A new and rapid genotypic assay for the detection of neuraminidase inhibitor resistant influenza A viruses of subtype H1N1, H3N2, and H5N1

Detection of Molecular Markers of Drug Resistance in 2009 Pandemic Influenza A (H1N1) Viruses by Pyrosequencing

“…The widespread use of NAIs can lead to the emergence of resistant viruses and is a serious public health concern. For influenza surveillance laboratories, pyrosequencing offers a valuable tool for the rapid highthroughput detection of signature mutations linked to drug resistance (15,16,19,38). We previously designed primers for the detection of the most common resistance markers in seasonal H1N1 viruses (16), but due to differences in the NA sequences between the seasonal H1N1 viruses and 2009 pandemic viruses (21), a new set of primers needed to be designed and validated.…”

“…Pyrosequencing has previously been shown to provide a rapid and high-throughput method for the detection of molecular markers of drug resistance in seasonal as well as highly pathogenic avian influenza viruses (4,8,15,16,19,30,31,38).…”

Influenza Antiviral Resistance Testing in New York and Wisconsin, 2006 to 2008: Methodology and Surveillance Data

“…In addition to the sequencing methods presented here for the detection of resistance mutations, real-time PCR methods sensitive enough to allow detection of the separate, singlebase-pair differences that confer resistance to influenza antivirals are needed; several new methods have recently been published (6,14). Real-time PCR methods capable of this type of detection will allow increasing numbers of laboratories to monitor resistance, since the instrumentation necessary is already available in a large number of clinical laboratories.…”

Novel Genotyping and Quantitative Analysis of Neuraminidase Inhibitor Resistance-Associated Mutations in Influenza A Viruses by Single-Nucleotide Polymorphism Analysis

“…The resistance markers in NA gene are most commonly analyzed by the Sanger method of DNA sequencing, but this method requires purification of PCR-amplified NA segments and is timeconsuming for a large number of samples. Two other methods, TaqMan real-time PCR-based assays and pyrosequencing, provide rapid, sensitive, and quantitative analysis of resistance markers in influenza viruses (7,9,20,23), but they require costly specific labeled primers or probes. Further, pyrosequencing requires specific equipment that is not widely available.…”