Application of internal control based on recombinant retroviral particles for increasing the PCR assay reliability

Abstract

Molecular diagnostic tests based on PCR preceded by reverse transcription (RT-PCR) are now used commonly for the detection of viral pathogens with RNA genomes. The application of internal controls to validate the entire process of these assays is necessary to prevent false-negative results caused by inhibition or inefficient extraction. In the present study a strategy to produce a new type of internal control for RT-PCR based on recombinant retroviral particles is described. Cell clones stably producing retroviral particles were established by transfecting GP+env-AM12 packaging cells with constructed MoMuLV-derived retroviral vector pLneo/gfp and subsequent cultivation on selective medium with G418. Theegfpgene was used as a target for primers and hybridization probe design for real-time RT-PCR assay and as a marker for flow cytometry analysis of eGFP expression by transfected cells. The developed internal control is stable and ribonuclease resistant, economical to produce, noninfectious and safe for routine use. It closely mimics the natural virus and could be successfully used to monitor all the stages of RT-PCR, including nucleic acid extraction, RNA reverse transcription and amplification.