Mycobacterial speciation and resistance typing using a PamChip; an example of rapid SNP detection

R.M. Anthony¹, A.R.J. Schuitema¹, L. Oskam¹, R. vanBeuningen², P.J. Boender², P.R. Klatser¹

¹KIT Biomedical Research, Meibergdreef 39, 1105 AZ Amsterdam, Nederland
²PamGene B.V., Burgemeester Loeffplein 70a, 5211 RX 's-Hertogenbosch, Nederland  

We have utilised a novel flow through array system, the 'PamChip', which allows real time monitoring of hybridisation in a flow through format, to speciate and detect mutations associated with rifampicine resistance in Mycobacterium tuberculosis. Un-purified labelled PCR products were directly applied to the PamChip and the resulting hybridisation detected using a fluorescent microscope with CCD camera. Direct fluorescent detection of the hybridisation allowed the species from which the PCR products were obtained to be determined within 20 minutes. Single nucleotide polymorphisms could be detected. The PamChip provides a rapid and accurate method of characterising a PCR product. Additionally, as the system is real time, temperature control of the hybridisation chamber allows the melting characteristics of each amplicon and probe combination to be studied. Allowing detailed study of probe performance. The effect of probe selection on the specificity and sensitivity of this type of assay will be outlined.