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• azidy toxicita   MeSH
• genetické inženýrství účinky léků   MeSH
• genetika účinky léků   MeSH
• glycerol analogy a deriváty   MeSH
• glykoly toxicita   MeSH
• mutageny MeSH
• vztah mezi dávkou a účinkem léčiva MeSH

• MeSH
• adenin analogy a deriváty   MeSH
• azidy toxicita   MeSH
• Escherichia coli genetika   MeSH
• mutageny toxicita   MeSH
• oprava DNA MeSH
• propylenglykoly toxicita   MeSH

• MeSH
• Escherichia coli genetika   MeSH
• methylnitronitrosoguanidin toxicita   MeSH
• mutace MeSH
• mutageny MeSH
• propylenglykoly toxicita   MeSH

A simple, cost effective, and fast gas chromatography method with flame ionization detection (GC-FID) for simultaneous measurement of ethylene glycol, 1,2-propylene glycol and glycolic acid was developed and validated for clinical toxicology purposes. This new method employs a relatively less used class of derivatization agents - alkyl chloroformates, allowing the efficient and rapid derivatization of carboxylic acids within seconds while glycols are simultaneously derivatized by phenylboronic acid. The entire sample preparation procedure is completed within 10 min. To avoid possible interference from naturally occurring endogenous acids and quantitation errors 3-(4-chlorophenyl) propionic acid was chosen as an internal standard. The significant parameters of the derivatization have been found using chemometric procedures and these parameters were optimized using the face-centered central composite design. The calibration dependence of the method was proved to be quadratic in the range of 50-5000 mg mL(-1), with adequate accuracy (92.4-108.7%) and precision (9.4%). The method was successfully applied to quantify the selected compounds in serum of patients from emergency units.

• MeSH
• chromatografie plynová metody   MeSH
• ethylenglykol krev   otrava   moč   MeSH
• glykoláty krev   otrava   moč   MeSH
• lidé MeSH
• plamínková ionizace metody   MeSH
• propylenglykol krev   otrava   moč   MeSH
• sérum chemie   MeSH
• studie případů a kontrol MeSH
• toxikologie metody   MeSH
• urgentní zdravotnické služby * MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

A series of twelve breast milk samples were analysed by gas chromatography-mass spectrometry (GC/MS) operated in selected ion monitoring mode for 3-chloropropane-1,2-diol (3-MCPD). Whilst none of the samples contained 3-MCPD above the limit of detection of 3 microg kg(-1) milk, all contained high amounts of 3-MCPD esterified with higher fatty acids. The levels of 3-MCPD released by hydrolysis of these esters (bound 3-MCPD) ranged from the limit of detection (300 microg kg(-1), expressed on a fat basis) to 2195 microg kg(-1); with a mean level of bound 3-MCPD of 1014 microg kg(-1), which corresponded to 35.5 microg kg(-1) milk. The presence of bound 3-MCPD was confirmed using orthogonal gas chromatography coupled with high-speed time-of-flight mass spectrometry analysis for four randomly selected breast milk samples. Six breast milks collected from one of the nursing mothers 14-76 days after childbirth contained bound 3-MCPD within the range of 328-2078 microg kg(-1) fat (mean 930 microg kg(-1) fat). The calculated bound 3-MCPD content of these samples was within the range of 6 and 19 microg kg(-1) milk (mean of 12 microg kg(-1) milk). The major types of 3-MCPD esters were the symmetric diesters with lauric, palmitic, and oleic acids, and asymmetric diesters with palmitic acid/oleic acid among which 3-chloro-1,2-propanediol 1,2-dioleate prevailed.

• MeSH
• alfa-chlorhydrin analýza   MeSH
• analýza potravin metody   MeSH
• dospělí MeSH
• financování organizované MeSH
• kontaminace potravin analýza   MeSH
• lidé MeSH
• lipidy analýza   MeSH
• mateřské mléko chemie   MeSH
• mladiství MeSH
• plynová chromatografie s hmotnostně spektrometrickou detekcí metody   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• ženské pohlaví MeSH

• MeSH
• farmaceutická chemie MeSH
• propylenglykol MeSH

BACKGROUND: Human milk oligosaccharides (HMOs) are one of the major glycan source of the infant gut microbiota. The two species that predominate the infant bifidobacteria community, Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum, possess an arsenal of enzymes including α-fucosidases, sialidases, and β-galactosidases to metabolise HMOs. Recently bifidobacteria were obtained from the stool of six month old Kenyan infants including species such as Bifidobacterium kashiwanohense, and Bifidobacterium pseudolongum that are not frequently isolated from infant stool. The aim of this study was to characterize HMOs utilization by these isolates. Strains were grown in presence of 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), 3'-sialyl-lactose (3'-SL), 6'-sialyl-lactose (6'-SL), and Lacto-N-neotetraose (LNnT). We further investigated metabolites formed during L-fucose and fucosyllactose utilization, and aimed to identify genes and pathways involved through genome comparison. RESULTS: Bifidobacterium longum subsp. infantis isolates, Bifidobacterium longum subsp. suis BSM11-5 and B. kashiwanohense strains grew in the presence of 2'-FL and 3'- FL. All B. longum isolates utilized the L-fucose moiety, while B. kashiwanohense accumulated L-fucose in the supernatant. 1,2-propanediol (1,2-PD) was the major metabolite from L-fucose fermentation, and was formed in equimolar amounts by B. longum isolates. Alpha-fucosidases were detected in all strains that degraded fucosyllactose. B. longum subsp. infantis TPY11-2 harboured four α-fucosidases with 95-99 % similarity to the type strain. B. kashiwanohense DSM 21854 and PV20-2 possessed three and one α-fucosidase, respectively. The two α-fucosidases of B. longum subsp. suis were 78-80 % similar to B. longum subsp. infantis and were highly similar to B. kashiwanohense α-fucosidases (95-99 %). The genomes of B. longum strains that were capable of utilizing L-fucose harboured two gene regions that encoded enzymes predicted to metabolize L-fucose to L-lactaldehyde, the precursor of 1,2-PD, via non-phosphorylated intermediates. CONCLUSION: Here we observed that the ability to utilize fucosyllactose is a trait of various bifidobacteria species. For the first time, strains of B. longum subsp. infantis and an isolate of B. longum subsp. suis were shown to use L-fucose to form 1,2-PD. As 1,2-PD is a precursor for intestinal propionate formation, bifidobacterial L-fucose utilization may impact intestinal short chain fatty acid balance. A L-fucose utilization pathway for bifidobacteria is suggested.

• MeSH
• alfa-L-fukosidasa klasifikace   genetika   metabolismus   MeSH
• beta-galaktosidasa metabolismus   MeSH
• Bifidobacterium longum enzymologie   genetika   metabolismus   MeSH
• Bifidobacterium enzymologie   genetika   metabolismus   MeSH
• DNA bakterií genetika   MeSH
• feces mikrobiologie   MeSH
• fukosa metabolismus   MeSH
• genom bakteriální MeSH
• kojenec MeSH
• kyseliny mastné těkavé metabolismus   MeSH
• kyseliny sialové metabolismus   MeSH
• laktosa analogy a deriváty   metabolismus   MeSH
• lidé MeSH
• mateřské mléko metabolismus   MeSH
• metabolické sítě a dráhy MeSH
• oligosacharidy metabolismus   MeSH
• propylenglykol metabolismus   MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvence nukleotidů MeSH
• střeva mikrobiologie   MeSH
• trisacharidy metabolismus   MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• erytrocyty fyziologie   MeSH
• hemolýza fyziologie   MeSH
• hornictví MeSH
• hypertonické roztoky chemie   MeSH
• lidé MeSH
• propylenglykol MeSH
• Check Tag
• lidé MeSH