Detection of HIV1 DNA in biological samples by an homogeneous assay: fluorescence measurement of double-stranded RNA synthesized from amplified DNA

A nonisotopic homogeneous detection of nucleic acid sequences after amplification is described. We show that a DNA fragment bearing T7 RNA polymerase promoters on each extremity is able to be transcribed in two complementary RNAs, leading to a high yield direct synthesis of double-stranded RNA (dsRNA). Thus, this dsRNA can be easily detected and quantified in solution by fluorescence in the presence of propidium iodide. This reaction, used as a postamplification step, has been associated with a nested polymerase chain reaction (PCR); the second PCR round allowing the incorporation of the T7 promoters. This leads to a very efficient homogeneous assay. The fluorescence signal is proportional to the concentration of PCR product and is highly specific. This method can be easily carried out with currently available reagents and with unsophisticated instrumentation. This homogeneous procedure has been evaluated for the detection of HIV1 in blood samples; the sensitivity and the specificity appear to be equivalent to that of the radioactive method.     

Chemiluminescence: sensitive detection technology for reporter gene assays

A series of enzyme-activated chemiluminescence-based assays of reporter gene expression, useful in many biomedical applications, has been developed. The chemiluminescence detection systems for beta-galactosidase, beta-glucuronidase (GUS), and secreted placental alkaline phosphatase (SEAP) reporter enzymes are all based on use of 1,2-dioxetane substrates. This detection technology also permits the combined luminescence detection of two different reporter enzymes in the same tube, e.g., a dual assay for beta-galactosidase and luciferase. The sensitivity of these chemiluminescence assays is several orders of magnitude greater than that of conventional colorimetric or fluorometric detection methods; e.g., the detection limit for beta-galactosidase by the chemiluminescence assay is 8 fg and by a fluorometric assay is 2 pg. Furthermore, chemiluminescence enables detection of beta-galactosidase, GUS, and SEAP enzyme concentrations over a dynamic range of more than five to six orders in magnitude. These assays offer highly sensitive, quantitative, rapid, nonisotopic detection of reporter enzymes that are widely used in both mammalian cells and plant cells.     

Effects of the myosin inhibitor 2,3-butanedione monoxime on the physiology of fission yeast

Bacterial infections that may be related to impaired phagocyte function often develop in patients infected with human immunodeficiency virus (HIV). We examined the oxidative capacity of circulating phagocytes in 78 HIV+ patients and 31 control subjects by measuring chemiluminescence with a whole blood assay. Phagocytes were stimulated with zymosan opsonized with human complement (hC-OPZ) or immunoglobulin (hI-OPZ) with or without exogenous primers. Patients with CD4+ < 500/microL showed reduced whole blood chemiluminescence at maximal opsonin receptor (MOR) activity after priming in response to hC-OPZ relative to control subjects, and the difference was significant for patients with CD4+ < 100/microL. Patients had lower absolute phagocyte counts; however, the chemiluminescence activity calculated per phagocyte count was significantly depressed in advanced HIV infection, indicating the impairment of phagocytic cell function and a reduction in number. Data were similar when hI-OPZ was used as a stimulus. The chemiluminescence of unprimed phagocytes at circulating opsonin receptor (COR) activity relative to maximally primed phagocytes (COR/MOR ratio) was significantly higher for HIV+ patients as compared with control subjects and indicates a defect in phagocyte priming. Alternatively, the phagocytes do not increase chemiluminescence with priming because they have already been primed or activated in vivo. In late-stage disease, decreased opsonin receptor-dependent respiratory burst activity contributes to the risk of secondary bacterial infections.     

New enhancers for the chemiluminescent peroxidase catalysed chemiluminescent oxidation of pyrogallol and purpurogallin

The effects of various boronate compounds, 4-biphenylboronic acid, 4-bromobenzene-boronic acid, trans-4-(3-propionic acid)phenylboronic acid and 4-iodophenylboronic acid, on the horseradish peroxidase (HRP) catalysed chemiluminescent oxidation of pyrogallol and purpurogallin by peroxide were investigated. trans-4-(3-Propionic acid)phenylboronic acid produced a 13.7-fold enhancement in the peak light emission from the chemiluminescent HRP catalysed pyrogallol reaction (detection limit for HRP < 1.25 fmol). At low enhancer concentration a single peak of light emission was observed and as the enhancer concentration increased the time to peak light emission became progressively longer. The chemiluminescence showed two peaks at higher concentrations (> 54.3 mumol/L) and the individual peak times depended upon the concentration of the enhancer. All of the boronates enhanced peak light emission in the chemiluminescent HRP catalysed purpurogallin reaction. 4-Biphenylboronic acid was the most effective and it enhanced peak light emission 314-fold. The practical detection limit for HRP (Type VIA) using this enhancer was 4.18 pmol (peak emission at 20 minutes). This compound also enhanced peak light emission 232-fold from a chemiluminescent HRP-purpurogallin reaction in which molecular oxygen replaced peroxide as the oxidant.     

Metastasis induced by overexpression of p185neu-T after orthotopic injection into a prostatic epithelial cell line (NbE)

The effects of the composition of bacteriological growth media on the light output in a chemiluminometric assay of beta-galactosidase in Escherichia coli using 1,2-dioxetane substrates has been studied. In this assay a basic conflict exists between conditions that promote optimal bacterial growth and those conducive to maximal chemiluminescence. Common medium ingredients such as yeast or beef extract, protein hydrolysates and lactose suppress light emission and/or lead to high backgrounds. Quenching of light emission is probably partly due to light absorption by medium ingredients such as oxgall, and partly to interference with the reaction triggering the chemiluminescent process. Elevated backgrounds are caused by the presence of high concentrations of protein hydrolysates, which interact with the alkali in the accelerator solution. Only two purposely developed media, i.e. ILM and Colicult are shown to reconcile the requirements of growth support with that of optimal luminescent properties.     

Bioethics of the refusal of blood by Jehovah''s Witnesses: Part 2. A novel approach based on rational non-interventional paternalism

Due to costs in using and disposing of radiochemicals and to health considerations, we have been developing applications which include non-isotopic detection of DNA and proteins using chemiluminescence. Our major interests are in the detection of viral nucleic acids and in the analysis of transgenic plants. Generally, probes were labelled with digoxigenin, either by the random priming method or by PCR, and then detected with CSPD or CDP-Star. We routinely use a tissue blotting protocol for diagnosing TYLCV, a plant virus becoming a post in the Mediterranean region. Test results were comparable with those using the same radiolabelled probe. When total nucleic acids are extracted from the plant samples and used in dot-blot or Southern blot assays, viral DNAs are promptly detected by chemiluminescence. In transgenic plants, chemiluminescence was used to detect the transgene on genomic Southern blots, the transgenic mRNAs on Northern blots, and the transgenic protein on Western blots. In Southern and Northern blots, the quality of the results obtained was usually satisfactory, but not as good as with a radiolabelled probe, the main problem being the signal-to-background ratio. Our goal is now to improve the quality of results in demanding applications such as genomic Southern blots, by reducing the background on membranes.     

1,2-Diarylethylenediamines as sensitive pre-column derivatizing reagents for chemiluminescence detection of catecholamines in HPLC

Chemiluminescence detection in high-performance liquid chromatography for derivatives of catecholamines (norepinephrine, epinephrine and dopamine) and isoproterenol was studied on the basis of the peroxyoxalate chemiluminescence reaction. The amines and isoproterenol, derivatized with 1,2-diarylethylenediamines, were separated on a reversed-phase HPLC column (TSK gel ODS-120T) with isocratic elution using a mixture of imidazole buffer (pH 5.8, 120 mM)-methanol-acetonitrile (6:2:9, v/v/v). The eluate was detected by a post-column chemiluminescence reaction system, using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl]oxalate and hydrogen peroxide. Of the 141,2-diarylethylenediamines investigated, it was found that 1,2-bis(3-chlorophenyl)ethylenediamine, 1,2-bis(3,4-dichlorophenyl)-ethylenediamine and 1,2-bis(4-chlorophyenyl)ethylenediamine were the most sensitive derivatives for all catecholamines. The derivatization and peroxyoxalate chemiluminescence reaction conditions were optimized for 1,2-bis(3-chlorophenyl)-ethylenediamine. The chromatographic detection limits for catecholamines were approximately 40-120 amol for an injection volume of 100 microliters (signal-to-noise ratio of 3).     

Class II MHC/peptide complexes on T cell antigen-presenting cells: agonistic antigen recognition inhibits subsequent antigen presentation

Conventional in situ hybridisation (ISH) usually requires the presence of at least 10-50 copies of the nucleic acid sequence in question per cell. In situ PCR has been proposed as an alternative method, which may yield single-copy sensitivity, but shows a relatively high rate of false-negative or even false-positive reactions. Very recently, possible alternatives have been described, which can be performed in routine laboratories without the need for expensive equipment. Streptavidin-Nanogold-Silver ISH is an easy-to-perform assay, which can be applied to detect low copy numbers of nucleic acid sequences in paraffin sections and cytological preparations. Its combination with labelled tyramides (TSATM = tyramide signal amplification, also known as CARD = catalysed reporter deposition) can achieve single gene copy sensitivity in detecting DNA viruses and also shows very high sensitivity for RNA detection. Possible applications include the early recognition of viral infection, cancer-associated genes, genetic diseases, and also the specific detection of mRNA.     

Comparison of the Amplicor HIV-1 monitor test and the nucleic acid sequence-based amplification assay for quantitation of human immunodeficiency virus RNA in plasma, serum, and plasma subjected to freeze-thaw cycles

The Amplicor HIV-1 Monitor test was compared to the nucleic acid sequence-based amplification (Nasba) assay system for the quantitation of human immunodeficiency virus (HIV) RNA in three different types of clinical samples: plasma, serum, and plasma subjected to freeze-and-thaw cycles. Each assay detected HIV RNA in the same 73 (90%) of 81 samples tested, and the quantitative results obtained with the two assays were significantly correlated. Both assays detected higher RNA levels in patients with CD4+ cell counts lower than 200 cells/mm3 than in patients with CD4+ cell counts higher than 200 cells/mm3. In addition, RNA levels in plasma higher than 5 logs predicted higher numbers of clinical events than did RNA levels in plasma lower than 5 logs. Quantitation of HIV RNA in paired plasma and serum samples showed lower HIV RNA content in serum than in the paired plasma sample, with mean differences between HIV RNA contents of plasma and serum of 0.54 and 0.28 log RNA copy/ml by the Nasba assay and the Amplicor HIV-1 Monitor assay, respectively. No significant loss of HIV RNA was detected with either assay in plasma samples subjected to multiple freeze-and-thaw cycles. These studies demonstrate that the Nasba and Amplicor assays perform similarly with plasma and serum samples. Further, the results indicate that freeze-and-thaw cycles do not result in significant loss of detectable HIV RNA.     

Post-column derivatization for fluorescence and chemiluminescence detection in capillary electrophoresis

Instrumental developments and applications of post-column derivatization for fluorescence and chemiluminescence detection in capillary electrophoresis (CE) are reviewed. Various systems to merge the reagent solution with the separation medium have been developed, including coaxial capillary reactors, gap reactors and free solution or end-column systems. For all reactor types the geometry of the system, as well as the method to propel the reaction mixture (by pressure or by voltage) appeared to be critical to preserve the separation efficiency. Plate numbers of over 100,000 could be realised with different reactors. The strict requirements on the rate of post-column derivatization reactions to be applied in CE limit the number of different reagents that have been used. For fluorescence detection, with laser or lamps as the excitation source, so far mainly o-phthalaldehyde and its naphthalene analogue have been used as reagent. Derivatization systems that are based on complexation reactions also showed good promise for application in CE. Detection limits could be obtained that were comparable to those obtained after pre-column derivatization. Various reagents for chemiluminescence detection (e.g. the luminol and peroxyoxalate systems) have been studied. The often complicated chemistry involved made application of these reagents in CE even more difficult. Results obtained so far, in terms of sensitivity, have not been up to expectation, with detection limits usually in the order of micromol l(-1).     

DNA probe hybridisation in microwells using a new bioluminescent system for the detection of PCR-amplified HIV-1 proviral DNA

A new bioluminescent detection system combined with a sandwich DNA hybridisation reaction in microwells has been developed for the detection of human immunodeficiency virus type 1 (HIV-1) provirus DNA amplified by the polymerase chain reaction (PCR). First, a fragment of the HIV-1 gag gene was amplified. The amplified DNA fragments were denatured and hybridised to a capture probe immobilised in microwells and to a biotinylated detection probe. A streptavidin-pyruvate kinase conjugate could then react on the biotinylated probe and the kinase activity detected by means of the luciferin-luciferase system, with production of a bioluminescent signal. This sandwich assay followed by a bioluminescent reaction detected as little as 7 amol of target DNA. The bioluminescent assay detected 5 HIV copies generated after one round of PCR, even if no band was seen on an agarose gel. The assay was applied to the detection of HIV-proviral DNA in peripheral blood mononuclear cells after one round of PCR and allowed to clearly identify a positive sample as compared to nested PCR.     

The clinical utility of viral quantitation using molecular methods

BACKGROUND: The quantitation of viral nucleic acids in biological fluids has become increasingly desirable over the past several years. To this end, a number of quantitative molecular procedures have been developed. OBJECTIVES: The objective was to review the current literature on the molecular techniques used in the quantitation of viral nucleic acids and to assess the appropriateness of these methods for clinical use. RESULTS: Assays involving both target and signal amplification are now available for the accurate and precise quantitation of viral burden in infected patients. These methods include quantitative polymerase chain reaction (PCR), branched chain signal amplification (bDNA), nucleic acid sequence-based amplification (NASBA) and the SHARP signal and hybrid capture systems. Our understanding of the natural history and pathogenesis of viruses such as the human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) may be greatly facilitated by accurate determinations of viral and infected cell burden. Quantitation of viral load in infected individuals may also be useful to assess disease progression, monitor the efficacy of therapy and to predict treatment failure and the emergence of drug-resistant viruses. CONCLUSION: Precise, accurate and reproducible quantitation of viral load is now feasible. Molecular assays for viral quantitation should have a considerable impact on medical research and clinical care.     

Detection of plant viruses--biotechnological and molecular advances

Recent advances in biotechnology and molecular biology have played a significant role in development of rapid, specific and sensitive assays for detection of plant viruses. Production of monospecific polyclonal antibodies, monoclonal antibodies have enabled to group isolates of viruses and distinction of closely related strains. In cDNA hybridization applications, there is an increasing interest to employ non-radioactive probes for detection of nucleic acids. Detection limit of nucleic acid is remarkably comparable to those of radioactive labelled probes. Application of polymerase chain reaction (PCR) has made it possible to amplify the low numbers of viral RNA/DNA molecules and their subsequent detection. Underlying principles, their advantages and disadvantages for application of monospecific polyclonal antibodies, hybridoma technology, molecular hybridization and PCR technology with reference to detection of plant viruses have been discussed in this review.     

The importance of urinary protein excretion during conservative management of severe preeclampsia

A chemiluminescence in situ hybridization method was developed for the search of B19 parvovirus DNA in bone marrow cells, employing digoxigenin-labeled B19 DNA probes, immunoenzymatically detected with a highly sensitive 1,2-dioxetane phosphate as chemiluminescent substrate. The light emitted from the in situ-hybridized probe was analyzed and measured by a high-performance luminograph connected to an optical microscope and to a personal computer for the quantification of the photon fluxes from the single cells and for image analysis. The chemiluminescence in situ hybridization was applied to bone marrow cell smears of patients with aplastic crisis or hypoplastic anemia, who had been previously tested by in situ hybridization with colorimetric detection, dot blot hybridization, and nested PCR. The chemiluminescent assay provided an objective estimation of the data, proved specific, and showed an increased sensitivity in detecting B19 DNA compared with in situ hybridization with colorimetric detection.     

Comparison of 5-hydroxy-2, 3-dihydrophthalazine-1, 4-dione and luminol as co-substrates for detection of horseradish peroxidase in enhanced chemiluminescent reactions

The utility of 5-hydroxy-2, 3-dihydrophthalazine-1, 4-dione (HDP) as a co-substrate for the chemiluminescent detection of horseradish peroxidase was assessed. Several substituted aryl boronic acid derivatives (4-phenyl, 4-iodo) acted as potent enhancers of the peroxidase catalyzed reaction. Addition of chelating agents (EDTA) and surfactants (Tween-20 and [poly (vinylbenzyl)tributylphosphonium chloride-poly (vinylbenzyl) trioctylphosphonium chloride copolymer]) modulated background light emission and the intensity and duration of the signal from both HDP and luminol. However, HDP was found to be inferior to luminol in the peroxidase assay. Comparative studies revealed that at 500 amol of peroxidase the S/B was ten-fold higher using a commercial luminol-based signal reagent as compared with an HDP-EDTA-Tween-20 reagent (S/B t = 0 min 21.8 vs 1.7, S/B t = 10 min 17.8 vs 2.0).     

Reevaluation of the sensitivity of screening assays for anti-HIV antibodies. The Retrovirus Work Group of the SFTS. French Society of Blood Transfusion

A comparative evaluation of the sensitivity of anti-HIV screening assays has been recently performed with a selected panel including all the actually known difficulties of HIV serology: Very recent seroconversions (per-seroconversions) and recent HIV1/M seroconversions, HIV1/M seropositive with a subtype different from B, HIV1 group 0 and HIV2. The criteria of sensitivity were to recognize as positive all the seropositive samples and at least 2 of the 8 per-seroconversions. The 18 combined ELISA fulfilled these criteria, except for one assay with a deficient HIV2 sensitivity. However, all these assays do not have the same level of sensitivity. The most important differences were observed with the pre-seroconversion samples, the number of positive results on such samples varying from 2 to 8. The capacity of a screening assay to early detect an HIV-infected subject is a supplementary criterium to retain, particularly in blood transfusion screening, in order to shorten the "window" period.     

Laboratory diagnosis of tuberculosis: past, present, and future

Resurgence of tuberculosis justifies extraordinary efforts to expedite TB diagnosis and susceptibility testing. This demands that laboratory support expand to a "second generation" of methods and procedures, including rapid availability of fluorochrome smears of concentrated specimens, faster techniques for detection (e.g., the BACTEC radiometric broth system and microcolony detection), quicker identification (e.g., high-pressure liquid chromatography, nonisotopic genetic probes), more rapid susceptibility testing methods (e.g., BACTEC), and reporting of these results as critical values. Guidelines have been established for turnaround time for results of smears, TB organism identification, and susceptibility testing to usual first-line drugs. A "third generation" of laboratory techniques soon will make testing not only more effective but also more efficient. These methods include direct testing of respiratory specimens through nonisotopic genetic probes as well as nucleic acid amplification techniques utilizing polymerase chain reaction (PCR) and other molecular procedures. These new procedures and protocols place heavy demands on laboratory test volume, technologist time and costs. For the healthcare system or clinical laboratory without the resources to deal with these new demands, referral of TB specimens represents a reasonable alternative, as long as transport is adequate to meet current CDC and other guidelines for turnaround time.