Competitive polymerase chain reaction assay for quantitation of HIV-1 DNA and RNA

Stieger, Martin; Démollière, C.; Ahlborn-Laake, L; Mous, Jan

doi:10.1016/0166-0934(91)90095-h

Cited by 55 publications

(18 citation statements)

References 26 publications

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“…As an alternative way to discriminate amplified target sequences from st sequences, recognition sites of a restriction enzyme or a DNA-binding protein present either in the st or the target sequence may be used (Fox et al, 1992;Lundeberg et al, 1991;Stieger et al, 1991). Without the use of labelled probes in post-PCR detection steps, however, formation of heteroduplexes between st and st amplimers during PCR may lead to significant errors (Becker-Andr~ & Hahlbrock, 1989).…”

Section: Discussionmentioning

confidence: 99%

Quantitative determination of human cytomegalovirus target sequences in peripheral blood leukocytes by nested polymerase chain reaction and temperature gradient gel electrophoresis

Schäfer

Braun

Möhring

et al. 1993

Journal of General Virology

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A competitive nested PCR-temperature gradient gel electrophoresis protocol (nPCR/TGGE) has been established for the quantification of human cytomegalovirus (HCMV) target sequences. The measurement was achieved by co-amplification of a defined copy number of an internal standard (st) and separation of st and wildtype (wt) amplimers by temperature gradient gel electrophoresis (TGGE). The number of HCMV target sequences could be precisely determined within wt/st ratios of 0.1 to 10. With 50 copies of the st sequence the detection limit of nPCR/TGGE was found to be five to l0 copies of the target sequence. Effects of sample preparation on quantitative HCMV PCR were minimized by the additional quantification offl-globin target sequences and calculation of the ratio of HCMV copies/fl-globin copies. Serial peripheral blood leukocyte specimens of 17 renal allograft recipients positive in a qualitative nested HCMV PCR were tested using nPCR/TGGE. Thirty healthy blood donors served as negative controls. Positive results were obtained by nPCR/TGGE in nine renal allograft recipients but in none of the healthy blood donors. Five of five patients with an HCMV pp65 antigenaemia and positive for HCMV IgM were positive in nPCR/TGGE. The highest HCMV/fl-globin ratios (10000 to 8000 copies HCMV/ 106 copies fl-globin) were found in transplant recipients experiencing acute clinically symptomatic HCMV infection. HCMV DNA levels in asymptomatic patients ranged from 900 to 200 copies HCMV/10 ~ fl-globin.

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Section: Discussionmentioning

confidence: 99%

Quantitative determination of human cytomegalovirus target sequences in peripheral blood leukocytes by nested polymerase chain reaction and temperature gradient gel electrophoresis

Schäfer

Braun

Möhring

et al. 1993

Journal of General Virology

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“…fluoresceinated primers and scanning laser densitometry to detect the products, is also possible. Since the control sequence can be cloned into riboprobe-type vectors, it will be possible to produce an RNA copy of the control sequence for quantification of infectious agents containing RNA genomes (for example, Steiger et al, 1991). Such systems will prove invaluable in investigations into the effects of antiviral agents on plasma levels of HIV in infected individuals.…”

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confidence: 99%

Quantification of human cytomegalovirus DNA using the polymerase chain reaction

Fox

Griffiths

Emery

1992

Journal of General Virology

119

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“…The Coulter ELISA recognizes all the core proteins equally (G. P. Toedter, personal communication) so a reduction in signal indicates a reduction in HIV particles, and not a loss in the signal due to non-cleavage of the p55 precursor molecule. Steiger et al (1991), proposed that an increase in intracellular viral RNA observed in chronically infected cells grown in 0.1 fLM concentration of this inhibitor, was probably caused by the inhibition of virion release. This would account for the reduced levels of core antigen detected in the culture medium.…”

Section: Discussionmentioning

confidence: 99%

Antiviral Properties of the HIV-1 Proteinase Inhibitor RO 31-8959

Galpin

Roberts

O’Connor³

et al. 1994

Antivir Chem Chemother

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The peptide derivative Ro 31-8959 has been shown to be a potent inhibitor of HIV proteinase with an IC50 of 2 × 10−9M, against HIV-1RF in acutely infected lymphoblastoid cells. This inhibition was not overcome by increasing the infectious dose or by extending the culture time. Similar antiviral activity was also obtained against HIV-2, SIV and several AZT-resistant strains of HIV-1. The time of addition of the inhibitor could be delayed for 22 h without significant loss of activity, supporting its mode of action as taking place late in the replication cycle of HIV-1. Ro 31-8959 also showed activity against chronically infected cells.

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Competitive polymerase chain reaction assay for quantitation of HIV-1 DNA and RNA

Cited by 55 publications

References 26 publications

Quantitative determination of human cytomegalovirus target sequences in peripheral blood leukocytes by nested polymerase chain reaction and temperature gradient gel electrophoresis

Quantitative determination of human cytomegalovirus target sequences in peripheral blood leukocytes by nested polymerase chain reaction and temperature gradient gel electrophoresis

Quantification of human cytomegalovirus DNA using the polymerase chain reaction

Antiviral Properties of the HIV-1 Proteinase Inhibitor RO 31-8959

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