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Detection of NASBA amplified bacterial tmRNA molecules on SLICSel designed microarray probes

2011; BioMed Central; Volume: 11; Issue: 1 Linguagem: Inglês

10.1186/1472-6750-11-17

1472-6750

Ott Scheler, Lauris Kaplinski, Barry Glynn, Priit Palta, Sven Parkel, Kadri Toome, Majella Maher, Thomas Barry, Maido Remm, Ants Kurg,

Molecular Biology Techniques and Applications

We present a comprehensive technological solution for bacterial diagnostics using tmRNA as a marker molecule. A robust probe design algorithm for microbial detection microarray is implemented. The probes were evaluated for specificity and, combined with NASBA (Nucleic Acid Sequence Based Amplification) amplification, for sensitivity. We developed a new web-based program SLICSel for the design of hybridization probes, based on nearest-neighbor thermodynamic modeling. A SLICSel minimum binding energy difference criterion of 4 kcal/mol was sufficient to design of Streptococcus pneumoniae tmRNA specific microarray probes. With lower binding energy difference criteria, additional hybridization specificity tests on the microarray were needed to eliminate non-specific probes. Using SLICSel designed microarray probes and NASBA we were able to detect S. pneumoniae tmRNA from a series of total RNA dilutions equivalent to the RNA content of 0.1-10 CFU. The described technological solution and both its separate components SLICSel and NASBA-microarray technology independently are applicative for many different areas of microbial diagnostics.