Flu Vaccine Example

 

The ability to detect and definitely identify multiple related pathogens can be demonstrated clearly with Influenza Virus Vaccine, which is an inactivated Influenza vaccine comprised of Influenza A and B virus, including subtypes, A/H1N1 and A/H3N2, with the specific strain composition of each subtype varying by flu season.

 

The image file of Influenza vaccine (2005-2006 flu season) hybridized onto the RPM-Flu microarray is shown in Figure 4. Fluorescent signal is clearly detected on that potion of the array containing probes complementary to Influenza gene sequences (tiles), although random weak signal is also present. By examining an area of background (green box), a portion of the microarray where Influenza tiles are not present, but non-Influenza tiles are located (i.e. probes complementary to other respiratory pathogen gene sequences), signal from false positive hybridization events can be seen (NOTE: the regularly-occurring checkerboards patterns seen on the microarray are signal from a hybridization control used for alignment of the scanning system).

 

If an area containing Influenza tiles (blue box) is examined, large amounts of strong signal, representing signal from true positive hybridization events, can be seen. Signal is converted into nucleotide sequence as a fasta file using a translation key, and a small region of positive signal (red box) generates 45 bases corresponding to the Influenza A hemagglutinin gene (HA), subtype HA1. Examination of the fasta output (Figure 5) clearly shows long contiguous stretches of called sequence from the Influenza A HA1 gene, but extremely little called sequence, indicated as N’s (no calls), from neighboring genes of other respiratory pathogens, such as Enterovirus 70 UTR and Influenza A HA10. If the entire sequence called from the HA1 tile is examined, the identity is revealed as A/New Caledonia/20/1999, the exact strain that was included in the 2005-2006 vaccine.

 

A few short stretches (3-5 bases) of called sequence can be seen in these neighboring genes, again representing false positive results, but clearly distinguishable from the true positive sequence results. Such false positive results indicate how frequently such non-specific hybridization may occur in nature, and the ensuing limitations of typical PCR assays.