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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors
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Published on: April 9, 2014

Substrate Recognition Governs Reverse Transcriptase Resistance to Diagnostic Inhibitors in RT-qPCR.

Inês F Costa1,2,3, Vânia O Fernandes1, Victor D Alves2,3

  • 1NZYtech-Genes & Enzymes, Campus do Lumiar, Building J, 1649-038 Lisbon, Portugal.

Diagnostics (Basel, Switzerland)
|June 26, 2026
PubMed
Summary
This summary is machine-generated.

Engineered reverse transcriptase (RT) variants show enhanced robustness against inhibitors, crucial for sensitive RNA diagnostics. Optimal inhibitor resistance occurs at moderate temperatures due to improved enzyme-nucleic acid interactions.

Keywords:
RT-qPCRclinical inhibitorsenzyme engineeringinhibitor resistancemolecular diagnosticsreverse transcriptasesubstrate recognition

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Diagnostic Development

Background:

  • Reverse transcription (RT) is vital for RNA diagnostics.
  • Enzyme inhibitors in samples often impede RT activity.
  • Engineered Moloney Murine Leukemia Virus RT variants (V1-V5) were studied for inhibitor resistance.

Purpose of the Study:

  • To investigate the molecular basis of inhibitor resistance in engineered RT variants.
  • To identify specific mutations conferring robustness against common inhibitors.
  • To optimize RT performance for sensitive diagnostic applications.

Main Methods:

  • Integrated structural analysis, thermal profiling, and diagnostic benchmarking.
  • Evaluated cDNA synthesis across a temperature range (40–70 °C) with 11 inhibitors.
  • Assessed 30 mutations across five engineered RT variants.

Main Results:

  • Specific mutations (E69K, E302K/R, W313F, N454K) enhanced RT robustness.
  • L435G mutation modulated enzyme flexibility, contributing to resistance.
  • Maximal inhibitor tolerance observed at moderate temperatures (≈40 °C).
  • Wild-type RT was inactivated by several inhibitors, unlike engineered variants.

Conclusions:

  • Enzyme-substrate engagement is a key factor in RT robustness.
  • Engineering RTs for inhibitor resistance is feasible for high-sensitivity diagnostics.
  • Moderate temperatures optimize RT performance under inhibitory conditions.