A novel non-radioactive assay for HIV-RT (RdDp) based on pyrosequencing for high-throughput drug screening

Zhang, Chang; Wu, Yang; Sun, Yuna; Hong, Chuan; Xiang, Kehui; Guo, Yu; Bartlam, Mark; Lou, Zhiyong

doi:10.1007/s13238-010-0031-0

Cited by 3 publications

(3 citation statements)

References 21 publications

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“…The same increase was, however, not seen with increased primer concentration, e.g. odT 20 . Instead, an increase in primer concentration resulted in higher raw fluorescence signal at the start but did not affect the overall fluorescence at the end of 60 min (data not shown).…”

Section: Optimization Of Primer Template and Dttp Concentrationsmentioning

confidence: 82%

“…The PERT assay has been used to study AZT drug resistance in viral vaccines [19]. Another recently developed HIV-1 RT activity measurement assay is based on the detection of released inorganic pyrophosphate (PPi) [20]. This assay has a better sensitivity compared to the previously described assays, but comes with the disadvantage that nonphosphorylated NRTIs cannot be studied if one plans to do phosphorylation step of inhibitor and extension of template in a single tube.…”

Section: Introductionmentioning

confidence: 99%

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SYBR Green II Dye-Based Real-Time Assay for Measuring Inhibitor Activity Against HIV-1 Reverse Transcriptase

et al. 2016

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There are arrays of in vitro assays to quantify the activity of HIV-1 reverse transcriptase (HIV-1 RT). These assays utilize either chemically customized/labelled nucleotides, or TaqMan probes, or radiolabeled nucleotides/primers. Although several real-time PCR assays exist commercially for measuring the RT activity, which are usually used for quantifying the viral titres, these assays are not optimized for measuring the inhibitory concentrations (IC50) of HIV-1 RT inhibitors. Moreover, a recently established inorganic pyrophosphate-coupled enzyme assay cannot be employed for studying nonphosphorylated nucleoside reverse transcriptase inhibitors (NRTIs). In the present study, we have developed a novel one-step assay with native nucleotide substrates and SYBR Green II dye to determine IC50 values of triphosphorylated NRTIs against HIV-1 RT. Using exact batches of wild-type and mutant RT, and triphosphorylated NRTIs, we showed that our method gave IC50 values for inhibitors similar to that of an earlier published colorimetric assay with BrdUTP substrate (CABS). Our assay should be suitable for high-throughput screening of antiretroviral drugs and could also be suitable for studying drug resistance profiles. Additionally, we also used our assay to study inhibition by AZT in its nonphosphorylated form by supplementing the reaction mixture with necessary kinases and ATP.

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Section: Optimization Of Primer Template and Dttp Concentrationsmentioning

confidence: 82%

Section: Introductionmentioning

confidence: 99%

SYBR Green II Dye-Based Real-Time Assay for Measuring Inhibitor Activity Against HIV-1 Reverse Transcriptase

et al. 2016

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“…Notably, a heterologous RNA as a template, the polymerase chain reaction (PCR), and various techniques for detection characterized most of this second generation of assays for the assessment of RT activity [ 5 , 6 , 7 , 8 , 9 , 10 ]. Interestingly, aside from being employed as a surrogate to estimate infectious HIV-1 [ 11 ], these methods were also adopted to assess the inhibitory activity of compounds towards HIV RT [ 12 , 13 , 14 ]. Moreover, due to the reliability of their results, RT-based assays have been proposed as a good alternative choice for monitoring patients living with HIV (PLWH) in resource-limited countries [ 15 , 16 ].…”

Section: Introductionmentioning

confidence: 99%

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

Marino-Merlo

Stefanizzi

Ragno

et al. 2022

Life

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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 × 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.

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Development and evaluation of a simple and effective RT-qPCR inhibitory assay for detection of the efficacy of compounds towards HIV reverse transcriptase

Marino-Merlo

Frezza

Papaianni

et al. 2017

Appl Microbiol Biotechnol

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Assessing the actual efficacy of compounds to directly inhibit HIV reverse transcriptase (RT) activity is a main goal in preclinical antiretroviral studies. Our previous studies demonstrated that the effects of inhibitor compounds towards HIV-RT could be efficiently assessed through a simple cell-free assay based on conventional reverse transcription PCR. In the present study, we describe a modified variant of our assay, termed RT real-time quantitative PCR inhibitory assay (RT-qPCR-IA), in which the ability of compounds to restrict the complementary DNA (cDNA) generation by HIV-RT using a specific RNA template is performed by the real-time technique, in order to improve both accuracy and sensitivity of the method. As specific RNA template, RNA extracted from stable transfectants ectopically expressing the herpes simplex virus 1 glycoprotein D gene was utilized. HIV-RT, of both commercial or house-made viral lysate origin, was employed for the assay. To assess the reliability of RT-qPCR-IA, we performed a comparative, quantitative analysis of the dose-dependent effect exerted by known nucleotide and non-nucleotide reverse-transcriptase inhibitors, using the SYBR Green dye chemistry as detection system. The results obtained with RT-qPCR-IA were compared to that obtained using a one-step PicoGreen technology-based commercial kit. The outcome of our study indicates that the development of the novel RT-qPCR-IA will provide rapid and accurate evaluation of the inhibitory efficacy of compounds towards HIV-RT activity. This evaluation could be very useful for large-scale screening of potential new anti-HIV drugs.

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A novel non-radioactive assay for HIV-RT (RdDp) based on pyrosequencing for high-throughput drug screening

Cited by 3 publications

References 21 publications

SYBR Green II Dye-Based Real-Time Assay for Measuring Inhibitor Activity Against HIV-1 Reverse Transcriptase

SYBR Green II Dye-Based Real-Time Assay for Measuring Inhibitor Activity Against HIV-1 Reverse Transcriptase

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

Development and evaluation of a simple and effective RT-qPCR inhibitory assay for detection of the efficacy of compounds towards HIV reverse transcriptase

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