Clementi M, Bagnarelli P, Valenza A, Menzo S; National Conference on Human Retroviruses and Related Infections.

Program Abstr First Natl Conf Hum Retrovir Relat Infect Natl Conf Hum Retrovir Relat Infect 1st 1993 Wash DC. 1993 Dec 12-16; 84.

Institute of Microbiology, University of Ancona, Ancona, Italy.

It has recently been observed that competitive reverse transcription polymerase chain (cRT-PCR) is the method of choice for quantifying RNA templates present at low amount in biological samples. However, several technical aspects require attention in this method including (i) correct selection of competitor templates; (ii) evaluation of competitor integrity (iii) precise quantitation of reaction products. To improve precision of competitive methods for the study of HIV-1 infection, we have developed a modified version of the cRT-PCR procedure optimized in our laboratory (Menzo et al., J Clin Microbiol 30, 1752, 1992; Bagnarelli et al., J Virol 66, 7328, 1992). In this modified procedure, the wild-type sequence (amplified using the SK38-SK39 primer set encompassing a 115 nt gag sequence) is co-amplified along with two different RNA competitors of differing length and added at differing concentration in the reaction mixture [competitor-A with an internal deletion of 18 bases (97 nt), and competitor-B, 28 bases longer than the wild type sequence (143 nt)]. In this case, competition among three sequences (two of which added at known amounts) is achieved. We optimized this modified procedure and observed that the additional control introduced in the assay (the competitor- A/competitor-B ratio) is important for the correct evaluation of cRT-PCR results.

Publication Types:

• Meeting Abstracts

Keywords:

• Base Sequence
• Evaluation Studies
• Genes, gag
• HIV Infections
• Polymerase Chain Reaction
• RNA
• RNA, Messenger
• RNA, Viral
• RNA-Directed DNA Polymerase
• Reverse Transcriptase Polymerase Chain Reaction
• analysis
• genetics
• methods

Other ID:

• 95921192

UI: 102214132

From Meeting Abstracts