Comparison of selective polymerase chain reaction primers and differential probe hybridization of polymerase chain reaction products for determination of relative amounts of codon 215 mutant and wild-type HIV-1 populations.

A mutation at codon 215 of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) gene results in decreased sensitivity to zidovudine (ZDV). In order to follow changes in codon 215 mutant (MUT) and wild-type (WT) populations in the plasma of patients during therapy, two polymerase chain reaction (PCR) procedures were investigated. The first was a nested, selective PCR, wherein a first round with viral-specific primers was followed by a second round with allele-specific primers. Although the procedure is relatively sensitive, some samples in the first round of PCR could not be amplified. In mixing experiments, mispriming of the MUT primer made relative determination of quantities subjective and difficult. Differential hybridization of PCR product with probes specific for codon 215 MUT or WT sequences was also investigated. A probe directed to a highly conserved region of the RT gene in the amplified PCR product was used to determine the total amount of PCR product analyzed. Differential hybridization was linear and reproducible over several logs of MUT:WT ratios, and determination of a 1:100 ratio of MUT:WT was readily achieved. When applied to longitudinal samples from three patients, dramatic changes in each population were readily apparent. These changes were evaluated with regard to viral load.

Rapid and precise quantification of HIV-1 RNA in plasma using a branched DNA signal amplification assay.

The level of human immunodeficiency virus type 1 (HIV-1) RNA in human plasma has been quantitated directly with use of a solid-phase nucleic acid hybridization assay, based on branched DNA (bDNA) signal amplification technology with chemiluminescent detection. Signal amplification is accomplished by the incorporation of sites for 1,755 alkaline phosphatase-labeled probes per genome of HIV-1, after successive hybridization of target-specific oligonucleotides and bDNA amplifier molecules. The assay is performed in microwells, much like an immunoassay, and is amenable to routine laboratory use. Reproducibility and specificity studies indicated that the bDNA method was precise and showed no reactivity with seronegative donors. HIV-1 RNA levels were quantitated for 348 seropositive specimens, with a detection rate of 83% for those specimens from patients with < 500 CD4+ T-cell counts. Plasma RNA levels were found to change with disease stage, and in response to antiviral therapy. Quantitation of HIV-1 RNA in the plasma of HIV-1-infected patients, with use of the bDNA assay, may be a useful method for monitoring HIV-1 disease progression and therapeutic response.

Direct and quantitative detection of HIV-1 RNA in human plasma with a branched DNA signal amplification assay.

AIM: To determine the relative effect of sample matrix on the quantitation of HIV RNA in plasma. METHOD: Two HIV-positive specimens were diluted into five and 10 different HIV-negative plasma samples, respectively. Branched DNA signal amplification technology and reverse-transcriptase polymerase chain reaction were used to measure the viral load. RESULTS: In one sample the viral load by polymerase chain reaction ranged from undetectable to 1.9 x 10(5) copies/ml, and the branched DNA results ranged from 2.6 x 10(4) to 4.2 x 10(4) HIV RNA equivalent/ml. In the other sample the corresponding figures were 6.3 x 10(4) to 5.5 x 10(5) copies/ml and 5.7 x 10(4) to 7.5 x 10(4) HIV RNA equivalents/ml. CONCLUSION: In contrast to reverse-transcriptase polymerase chain reaction the branched DNA signal amplification assay does not require a separate extraction step or enzymatic amplification of the target. Therefore this measurement is less affected by the sample matrix and the signal generated is directly proportional to the viral load.

Patient-side immunoassay system with a single-use cartridge for measuring analytes in blood.

We describe an immunoassay system suited to patient-side assay of therapeutic drugs and blood proteins. The system consists of an electronic monitor and single-use plastic cartridges containing dry reagents and liquid diluents. The monitor is turned on by insertion of a cartridge. To run the test, the user applies an unmeasured drop of blood to the cartridge when prompted by the monitor. All subsequent steps are performed without further user intervention and results are provided in less than 3 min. The system hemolyzes and precisely dilutes the blood. Hemoglobin concentration is measured, then the diluted blood is precisely diluted further and mixed with two dry reagents. The drug concentration is measured by a turbidimetric latex agglutination inhibition reaction. Theophylline and hemoglobin assay results for clinical samples correlate well with results of widely used comparison methods.

Application of a rapid non-radioisotopic nucleic acid analysis system to the detection of sexually transmitted disease-causing organisms and their associated antimicrobial resistances.

We devised a versatile method for detecting nucleic acids in crude lysates of biological samples. A controlled network of nucleic acid hybrids composed of the target fragment, several oligonucleotide probes, branched DNA amplifiers, and labeled oligonucleotides is produced on a solid phase to ultimately incorporate 60 to 300 molecules of alkaline phosphatase, which are detected with a chemiluminescent substrate. The visible light output can be recorded on a luminometer or on instant black-and-white film. Assays have been developed for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and for genes conferring penicillin and tetracycline resistance. Conducted much like ELISAS, the assays are performed in about 4 h (for 96 samples) in microliter dishes. The molecular detection limit of approximately 50,000 molecules of double-stranded DNA has permitted us to detect 1 to 10 x 10(3) of C. trachomatis and N. gonorrhoeae with specific probe sequences. Both plasmid and genomic target sequences can be detected by the same procedure. All of the assay components, except for a set of unmodified oligonucleotide probes, are universally applicable for all targets.

The specificity of pilin DNA sequences for the detection of pathogenic Neisseria.

A nucleic acid hybridization assay for the detection of the pilin gene of Neisseria gonorrhoeae has been devised. The method involves solution hybridization of pilin specific synthetic oligonucleotide probes to genomic DNA in crude cell lysates. This is followed by capture of the probe-target complex onto a microtitre dish well, signal amplification and labelling based on horseradish peroxidase conjugated to oligonucleotides. Detection is achieved with a chemiluminescent enzyme substrate. With a detection limit of about 20,000 cells, the 4-h assay is as sensitive as a radioactive dot-blot method. Over 150 strains of Neisseria gonorrhoeae collected from a variety of sources were detected with the assay. Several N. meningitidis serogroups were also found to react positively. No reactivity was observed with non-pathogenic Neisseria spp. or with other known pathogenic or normal microbial inhabitants of the human urogenital tract.

Method for detecting antibody formation to Neisseria gonorrhoeae.

The use of Concanavalin A as a coupling agent for sensitization of sheep erythrocytes with an alkaline extract of Neisseria gonorrhoeae organisms is described in this paper. The sensitized erythrocytes were used in a plaque assay for enumeration of antibody-producing cells and in hemagglutination and hemolysin tests for determination of serum antibody. Rabbits were immunized intraperitoneally, intravenously, or via the footpad with viable N. gonorrhoeae organisms. Plaque-forming cells were found in draining lymph nodes and in the spleens of these rabbits. The cells from some rabbits also lysed sheep erythrocytes coated with an alkaline extract prepared from Neisseria meningitidis organisms. No antibody-producing cells were found in the lymphoid tissues of normal rabbits. Rabbits immunized by all three routes developed high hemagglutination and hemolysin titers. Hemolysin titers were significantly higher than hemagglutination titers when both tests were performed on the same serum samples. Pooled antiserum from these rabbits also lysed erythrocytes coated with a meningococcal antigen, but titers for the specific immunizing species were higher.

Nonspecific resistance to intraocular infection. I. Elicitation of resistance by Neisseria meningitidis.

Both eyes of rabbits whose right eyes had previously been injected with Neisseria meningitidis were injected with Diplococcus pneumoniae. The pneumococci failed to grow in the right eye of most rabbits challenged 2 and 4 days after the injection of the meningococci, but grew well in all left eyes. The "protective" effect was less pronounced in the rabbits challenged on days 6 and 9 and virtually absent when the interval between the two injections was 14 or 22 days. Pneumococci grew equally well in both eyes of rabbits that had received nitrogen mustard treatment prior to the intravitreal injection of meningococci, indicating a possible role for the polymorphonuclear leukocyte in the protective effect.