Typing and subtyping influenza virus using DNA microarrays and multiplex reverse transcriptase PCR

A model DNA microarray has been prepared and shown to facilitate typing and subtyping of human influenza A and B viruses. Reverse transcriptase PCR wa s used to prepare cDNAs encoding similar to 500 bp influenza virus gene fra gments, which mere then cloned, sequenced, reamplified, and spotted to form a glass-bound microarray. These target DNAs included multiple fragments of the hemagglutinin, neuraminidase, and matrix protein genes. Cy3- or Cy5-la beled fluorescent probes were then hybridized to these target DNAs, and the arrays were scanned to determine the probe binding site(s), The hybridizat ion pattern agreed perfectly with the known grid location of each target, a nd the signal-to-background ratio varied from 5 to 30. No crosshybridizatio n could be detected beyond that expected from the limited degree of sequenc e overlap between different probes and targets. At least 100 to 150 bp of h omology was required for hybridization under the conditions used in this st udy. Combinations of Cy3- and Cy5-labeled DNAs can also be hybridized to th e same chip, permitting further differentiation of amplified molecules in c omplex mixtures, In a more realistic test of the technology, several sets o f multiplex PCR primers that collectively target influenza A and B virus st rains were identified and were used to type and subtype several previously unsequenced influenza virus isolates. The results show that DNA microarray technology provides a useful supplement to PCR-based diagnostic methods.