Development of a real-time RT-PCR for the detection of swine-lineage influenza A (H1N1) virus infections.
BACKGROUND
A novel influenza A virus, subtype H1N1 of swine-lineage (H1N1 swl) has transmitted rapidly to many regions of the world with evidence of sustained transmission within some countries. Rapid detection and differentiation from seasonal influenza is essential to instigate appropriate patient and public health management and for disease surveillance.


OBJECTIVE
To develop a rapid and sensitive real-time reverse transcriptase polymerase chain reaction (rtRT-PCR) for confirmation of H1N1 swl.


METHODS
A one-step rtRT-PCR approach was employed to target the matrix gene of the novel influenza A/H1N1 swl and validated against a panel of seasonal influenza A (H1N1 and H3N2), swine influenza A/H1N1 and avian influenza A/H5N1 viruses. The assay following validation was then used prospectively to detect H1N1 swl positive specimens from the recent outbreaks in the UK and the Republic of Ireland.


RESULTS
The one-step H1N1 swl matrix rtRT-PCR successfully detected H1N1 swl clinical specimens and did not cross-react with seasonal influenza A, subtypes H1N1 and H3N2 viruses and swine influenza A (H1N1). The H1N1 swl matrix assay did cross react with H5N1. The H1N1 swl matrix assay was then compared to two other assays using a dilution series and a panel of untyped influenza A positive clinical samples. These experiments found the assay to have a comparable sensitivity to the established universal influenza A rtRT-PCR and was more sensitive than the H1N1 swl specific assay that targeted the H1 region.


CONCLUSIONS
The results demonstrate that the rtRT-PCR is sensitive and should be used alongside existing universal influenza A assays to rapidly detect the novel H1N1 swl virus.
Carr
Michael J
MJ
National Virus Reference Laboratory, University College Dublin, Dublin, Ireland.
Gunson
Rory
R
Maclean
Alasdair
A
Coughlan
Suzie
S
Fitzgerald
Margaret
M
Scully
Mary
M
O'Herlihy
Brian
B
Ryan
John
J
O'Flanagan
Darina
D
Connell
Jeff
J
Carman
William F
WF
Hall
William W
WW
D000818:Animals; D004196:Disease Outbreaks; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000453:epidemiology / Q000821:virology*; D007494:Ireland / Q000453:epidemiology; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics; D016865:Reassortant Viruses / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013552:Swine; D006113:United Kingdom / Q000453:epidemiology; D014763:Viral Matrix Proteins / Q000235:genetics
C488936:M1 protein, Influenza A virus; D012367:RNA, Viral; D014763:Viral Matrix Proteins