Materials 21 Cell Lines

THP-1, human acute monocytic leukemia cell line (Centro Substrati Cellulari, Brescia, Italy, and American Type Culture Collection ATCC). These cells have Fc and C3b receptors and lack surface and cytoplasmic immunoglobulins they stain positive for alfa-naphthyl butyrate esterase, produce lysozyme, and are phagocytic (52). 2. P3 88 D1 mouse-monocyte-macrophage and U937 human histiocytic lymphoma cell lines (American Type Culture Collection ATCC). 3. The Mono Mac 6 (MM6) human monocytic cell line...

Introduction

Staphylococcus aureus is one of the most significant human pathogens producing nosocomial and community-acquired infections. The wide spectrum of clinical manifestations include superficial, deep-skin, and soft-tissue infections, osteomyelitis, pneumonia, endocarditis, and septicemia as well as a variety of toxin-mediated diseases, including food-borne gastroenteritis, staphylococcal-scalded skin syndrome, and toxic shock syndrome, with -lactam antibiotics being the drugs of choice for therapy...

Real Time PCR

Oligonucleotide primers and fluorescence-labeled hybridization probes are designed for amplification and sequence-specific detection of both a 188-bp fragment within the mecA gene and a 178-bp fragment within the S. aureus-specific Sa442 gene (see Note 5). Nucleotide sequences and positions are listed in Table 1. Lyophilized primers are diluted in PCR-grade water (see Note 6). Concentrations of the stock solution are listed in Table 2. 1. Place the required number of LightCycler Capillaries...

Interpretation of Results

HOOF-print genotypes consist of the specific alleles identified from 10 polymorphic genomic loci. Alleles are named for the calculated number of complete repeat units at each locus because each locus also contains a characteristic partial repeat in addition to the complete repeat units. Using Locus-1 as an example, Allele 2 has 2 and 3 8 repeat units, Allele 3 has 3 and 3 8 repeat units and so on. The sizes of the respective partial repeats for the 10 loci are Locus-9 contains two series of...

Geert Jannes and Daniel De

Nucleic acid-based diagnostics gradually are replacing or complementing culture-based, biochemical, and immunological assays in routine microbiology laboratories. Similar to conventional tests, the first-generation deoxyribonucleic acid assays determined only a single analyte. Recent improvements in detection technologies have paved the way for the development of multiparameter assays using macroarrays or micro-arrays, while the introduction of closed-tube real-time polymerase chain reaction...

DNA Purification

The classical way of purifying nucleic acids from complex-, inhibitor-, and protein-containing solutions is to apply organic solvents such as phenol chloroform (33). Other organic solvents such as chloroform or ether can be used to separate, for instance, fat from the aqueous DNA-containing phase. For algal and plant materials where co-purification of polysaccharides together with DNA may be a problem, the polysaccharides can be selectively precipitated with cetyl trimethyl ammonium bromide...

Extraction of Genomic DNA

Using an aliquot of 50 pL of sample, one should largely follow the manufacturer's instructions for extracting genomic DNA using the Promega Wizard SV 96 System, but modifications can be made according to the requirements for automation see Note 5 . 2. Add 100 pL of bacterial culture or body fluid to 200 pL of SV RNA lysis buffer and vortex to mix. 3. Program the robot to automatically place the SV96 DNA binding plate on top of the vacuum manifold. 4. Add 150 pL of blood lysate to the wells...

Diagnosis of Bloodstream Infections

Bloodstream infections BSIs are a major cause of morbidity and mortality worldwide. It has been shown that an increased time to detection of BSIs was associated with longer hospital stay and higher cost 55 . The detection of bacterial or fungal growth in blood cultures currently is considered as the gold standard. Despite improvements in growth media and instrumentation, blood culture is too slow and has a poor diagnostic sensitivity and specificity. Molecular techniques for pathogen...

Sizing of Amplicons by Capillary Electrophoresis With Fluorescent Detection

Identification of alleles that differ by 8 bp requires a size analysis method that is capable of very precise measurements. Capillary electrophoresis 21 with fluorescent detection has this capability. The high sensitivity allows minute amounts of sample to be sharply resolved by differential migration through a 36-cm electric field. Samples should be diluted in PCR-grade water, typically in the range of 1 4 through 1 200, depending on the amplicon concentration, which can be estimated from the...

Troubleshooting Potential Problems With the Experimental Data

Allelic Ladder Plex

There are a number of things that can affect data production or data interpretation. The following section describes some of the more common problems encountered when performing the HOOF-print assay. This section also shows that it is essential to carefully examine the data before reaching a final determination. One particular danger is the overreliance on allele-calling software that automatically assigns the data into bins alleles based on size. These programs may not be able to distinguish...