Reverse Transcription Quantitative Polymerase Chain Reaction for Detection of and Differentiation Between RNA and DNA of HIV-1-Based Lentiviral Vectors

Hum Gene Ther Methods. 2017 Aug;28(4):215-221. doi: 10.1089/hgtb.2017-081.

Abstract

The purpose of the described method is the detection of and differentiation between RNA and DNA of human immunodeficiency virus (HIV)-derived lentiviral vectors (LV) in cell culture supernatants and swab samples. For the analytical surveillance of genetic engineering, operations methods for the detection of the HIV-1-based LV generations are required. Furthermore, for research issues, it is important to prove the absence of LV particles for downgrading experimental settings in terms of the biosafety level. Here, a quantitative polymerase chain reaction method targeting the long terminal repeat U5 subunit and the start sequence of the packaging signal ψ is described. Numerous controls are included in order to monitor the technical procedure.

Keywords: qRT-PCR; recombinant lentiviral vector; surveillance.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Cells, Cultured
  • Containment of Biohazards / standards
  • DNA, Viral / chemistry
  • DNA, Viral / genetics*
  • Genetic Therapy / methods*
  • Genetic Vectors / genetics*
  • HIV Long Terminal Repeat / genetics
  • HIV-1 / genetics*
  • Humans
  • RNA, Viral / chemistry
  • RNA, Viral / genetics*
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Reverse Transcriptase Polymerase Chain Reaction / standards

Substances

  • DNA, Viral
  • RNA, Viral