A broad spectrum, one-step reverse-transcription PCR amplification of the neuraminidase gene from multiple subtypes of influenza A virus.
BACKGROUND
The emergence of high pathogenicity strains of Influenza A virus in a variety of human and animal hosts, with wide geographic distribution, has highlighted the importance of rapid identification and subtyping of the virus for outbreak management and treatment. Type A virus can be classified into subtypes according to the viral envelope glycoproteins, hemagglutinin and neuraminidase. Here we review the existing specificity and amplification of published primers to subtype neuraminidase genes and describe a new broad spectrum primer pair that can detect all 9 neuraminidase subtypes.


RESULTS
Bioinformatic analysis of 3,337 full-length influenza A neuraminidase segments in the NCBI database revealed semi-conserved regions not previously targeted by primers. Two degenerate primers with M13 tags, NA8F-M13 and NA10R-M13 were designed from these regions and used to generate a 253 bp cDNA product. One-step RT-PCR testing was successful in 31/32 (97%) cases using a touchdown protocol with RNA from over 32 different cultured influenza A virus strains representing the 9 neuraminidase subtypes. Frozen blinded clinical nasopharyngeal aspirates were also assayed and were mostly of subtype N2. The region amplified was direct sequenced and then used in database searches to confirm the identity of the template RNA. The RT-PCR fragment generated includes one of the mutation sites related to oseltamivir resistance, H274Y.


CONCLUSIONS
Our one-step RT-PCR assay followed by sequencing is a rapid, accurate, and specific method for detection and subtyping of different neuraminidase subtypes from a range of host species and from different geographical locations.
Alvarez
Alejandra Castillo
AC
Biochip Innovations Pty Ltd,, 8 Mile Plains, Queensland, Australia. alejandra.castillo@biochips.com.au
Brunck
Marion E G
ME
Boyd
Victoria
V
Lai
Richard
R
Virtue
Elena
E
Chen
Wenbin
W
Bletchly
Cheryl
C
Heine
Hans G
HG
Barnard
Ross
R
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D046091:Charadriiformes / Q000821:virology; D002645:Chickens / Q000821:virology; D002648:Child; D002675:Child, Preschool; D017931:DNA Primers; D004372:Ducks / Q000821:virology; D005260:Female; D006736:Horses / Q000821:virology; D006801:Humans; D007223:Infant; D053127:Influenza A Virus, H9N2 Subtype; D009980:Influenza A virus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D005585:Influenza in Birds / Q000821:virology; D007251:Influenza, Human / Q000821:virology; D008297:Male; D008875:Middle Aged; D009305:Nasopharynx / Q000821:virology; D009439:Neuraminidase / Q000235:genetics*; D009976:Orthomyxoviridae Infections / Q000662:veterinary / Q000821:virology; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D012367:RNA, Viral; D009439:Neuraminidase