design of multiplexed detection assays for identification of avian influenza a virus subtypes pathogenic to humans by SmartCycler real-time reverse transcription-PCR.
Influenza A virus (IAV) epidemics are the result of human-to-human or poultry-to-human transmission. Tracking seasonal outbreaks of IAV and other avian influenza virus (AIV) subtypes that can infect humans, aquatic and migratory birds, poultry, and pigs is essential for epidemiological surveillance and outbreak alerts. In this study, we performed four real-time reverse transcription-PCR (rRT-PCR) assays for identification of the IAV M and hemagglutinin (HA) genes from six known AIVs infecting pigs, birds, and humans. IAV M1 gene-positive samples tested by single-step rRT-PCR and a fluorogenic Sybr green I detection system were further processed for H5 subtype identification by using two-primer-set multiplex and Sybr green I rRT-PCR assays. H5 subtype-negative samples were then tested with either a TaqMan assay for subtypes H1 and H3 or a TaqMan assay for subtypes H2, H7, and H9 and a beacon multiplex rRT-PCR identification assay. The four-tube strategy was able to detect 10 RNA copies of the HA genes of subtypes H1, H2, H3, H5, and H7 and 100 RNA copies of the HA gene of subtype H9. At least six H5 clades of H5N1 viruses isolated in Southeast Asia and China were detected by that test. Using rRT-PCR assays for the M1 and HA genes in 202 nasopharyngeal swab specimens from children with acute respiratory infections, we identified a total of 39 samples positive for the IAV M1 gene and subtypes H1 and H3. When performed with a portable SmartCycler instrument, the assays offer an efficient, flexible, and reliable platform for investigations of IAV and AIV in remote hospitals and in the field.
Wang
Wei
W
Chinese Academy of Sciences, Institut Pasteur of Shanghai, Shanghai Institute of Biological Sciences, 411 Hefei Road, 200025 Shanghai, People's Republic of China.
Ren
Peijun
P
Mardi
Sek
S
Hou
Lili
L
Tsai
Cheguo
C
Chan
Kwok Hung
KH
Cheng
Peter
P
Sheng
Jun
J
Buchy
Philippe
P
Sun
Bing
B
Toyoda
Tetsuya
T
Lim
Wilina
W
Peiris
J S Malik
JS
Zhou
Paul
P
Deubel
Vincent
V
D000293:Adolescent; D052160:Benzothiazoles; D002648:Child; D002675:Child, Preschool; D017931:DNA Primers; D003959:Diamines; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D006801:Humans; D007223:Infant; D009980:Influenza A virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology*; D008969:Molecular Sequence Data; D009305:Nasopharynx / Q000821:virology; D009930:Organic Chemicals; D011804:Quinolines; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods; D014763:Viral Matrix Proteins / Q000235:genetics
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D019267:Hemagglutinin Glycoproteins, Influenza Virus; C488936:M1 protein, Influenza A virus; D009930:Organic Chemicals; D011804:Quinolines; D014763:Viral Matrix Proteins; C109963:hemagglutinin, avian influenza A virus; C098022:SYBR Green I