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J Virol Methods. 2008 Dec 2. [Epub ahead of print]
Evaluation of Nanogen MGB Alert((R)) Detection Reagents in a multiplex real-time PCR for influenza virus types A and B and respiratory syncytial virus.
Hymas WC, Hillyard DR. - Associated Regional and University Pathologists (ARUP), Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108, United States.
A multiplex real-time RT-PCR assay that detects influenza A, influenza B and respiratory syncytial virus (RSV) using the MGB Alert((R)) 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.
PMID: 19061916 [PubMed - as supplied by publisher]
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Evaluation of Nanogen MGB Alert((R)) Detection Reagents in a multiplex real-time PCR for influenza virus types A and B and respiratory syncytial virus.
Hymas WC, Hillyard DR. - Associated Regional and University Pathologists (ARUP), Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108, United States.
A multiplex real-time RT-PCR assay that detects influenza A, influenza B and respiratory syncytial virus (RSV) using the MGB Alert((R)) 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.
PMID: 19061916 [PubMed - as supplied by publisher]
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