title: Evaluation of Nanogen MGB Alert Detection Reagents in a multiplex real-time PCR for influenza virus types A and B and respiratory syncytial virus.
abstract: A multiplex real-time RT-PCR assay that detects influenza A, influenza B and respiratory syncytial virus (RSV) using the MGB Alert Influenza A&B/RSV Detection Reagent RUO (Nanogen, San Diego, CA) was developed. The Nanogen detection reagents consist of PCR primers and minor groove binder-conjugated hybridization probes for each virus and an internal control. Virus typing was determined by post-PCR melt curve analysis. A non-competitive armored RNA internal control was co-extracted with each sample to monitor nucleic acid extraction and RT-PCR. The assay was evaluated using a collection of culture, DFA and RT-PCR (Hexaplex, Prodesse, Waukesha, WI) positive and negative samples. The real-time multiplex assay detected 236 of 237 positive specimens for a 99% correlation. Of 30 Hexaplex negative samples tested, the multiplex real-time assay detected an additional 7 positives confirmed using additional PCR assays. Melt curve analysis for each virus produced average melting peaks of 60.4 degrees C, 66.7 degrees C and 69.4 degrees C for influenza A, influenza B and RSV respectively. Sequence analysis of 7 influenza A samples producing aberrant melt curves, confirmed the presence of a single nucleotide polymorphism beneath the influenza A probe. The limit of detection of each virus in 4 different sample types was measured to be between 7 and 806 copies. Overall, the multiplexed real-time RT-PCR assay was sensitive, robust and easy to use.
authors.affiliation: Associated Regional and University Pathologists (ARUP), Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108, USA. hymasw@aruplab.com
authors.forename: Weston C
authors.initials: WC
authors.lastname: Hymas
authors.forename: David R
authors.initials: DR
authors.lastname: Hillyard
mesh_terms: D017931:DNA Primers; D006801:Humans; D007202:Indicators and Reagents / Q000032:analysis; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D009981:Influenza B virus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis; D018113:Respiratory Syncytial Virus, Human / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D007202:Indicators and Reagents; D012367:RNA, Viral