Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2013

Validation of a Novel RNA Internal Control for Monitoring RNA Extraction in a Routine Clinical Microbiology Laboratory (#232)

Samuel Maloney 1 , Fleur Francis 1 , Cheryl Bletchly 2 , Robert Norton 3
  1. Pathology Queensland, Townsville, QLD, Australia
  2. Pathology Queensland, Brisbane, QLD, Australia
  3. Townsville Hospital, DOUGLAS, QLD, Australia

Detection of viral ribonucleic acid (RNA) by reverse transcriptase (RT) real-time (q) polymerase chain reaction (PCR) has largely replaced viral isolation and characterisation by tissue culture and immunologic methods in clinical virology laboratories. RT qPCR is impaired by the presence of substances in some clinical samples that inhibit the reaction. These inhibitors may be incompletely removed by processing of clinical specimens and extraction of RNA. Additionally processing and extraction techniques may result in decreased RNA concentration in the analyte.

 Several different internal controls (IC) have been used as a method to monitor RT qPCR in an attempt to detect when inhibition or inefficient extraction has occurred. However identification of an appropriate control is challenging. RNAase is ubiquitous in the environment and leads to degradation of naked control RNA. RT PCR chemistry differs from traditional PCR as it involves a reverse transcription step. Optimal extraction of RNA differs from that of DNA making stable DNA controls sub-optimal as a marker of RNA extraction. “Armoured” RNA molecules have a protein coat that renders them resistant to degradation by RNAase however they have defined limitations.

We report the performance of novel RNA control particle. The particle contains control RNA contained in a lipid envelope. We evaluate its use as an amplification control alone and as a combined extraction/amplification control. The novel IC is assessed on several extraction platforms in the presence of known and unknown quantities of inhibitors and compared with an equine herpes virus (EHV) IC.

 We also assessed the particle in clinical assays as non-competitive IC of both extraction and amplification compared with the currently DNA IC.