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Titolo:
Influence of preassay and sequence variations on viral load determination by a multiplex real-time reverse transcriptase-polymerase chain reaction for feline immunodeficiency virus
Autore:
Klein, D; Leutenegger, CM; Bahula, C; Gold, P; Hofmann-Lehmann, R; Salmons, B; Lutz, H; Gunzburg, WH;
Indirizzi:
Univ Vet Sci, Inst Virol, A-1210 Vienna, Austria Univ Vet Sci Vienna Austria A-1210 i, Inst Virol, A-1210 Vienna, Austria Univ Zurich, Dept Vet Internal Med, Clin Lab, Zurich, Switzerland Univ Zurich Zurich Switzerland ernal Med, Clin Lab, Zurich, Switzerland Bavarian Nord Res Inst, Martinsried, Germany Bavarian Nord Res Inst Martinsried Germany s Inst, Martinsried, Germany
Titolo Testata:
JOURNAL OF ACQUIRED IMMUNE DEFICIENCY SYNDROMES
fascicolo: 1, volume: 26, anno: 2001,
pagine: 8 - 20
SICI:
1525-4135(20010101)26:1<8:IOPASV>2.0.ZU;2-3
Fonte:
ISI
Lingua:
ENG
Soggetto:
IMMUNE FUNCTION; FIV INFECTION; MODEL; PLASMA; AIDS; PCR; QUANTIFICATION; VACCINATION; CATS; QUANTITATION;
Keywords:
real-time PCR; TaqMan; FIV; quantitative RT-PCR; viral load;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Clinical Medicine
Life Sciences
Citazioni:
26
Recensione:
Indirizzi per estratti:
Indirizzo: Klein, D Univ Vet Sci, Inst Virol, Vet Pl 1, A-1210 Vienna, Austria Univ Vet Sci Vet Pl 1 Vienna Austria A-1210 1210 Vienna, Austria
Citazione:
D. Klein et al., "Influence of preassay and sequence variations on viral load determination by a multiplex real-time reverse transcriptase-polymerase chain reaction for feline immunodeficiency virus", J ACQ IMM D, 26(1), 2001, pp. 8-20

Abstract

Determination of retroviral load is an important tool in the investigationof the success of therapeutic or vaccination trials in patients infected with lentiviruses such as HIV, or with their simian (SIV) or feline (FIV) counterparts. We have developed an one-tube quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay based on the ABI Prism 7700 Sequence Detection System (TaqMan) to quantify the viral load of FIV-infected cats. Two different primer/probe systems were designed to detect a broad range of cladeA FIV isolates. Both systems are characterized by excellent reproducibility, high sensitivity, and a wide range of quantification. As a consequence of this improved precision in the quantitative RT-PCR, preassay variations have greater impact on the accuracy of the viral load estimation. To compensate for these variations, we improved the assay and developed a multiplex real-time RT-PCR, which allows simultaneous calculation of the viral copy number and the individual recovery rate in an one-tube reaction. This enablesthe rapid and accurate calculation of copy number independent of preassay variations. In further studies, two additional real-time RT-PCR assays weredesigned and used to investigate the influence of sequence variations in the binding regions for either the primers or probe. Sequence mismatches in this region had a significant effect (up to 4 logarithmic decades) on reaction efficiency. In view of the inherent variability of retroviral sequences, these results underline the necessity to check reaction efficiencies before determining viral load.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 18/01/20 alle ore 21:33:29