Hybridization Assays Using an Expressible DNA Fragment Encoding Firefly Luciferase as a Label

We report the use of a new label, an expressible enzymecoding DNA fragment, for nucleic acid hybridization assays. The DNA label contains a firefly luciferase coding sequence downstream from a T7 RNA polymerase promoter. The target DNA (200 bp) is denatured and hybridized simultaneously with two oligonucleotide probes. One of the probes is immobilized in microtiter wells, via the digoxigenin/anti-digoxigenin interaction, and the other probe is biotinylated. After completion of the hybridization, the hybrids are reacted with a streptavidin-luciferase DNA complex. Subsequently, the solid-phase bound DNA is expressed by coupled transcription/ translation. The synthesized luciferase catalyzes the luminescent reaction of luciferin with O 2 and ATP. The luminescence is linearly related to the amount of target DNA in the range of 5-5000 amol. The CVs obtained for 20 and 100 amol of target are 6.5% and 10.8%, respectively (n ) 4). The specific and strong interaction between two complementary nucleic acid strands forms the basis for the development of hybridization assays. Hybridization methodology is emerging as the most promising area in laboratory medicine and has transformed the way clinical testing is realized. Previous tests have been based on the monitoring of gene products, i.e., phenotypic markers, such as oncoproteins, viral antigens, etc. In contrast, current laboratory tests that are based on hybridization allow the analysis of disease at the nucleic acid level. Thus, pre-or postnatal diagnosis of genetic disease can be accomplished by hybridization of the patient's DNA with allele-specific oligonucleotide probes that recognize mutations, deletions, or insertions causing the disease. Also, the various infectious agents can be measured in biological fluids by hybridization with specific probes. In forensic science, hybridization of DNA with minisatellite probes allows the unique identification of individuals (DNA fingerprinting). 1,2 Radioactive probes (usually labeled with 32 P), in combination with autoradiographic detection, dominated in the field of hybridization assays for more than 2 decades and provide the highest sensitivities. However, the short half-life of 32 P, the health hazards and problems associated with its use and disposal, and the long exposure times (many hours to days) required for detection have placed limitations on the routine use of hybridization assays in the clinical laboratory. The current trend in this area is toward novel nonradioactive alternatives. 2,3 The labels can be incorporated into the probes either enzymatically (e.g., using DNA polymerase or deoxynucleotidyl transferase and modified deoxynucleoside triphosphates) or by chemical conjugation (e.g., introduction of NH 2 groups into the probe via cytidine transamination and then conjugation to the reporter molecule). 4 Nonisotopic hybridization assays based on fluorescent, chemiluminescent, or enzyme labels have been developed. Generally, there are two strategies for the analysis of hybrids. Either the reporter molecule is directly conjugated to the probe, 5,6 or a ligand is attached to the probe and the hybrids are measured in a subsequent step by adding a specific, labeled binding protein. The ligand may be biotin or a hapten (e.g., digoxigenin). Labeled (strept)avidin or antihapten antibodies may then be employed for detection. 7,8 Enzymes (such as alkaline phosphatase and horseradish peroxidase) are the most widely used nonradioisotopic labels because they provide amplification through the high turnover of their substrates to detectable products. 2 Recently, we reported 9 that a DNA fragment (DNA template) coding for an enzyme can be used as a novel label for the development of highly sensitive immunoassays (expression immunoassays). In these assays, after completion of the immunoreaction, the DNA template (a luciferase-coding DNA) is expressed by in vitro transcription/translation and the activity of the synthesized enzyme is measured. Furthermore, it was estimated that 12-14 luciferase molecules were synthesized from each DNA template molecule. In the present work, we extend our investigation in the area of hybridization assays. EXPERIMENTAL SECTION Instrumentation. Luminescence measurements were carried out using a liquid scintillation counter (Model LS-6500, Beckman Instruments Inc., Fullerton, CA) in the single photon monitoring mode. Fluorescence measurements were performed with th

Exploring the use of internal and externalcontrols for assessing microarray technical performance

<p>Abstract</p> <p>Background</p> <p>The maturing of gene expression microarray technology and interest in the use of microarray-based applications for clinical and diagnostic applications calls for quantitative measures of quality. This manuscript presents a retrospective study characterizing several approaches to assess technical performance of microarray data measured on the Affymetrix GeneChip platform, including whole-array metrics and information from a standard mixture of external spike-in and endogenous internal controls. Spike-in controls were found to carry the same information about technical performance as whole-array metrics and endogenous "housekeeping" genes. These results support the use of spike-in controls as general tools for performance assessment across time, experimenters and array batches, suggesting that they have potential for comparison of microarray data generated across species using different technologies.</p> <p>Results</p> <p>A layered PCA modeling methodology that uses data from a number of classes of controls (spike-in hybridization, spike-in polyA+, internal RNA degradation, endogenous or "housekeeping genes") was used for the assessment of microarray data quality. The controls provide information on multiple stages of the experimental protocol (e.g., hybridization, RNA amplification). External spike-in, hybridization and RNA labeling controls provide information related to both assay and hybridization performance whereas internal endogenous controls provide quality information on the biological sample. We find that the variance of the data generated from the external and internal controls carries critical information about technical performance; the PCA dissection of this variance is consistent with whole-array quality assessment based on a number of quality assurance/quality control (QA/QC) metrics.</p> <p>Conclusions</p> <p>These results provide support for the use of both external and internal RNA control data to assess the technical quality of microarray experiments. The observed consistency amongst the information carried by internal and external controls and whole-array quality measures offers promise for rationally-designed control standards for routine performance monitoring of multiplexed measurement platforms.</p

Linoleic Acid-Induced Ultra-Weak Photon Emission from Chlamydomonas reinhardtii as a Tool for Monitoring of Lipid Peroxidation in the Cell Membranes

Reactive oxygen species formed as a response to various abiotic and biotic stresses cause an oxidative damage of cellular component such are lipids, proteins and nucleic acids. Lipid peroxidation is considered as one of the major processes responsible for the oxidative damage of the polyunsaturated fatty acid in the cell membranes. Various methods such as a loss of polyunsaturated fatty acids, amount of the primary and the secondary products are used to monitor the level of lipid peroxidation. To investigate the use of ultra-weak photon emission as a non-invasive tool for monitoring of lipid peroxidation, the involvement of lipid peroxidation in ultra-weak photon emission was studied in the unicellular green alga Chlamydomonas reinhardtii. Lipid peroxidation initiated by addition of exogenous linoleic acid to the cells was monitored by ultra-weak photon emission measured with the employment of highly sensitive charged couple device camera and photomultiplier tube. It was found that the addition of linoleic acid to the cells significantly increased the ultra-weak photon emission that correlates with the accumulation of lipid peroxidation product as measured using thiobarbituric acid assay. Scavenging of hydroxyl radical by mannitol, inhibition of intrinsic lipoxygenase by catechol and removal of molecular oxygen considerably suppressed ultra-weak photon emission measured after the addition of linoleic acid. The photon emission dominated at the red region of the spectrum with emission maximum at 680 nm. These observations reveal that the oxidation of linoleic acid by hydroxyl radical and intrinsic lipoxygenase results in the ultra-weak photon emission. Electronically excited species such as excited triplet carbonyls are the likely candidates for the primary excited species formed during the lipid peroxidation, whereas chlorophylls are the final emitters of photons. We propose here that the ultra-weak photon emission can be used as a non-invasive tool for the detection of lipid peroxidation in the cell membranes

Epidemiology of intra-abdominal infection and sepsis in critically ill patients: “AbSeS”, a multinational observational cohort study and ESICM Trials Group Project

Purpose: To describe the epidemiology of intra-abdominal infection in an international cohort of ICU patients according to a new system that classifies cases according to setting of infection acquisition (community-acquired, early onset hospital-acquired, and late-onset hospital-acquired), anatomical disruption (absent or present with localized or diffuse peritonitis), and severity of disease expression (infection, sepsis, and septic shock). Methods: We performed a multicenter (n = 309), observational, epidemiological study including adult ICU patients diagnosed with intra-abdominal infection. Risk factors for mortality were assessed by logistic regression analysis. Results: The cohort included 2621 patients. Setting of infection acquisition was community-acquired in 31.6%, early onset hospital-acquired in 25%, and late-onset hospital-acquired in 43.4% of patients. Overall prevalence of antimicrobial resistance was 26.3% and difficult-to-treat resistant Gram-negative bacteria 4.3%, with great variation according to geographic region. No difference in prevalence of antimicrobial resistance was observed according to setting of infection acquisition. Overall mortality was 29.1%. Independent risk factors for mortality included late-onset hospital-acquired infection, diffuse peritonitis, sepsis, septic shock, older age, malnutrition, liver failure, congestive heart failure, antimicrobial resistance (either methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, extended-spectrum beta-lactamase-producing Gram-negative bacteria, or carbapenem-resistant Gram-negative bacteria) and source control failure evidenced by either the need for surgical revision or persistent inflammation. Conclusion: This multinational, heterogeneous cohort of ICU patients with intra-abdominal infection revealed that setting of infection acquisition, anatomical disruption, and severity of disease expression are disease-specific phenotypic characteristics associated with outcome, irrespective of the type of infection. Antimicrobial resistance is equally common in community-acquired as in hospital-acquired infection