Rapid detection of the pandemic 2009 H1N1 virus M gene by real-time and gel-based RT-PCR assays.
BACKGROUND
Since the first pandemic 2009 H1N1 (pH1N1) virus was isolated from humans, it has also been detected in other mammalian (pigs, cats, dogs, ferrets) and avian (turkey) species, most likely because of cross-species transmission from humans. The pH1N1 contains six genes derived from swine influenza viruses (SIVs) currently circulating in North America of human- (PB1), avian- (PB2, PA), and swine- (HA, NP, and NS) origin and two genes (NA and M) derived from Eurasian SIVs. The novel genetic composition of pH1N1 necessitates development of novel molecular and serological assays to differentiate the pH1N1 virus from circulating human, swine, turkey, canine, and feline influenza viruses.


METHODS
To detect and discriminate the pH1N1 from currently circulating SIVs in North America, we developed and evaluated a TaqMan probe-based real-time and a gel-based RT-PCR assay, both targeting the pH1N1 matrix gene.


RESULTS
The real-time and gel-based RT-PCR assays were able to specifically detect the pH1N1 M gene and differentiate it from SIVs circulating in North America, including the classical and novel human-like H1N1 influenza virus as well as H1, H2, and H3 subtype triple reassortant SIVs. Both assays were highly sensitive and specific for the pH1N1 virus.


CONCLUSIONS
The newly developed pH1N1-specific real-time and gel-based RT-PCR assays can be used to detect and differentiate the pH1N1 virus from currently circulating SIVs in North America. We suggest a combinational diagnostic approach where the real-time RT-PCR is used for high-throughput detection of influenza positive or suspect samples and the gel-based RT-PCR for confirmation and sequencing of the M-gene.
Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA.
Wenjun
W
Ma
Richard
R
Oberst
Xi
X
Li
Deborah
D
Clouser
Richard
R
Hesse
Raymond
R
Rowland
Juergen A
JA
Richt
D000818:Animals; D017931:DNA Primers / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D015345:Oligonucleotide Probes / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014763:Viral Matrix Proteins / Q000235:genetics*; D014773:Virology / Q000379:methods*
D017931:DNA Primers; C031540:M-protein, influenza virus; D015345:Oligonucleotide Probes; D014763:Viral Matrix Proteins