Based on previous experience in our laboratory, we developed a real-time reverse transcriptase (RT) quantitative PCR (RT-qPCR) assay for the assessment of very low levels of HIV-1 RT activity. The RNA, acting as a template for reverse transcription into cDNA by HIV-1 RT, consisted of a synthetic RNA ad hoc generated by in vitro transcription and included a coding sequence for HSV-1 gD (gD-RNA-synt). Different conditions of variables involved in the RT-qPCR reaction, notably different amounts of gD-RNA-synt, different mixes of the reaction buffer, and different dNTP concentrations, were tested to optimize the assay. The results indicated that the gD-RNA-synt-based RT assay, in its optimized formulation, could detect a specific cDNA reverse transcription even in the presence of 1 x 10(-9) U of HIV RT. This achievement greatly improved the sensitivity of the assay over previous versions. In summary, this constructed RT-qPCR assay may be considered a promising tool for providing accurate information on very low HIV-1 RT activity.

Marino-Merlo, F., Stefanizzi, V., Ragno, A., Piredda, L., Grelli, S., Macchi, B., et al. (2022). Quantitative Evaluation of Very Low Levels of HIV-1 Reverse Transcriptase by a Novel Highly Sensitive RT-qPCR Assay. LIFE, 12(8), 1130-1145 [10.3390/life12081130].

Quantitative Evaluation of Very Low Levels of HIV-1 Reverse Transcriptase by a Novel Highly Sensitive RT-qPCR Assay

Piredda, Lucia;Grelli, Sandro;Macchi, Beatrice;
2022

Abstract

Based on previous experience in our laboratory, we developed a real-time reverse transcriptase (RT) quantitative PCR (RT-qPCR) assay for the assessment of very low levels of HIV-1 RT activity. The RNA, acting as a template for reverse transcription into cDNA by HIV-1 RT, consisted of a synthetic RNA ad hoc generated by in vitro transcription and included a coding sequence for HSV-1 gD (gD-RNA-synt). Different conditions of variables involved in the RT-qPCR reaction, notably different amounts of gD-RNA-synt, different mixes of the reaction buffer, and different dNTP concentrations, were tested to optimize the assay. The results indicated that the gD-RNA-synt-based RT assay, in its optimized formulation, could detect a specific cDNA reverse transcription even in the presence of 1 x 10(-9) U of HIV RT. This achievement greatly improved the sensitivity of the assay over previous versions. In summary, this constructed RT-qPCR assay may be considered a promising tool for providing accurate information on very low HIV-1 RT activity.
Pubblicato
Rilevanza internazionale
Articolo
Esperti anonimi
Settore BIO/19
Settore CHIM/08 - Chimica Farmaceutica
English
Con Impact Factor ISI
human immunodeficiency virus
in vitro transcription
quantitative PCR assay
reverse transcriptase
Marino-Merlo, F., Stefanizzi, V., Ragno, A., Piredda, L., Grelli, S., Macchi, B., et al. (2022). Quantitative Evaluation of Very Low Levels of HIV-1 Reverse Transcriptase by a Novel Highly Sensitive RT-qPCR Assay. LIFE, 12(8), 1130-1145 [10.3390/life12081130].
Marino-Merlo, F; Stefanizzi, V; Ragno, A; Piredda, L; Grelli, S; Macchi, B; Mastino, A
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/307475
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