title: Evaluation of a commercially available synthetic RNA lipid enveloped control molecule.
abstract: Detection of viral ribonucleic acid with RT PCR is a useful tool for viral detection. One of the drawbacks of this technique is the difficulty in including an internal control molecule to ensure the validity of the extraction and amplification process. In this study the potential usefulness of a novel lipid enveloped commercially available RNA control molecule is investigated. Initial optimisation of the detection assay was performed by amplification of IC (internal control) spiked into PCR water. Thirty-two clinical respiratory samples were spiked with the IC before and after extraction and RT PCR was then performed. Inefficient extraction was simulated. Inhibition of the RT PCR was achieved by serial dilution of heparin sulfate into samples post extraction. No Targets that matched the IC (Internal Control) primers were identified in 32 extracted sputum samples as determined by the absence of non specific amplification curves. The unextracted IC had an increased CT (cycle threshold) value compared to IC that had been extracted. Inefficient extraction was detected by an increased CT. Increasing concentrations of heparin inhibited the PCR in a predictable fashion. The Bioline IC molecule provides a stable RNA IC that has acceptable performance characteristics.
authors.affiliation: The Townsville Hospital, Townsville, Queensland, Australia; Microbiology, Pathology Queensland, Townsville, Queensland 4814, Australia. Electronic address: sam.maloney@dhhs.tas.gov.au.
authors.forename: Samuel
authors.initials: S
authors.lastname: Maloney
authors.affiliation: Microbiology, Pathology Queensland, Townsville, Queensland 4814, Australia.
authors.forename: Fleur
authors.initials: F
authors.lastname: Francis
authors.affiliation: Molecular Diagnostic Unit, Pathology Queensland, Brisbane, Queensland 4006, Australia.
authors.forename: Cheryl
authors.initials: C
authors.lastname: Bletchly
authors.affiliation: The Townsville Hospital, Townsville, Queensland, Australia; Microbiology, Pathology Queensland, Townsville, Queensland 4814, Australia. Electronic address: robert.norton@health.qld.gov.au.
authors.forename: Robert
authors.initials: R
authors.lastname: Norton
mesh_terms: D006801:Humans; D008081:Liposomes / Q000138:chemical synthesis; D025202:Molecular Diagnostic Techniques / Q000379:methods / Q000592:standards*; D012313:RNA / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D012328:RNA Viruses / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D013183:Sputum / Q000821:virology
chemical_list: D008081:Liposomes; D012313:RNA
keywords: Amplification control; Extraction control; Internal control; RNA control; Real-time PCR; Reverse transcriptase PCR