Figure 1.
Schematic representation of experimental procedure.
Multiplex PCR amplifies up to 12 genes and is performed outside of the INFINITI system. Amplification products are loaded in the INFINITI system at the primer extension step. Primer extension oligonucleotides include a tag sequence that will hybridize to the microarray and a specific detection sequence that allows for primer extension of S. pneumoniae genes. Fluorescent nucleotides are incorporated during primer extension. Once the reaction is complete, the INFINITI system automatically transfers the labeled products to the microarray for hybridization. Tag sequences hybridize to anti-tags located on the microarray. Microarrays are then washed, dried and loaded into the integrated confocal scanner where fluorescence is measured. The report generated by the instruments is analyzed off-line using the Pneumotyper software.
Figure 2.
Algorithm for results analysis.
Samples were assessed for the presence of S. pneumoniae by detecting the pneumolysin and the autolysin genes. If S. pneumoniae was detected in the sample, the serotyping probes were analyzed in order to identify the serotype.
Figure 3.
Capsular operon of S. pneumoniae serotype 19A.
Expected amplicons are shown under the sequence, with genotyped positions marked with vertical lines. The expected genotype for serotype 19A is in large characters while the other possible genotypes are shown in smaller type. Positions are in nucleotides.