Abstract
BackgroundA cost-effective, accurate and rapid simultaneous multiplex assay is required for testing and diagnoses of conventional and emerging viruses in clinical virology laboratories. We developed and optimized a dual priming oligonucleotide (DPO) multiplex PCR assay for detecting influenza viruses including seasonal H1N1, 2009 pandemic H1N1, H3N2, influenza B and H5N1.MethodsThe optimized multiplex DPO PCR was used to detect 233 clinical human samples. The results were compared to those obtained with RT-qPCR, conventional PCR and immunochromatographic assay.ResultsSpecificity analysis revealed that the DPO PCR assay amplified each target virus without any cross-amplification. Statistical analysis demonstrated that the multiplex DPO-PCR sensitivity was higher than for the immunochromatographic assay and lower than for qPCR, while no significant difference was observed compared with conventional PCR, when detecting influenza A and B. Additional experiments using the same sample panel indicated no significant differences between the number of positive samples detected by multiplex DPO PCR and RT-qPCR when applying a Cq with a value lower than 30.ConclusionsThe five-targeted simultaneous multiplex DPO PCR assay could be easily adopted into routine practice. This approach is cost effective with a short running time, low technical requirements for the detection of influenza virus and early diagnosis in clinical laboratories.
Highlights
A cost-effective, accurate and rapid simultaneous multiplex assay is required for testing and diagnoses of conventional and emerging viruses in clinical virology laboratories
The results suggested that the dual priming oligonucleotide (DPO) based multiplex polymerase chain reaction (PCR) system had a higher sensitivity and specificity than the other methods tested [4,5,6,7]
The purpose of this study was to develop and optimize a DPO multiplex PCR assay for detection of influenza A and B viruses, including the influenza A subtypes, such as seasonal H1N1, 2009 pandemic H1N1, H3N2, and avian influenza H5N1
Summary
A cost-effective, accurate and rapid simultaneous multiplex assay is required for testing and diagnoses of conventional and emerging viruses in clinical virology laboratories. Molecular assays are highly sensitive and specific for detecting influenza viruses such as rapid immune colloidal gold diagnostic tests, immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and viral culture. Nucleic acid based tests for respiratory viruses are currently used as a rapid and sensitive diagnostic approach for clinical specimens. A cost-effective, accurate and rapid multiplex assay is highly desirable in clinical virology laboratories for testing and diagnosing common and emerging viruses. Even though the multiplex real time polymerase chain reaction (PCR) is a rapid and sensitive method for the detection of respiratory viruses, such assays are limited to a maximum of four multiplexed targets, and the high cost
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