sybr-green-i and Cystic-Fibrosis

Mixed bacterial communities are commonly encountered in microbial infections of humans. Knowledge on the composition of species and viability of each species in these communities allows for a detailed description of the complexity of interspecies dynamics and contributes to the assessment of the severity of infections. Several assays exist for quantification of specific species in mixed communities, including analysis of quantitative terminal restriction fragment length polymorphisms. While this method allows for species-specific cell enumeration, it cannot provide viability data. In this study, flow cytometry was applied to assess the viability of Staphylococcus aureus and Burkholderia cepacia in mixed culture by membrane integrity analysis using SYBR® Green I and propidium iodide staining. Both bacteria are relevant to pulmonary infections of cystic fibrosis patients. Fluorescence staining was optimized separately for each species in pure culture due to differences between species in cell wall structure and metabolic capabilities. To determine viability of species in mixed culture, a protocol was established as a compromise between optimum conditions determined before for pure cultures. This protocol allowed the detection of viable and dead cells of both species, exhibiting an intact and a permeabilized membrane, respectively. To discriminate between S. aureus and B. cepacia, the protocol was combined with Gram-specific fluorescent staining using wheat germ agglutinin. The established three-color staining method was successfully tested for viability determination of S. aureus and B. cepacia in mixed culture cultivations. In addition, growth of both species was monitored by quantitative terminal restriction fragment length polymorphisms. The obtained data revealed alterations in viability during cultivations for different growth phases and suggest interspecies effects in mixed culture. Overall, this method allows for rapid simultaneous Gram-differentiation and viability assessment of bacterial mixed cultures and is therefore suitable for the analysis of dynamics of mixed communities of medical, environmental, and biotechnological relevance.

Topics: Bacterial Load; Bacteriological Techniques; Benzothiazoles; Burkholderia cepacia; Burkholderia Infections; Cell Membrane; Cystic Fibrosis; Diamines; Flow Cytometry; Humans; Microbial Viability; Microscopy, Fluorescence; Organic Chemicals; Polymorphism, Restriction Fragment Length; Propidium; Quinolines; Species Specificity; Staphylococcal Infections; Staphylococcus aureus; Wheat Germ Agglutinins

This chapter describes methodology for the labeling, hybridization, and detection of amplicon target DNA to arrays of oligonucleotide probes attached to plastic substrates. A systematic approach to target discrimination based on both hybridization and wash stringency is provided.

Topics: Benzothiazoles; Biotin; Cystic Fibrosis; Diamines; Electrophoresis; Humans; Mutation; Oligonucleotide Array Sequence Analysis; Oligonucleotide Probes; Organic Chemicals; Polymerase Chain Reaction; Quinolines; Staining and Labeling

Common methods for identification of DNA sequence variants use gel electrophoresis or column separation after PCR.. We developed a method for sequence variant analysis requiring only PCR and amplicon melting analysis. One of the PCR primers was fluorescently labeled. After PCR, the melting transition of the amplicon was monitored by high-resolution melting analysis. Different homozygotes were distinguished by amplicon melting temperature (T(m)). Heterozygotes were identified by low-temperature melting of heteroduplexes, which broadened the overall melting transition. In both cases, melting analysis required approximately 1 min and no sample processing was needed after PCR.. Polymorphisms in the HTR2A (T102C), beta-globin [hemoglobin (Hb) S, C, and E], and cystic fibrosis (F508del, F508C, I507del, I506V) genes were analyzed. Heteroduplexes produced by amplification of heterozygous DNA were best detected by rapid cooling (>2 degrees C/s) of denatured products, followed by rapid heating during melting analysis (0.2-0.4 degrees C/s). Heterozygotes were distinguished from homozygotes by a broader melting transition, and each heterozygote had a uniquely shaped fluorescent melting curve. All homozygotes tested were distinguished from each other, including Hb AA and Hb SS, which differed in T(m) by <0.2 degrees C. The amplicons varied in length from 44 to 304 bp. In place of one labeled and one unlabeled primer, a generic fluorescent oligonucleotide could be used if a 5' tail of identical sequence was added to one of the two unlabeled primers.. High-resolution melting analysis of PCR products amplified with labeled primers can identify both heterozygous and homozygous sequence variants.

Topics: Base Sequence; Benzothiazoles; Blood Proteins; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Diamines; DNA; Fluorescent Dyes; Genotype; Globins; Heteroduplex Analysis; Heterozygote; High-Temperature Requirement A Serine Peptidase 2; Homozygote; Humans; Mitochondrial Proteins; Oligonucleotide Probes; Organic Chemicals; Polymerase Chain Reaction; Polymorphism, Genetic; Quinolines; Serine Endopeptidases; Temperature

A simple and practical screening method, allowing the mass detection of targeted DNA mutations, was developed by combined use of allele specific PCR (ASP) and subsequent fluorogenic intercalation to the amplicon.. Crude DNAs were extracted from dried blood spots (DBS) by a simple boil method. Highly specific polymerase chain reaction (PCR) amplification was achieved by adopting Taq DNA polymerase (Amersham Pharmacia) modified with TaqStart Antibody (Clontech). A fluorogenic DNA intercalator, SYBR Green I (Molecular Probes), was directly added to the PCR products and the resultant fluorescence was measured by a conventional fluorometric microplate reader, microfluorometry (MFL).. The most common mutation in cystic fibrosis (CF), del F508, was successfully detected with clear differentiation as homozygotes (n=4) and heterozygotes (n=9) from control subjects (n=18). Fluorescence intensities, with mean+/-S.D. in arbitrary unit, were 895+/-249, 900+/-184 and 257+/-53, respectively. Those from control newborns (n=352) were 250+/-27 with the range of 188-475.. The proposed ASP/MFL provides a simple, objective and economical detection of known mutations or single nucleotide polymorphisms (SNPs). The usefulness of this method was clearly shown in the detection of del F508 in CF.

Topics: Adolescent; Adult; Alleles; Benzothiazoles; Blood Specimen Collection; Case-Control Studies; Child; Child, Preschool; Costs and Cost Analysis; Cystic Fibrosis; Cytophotometry; Diamines; DNA Mutational Analysis; Fluorescent Dyes; Genetic Testing; Heterozygote; Homozygote; Humans; Infant; Infant, Newborn; Mutation; Organic Chemicals; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Quinolines; Sequence Deletion