Economic high-throughput-identification of influenza A subtypes from clinical specimens with a DNA-oligonucleotide microarray in an outbreak situation.
Influenza A surface proteins H (haemagglutinin) and N (neuraminidase) occur in sixteen and nine distinct genotypes, respectively. The need for a timely production of vaccinations in case of pandemics or seasonal epidemics requires rapid typing methods for the determination of these alleles. The aim of the present study was to develop and improve a rapid and economic assay for determining H and N subtypes of influenza A from patient samples. The assay is based on the hybridisation of labelled amplicons from H and N reverse transcriptase-PCRs using consensus primer pairs to subtype-specific probes on microtiterstripe-mounted DNA-microarrays. An algorithm for semi-automatic data interpretation of raw data and assignment to H and N subtypes was proposed. Altogether, 191 samples were genotyped. This included 134 patient and 44 reference samples as well as controls. Under routine conditions sensitivity and specificity proved to be comparable to conventional nested or real-time PCRs. At least 130 out of 147 array-positive samples were unambiguously assignable. This included all sixteen variants of H as well as all nine variants of N. Furthermore, eighty-two samples from the 2009/2010 "novel H1N1/swine flu" (SF)-outbreak were correctly identified.
Institute for Medical Diagnostics Oderland, Frankfurt (Oder), Germany.
Dirk
D
Bandt
Stefan
S
Monecke
Carly
C
Scott
Astrid
A
Gall
Bernd
B
Hoffmann
Ralf
R
Ehricht
D000465:Algorithms; D004196:Disease Outbreaks / Q000145:classification*; D005838:Genotype; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000032:analysis* / Q000235:genetics; D057166:High-Throughput Screening Assays; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics* / Q000302:isolation & purification; D007252:Influenza Vaccines / Q000235:genetics; D007251:Influenza, Human / Q000453:epidemiology* / Q000821:virology*; D009439:Neuraminidase / Q000032:analysis* / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012680:Sensitivity and Specificity
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D009439:Neuraminidase