The polymerase chain reaction: a new tool for the understanding and diagnosis of HIV-1 infection at the molecular level.

The polymerase chain reaction (PCR) is at present the most powerful analytical tool for detection of specific nucleic acid sequences. The method is based on the in vitro amplification of DNA segments before detection with conventional hybridization techniques or visualization following electrophoresis and staining. The current diagnostic methods for HIV-1 do not allow easy identification of subgroups of infected patients including infants born to seropositive mothers, individuals with delayed serological responses to the virus, infected patients with indeterminate serology results, and patients with dual retroviral infections.

Detection of transcripts of human papillomaviruses 16 and 18 in cancer-derived cell lines and cervical biopsies by enzyme immunoassay for DNA-RNA hybrids following solution hybridization.

mRNAs of human papillomaviruses (HPV) 16 and 18 were detected in cancer-derived cell lines and genital tract biopsy specimens by a novel hybridization assay. Biotinylated whole genomic HPV DNA probes were hybridized in solution to extracted total nucleic acids. Hybrids between the labeled probes and RNA transcripts were captured on a microplate coated with an antibiotin antibody.

Diagnosis of Chlamydia trachomatis cervical infection by detection of amplified DNA with an enzyme immunoassay.

A sensitive and specific system for detection of amplified Chlamydia trachomatis DNA from cervical specimens by fluorometric quantitation in an enzyme immunoassay (EIA) format (polymerase chain reaction [PCR]-EIA) is described. The primers selected for PCR-amplified DNA were from the 15 serovars of C. trachomatis and two strains of Chlamydia pneumoniae (TWAR).

Quantitative detection of messenger RNA by solution hybridization and enzyme immunoassay.

A novel nucleic acid detection technique is described for the quantitative measurement of eukaryotic mRNA in biological samples. The procedure involves two steps: a hybridization reaction in solution with a biotinylated cDNA probe, and a conventional enzyme immunoassay that uses a monoclonal antibody for DNA.RNA hybrids to detect the specific mRNA.

Enzyme immunoassay for detection of hybrids between PCR-amplified HIV-1 DNA and a RNA probe: PCR-EIA.

An enzyme immunoassay was developed to detect human immunodeficiency virus type 1 (HIV-1) DNA amplified by polymerase chain reaction (PCR-EIA). A set of primers (outer set) was used in PCR to amplify a segment of the HIV-1 gag gene from peripheral blood mononuclear cells. Hybrids between the amplified DNA and a RNA probe were measured in a microtiter plate immunoassay using a beta-D-galactosidase-conjugated monoclonal antibody to DNA-RNA hybrids and a fluorescent substrate.

The evolving pattern of pediatric endocarditis from 1960 to 1985.

A diagnosis of endocarditis was made in 37 patients (three days to 21 years old) on the basis of the following: histology in 11; at least two positive blood cultures in patients with underlying cardiac disease in 22; less than two positive blood cultures, vegetations seen at echocardiography and a suggestive clinical syndrome in four. Twenty-six patients had primary endocarditis (17 with pre-existing cardiopathy, nine with normal hearts). The 11 others developed secondary endocarditis following heart surgery (early onset in six, late onset in five).

Immunodetection of DNA with biotinylated RNA probes: a study of reactivity of a monoclonal antibody to DNA-RNA hybrids.

A quantitative, nonisotopic hybridization assay which measures specific DNA-RNA hybrids is described. A biotinylated RNA probe is reacted in solution with a DNA target and the labeled hybrids are immobilized onto a solid phase surface with an antibiotin antibody. Bound hybrids are detected with a beta-galactosidase-labeled monoclonal antibody against DNA-RNA hybrids and are quantitated with the addition of a fluorogenic substrate.

Nonisotopic detection of RNA in an enzyme immunoassay using a monoclonal antibody against DNA-RNA hybrids.

A sensitive nonisotopic solution hybridization assay for detection of RNA is described and characterized using a pSP65 plasmid model system. The assay procedure is based on a hybridization reaction in solution between a biotinylated DNA probe and a target RNA. The biotin-labeled hybrids are captured on a microtiter plate coated with an antibody to biotin.

Comparison of colorimetric, fluorescent, and enzymatic amplification substrate systems in an enzyme immunoassay for detection of DNA-RNA hybrids.

The monoclonal antibody solution hybridization assay is a novel enzyme immunoassay for detection of RNA with a biotinylated DNA probe. To increase the sensitivity of this test, a fluorescent substrate and an enzymatic amplification cycling system were compared with a conventional colorigenic substrate for alkaline phosphatase. The fluorescent, cycling, and colorigenic substrates detected, respectively, 10, 10, and 100 amol of unbound alkaline phosphatase in 2 h.

New prospects for the diagnosis of viral infections.

The diagnosis of viral infections is important for the accurate management of patients with infectious diseases and for the monitoring of the course of epidemics in susceptible populations. The utility of traditional viral diagnostic assays is limited by the time, expense, and expertise required for the performance of tissue culture techniques. Similarly, the application of immunoassay techniques has been inhibited by the limited degrees of sensitivity and specificity which can be attained by most immunoassay methods.

Value of direct catheter staining in the diagnosis of intravascular-catheter-related infection.

Ninety-nine intravascular catheters were evaluated by a semiquantitative culture and Gram and acridine orange direct stains. A diagnosis of catheter-related infection was determined by a retrospective review of clinical records. Compared with the culture method, direct examination of catheters lacked sensitivity.

Effect of phosphoenolpyruvate carboxykinase inhibition on renal metabolism of glutamine: in vivo studies in the dog and rat.

The effect of in vivo administration of mercaptopicolinate (MCP), a potent inhibitor of phosphoenolpyruvate carboxykinase (PEPCK) and of gluconeogenesis, on renal ammonia production was studied in dog and rat. In the dog, MCP depressed only slightly renal ammonia production, but increased strikingly renal glutamine extraction. Aspartate and alanine synthesis by the kidney were also considerably enhanced.