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

Methodology article Open Access Detection of NASBA amplified bacterial tmRNA molecules on SLICSel designed microarray probes Ott Scheler1, 2Email author, Lauris Kaplinski2, 3, Barry Glynn4, Priit Palta2, 3, Sven Parkel1, 2, Kadri Toome1, Majella Maher4, Thomas Barry4, Maido Remm2, 3 and Ants Kurg1, 2 BMC Biotechnology201111:17 https://doi.org/10.1186/1472-6750-11-17 ©  Scheler et al; licensee BioMed Central Ltd. 2011 Received: 12 November 2010Accepted: 28 February 2011Published: 28 February 2011 Abstract Background 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. Results 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. Conclusions The described technological solution and both its separate components SLICSel and NASBA-microarray technology independently are applicative for many different areas of microbial diagnostics. Keywords Microarray ProbeMarker MoleculeNonspecific HybridizationNucleic Acid Sequence Base AmplificationBinding Energy Difference Background The ssrA gene which encodes the tmRNA molecule has been identified in all known bacterial phyla [1, 2]. The term tmRNA describes the dual "transfer" and "messenger" properties of this RNA molecule. In bacteria, the function of the tmRNA molecules is to release ribosomes that have become stalled during protein synthesis and to tag incomplete and unnecessary peptides for proteolysis. A typical tmRNA is between 300-400 nucleotides in size and is present in cells in relatively high copy number around 1000 copies per cell [3]. tmRNA molecules contain both conserved as well as variable regions between different species; complementary 3' and 5' ends fold together into a tRNA like structure that permits the entry to the ribosome when needed. Proteolysis-coding mRNA part and structural domains usually make up for the rest of the molecule. All those characteristics make the tmRNA transcript (and its ssrA gene) a suitable tool as a target marker molecule for phylogenetical analysis and species identification in microbial diagnostics. Over the last 10 years tmRNA and its corresponding gene have been used for species identification in several methods including fluorescence in situ hybridization (FISH) detection of specific bacteria [4], real-time PCR [5] and real-time NASBA [6] analysis of food and dairy contaminants and pathogen detection using biosensors [7]. Combining the capabilities of tmRNA for species identification with DNA microarray technology offers the potential to investigate samples simultaneously for large numbers of different target tmRNA molecules. DNA microarrays have found several practical applications in microbial diagnostics such as composition analysis and species identification of different environmental and medical samples as well as in microbial diversity investigation [8–10]. Depending on the experiment setup and specific probe design, precise detection of one specific microbe [11] or more complex analysis of microbial taxa can be performed [12]. The design of microarray probes for the detection of bacterial RNA poses unique challenges, because certain RNA/DNA or RNA/RNA mismatches have almost as strong binding affinity as matches [13]. The nearest-neighbor thermodynamic modeling (NN) approach should therefore be used to calculate the hybridization affinities (ΔG) of probes [14–16]. The hybridization on microarray surface is more complex then hybridization in solution and the NN model should include surface and positional parameters for more accurate modeling [17, 18]. Although there are many recent studies of surface hybridization thermodynamics [19], the exact hybridization properties of microarray probes cannot be precisely modelled and experimental verification is still needed [20, 21]. A common feature of many microarray analysis protocols is that the nucleic acid sequences of interest are amplified and labeled prior to the hybridization experiment. Hybridization protocols may involve labeled cDNA [22], cRNA [23] or (RT-)PCR products [24]. RNA molecules can also be amplified by Nucleic Acid Sequence Based Amplification (NASBA) [25]. Although not as common as RT-PCR, NASBA is less prone to genomic DNA contamination and therefore more suitable for applications where the testing of microbial viability is important [26]. Several methods have recently been published that describe different NASBA product labeling methods for the purpose of microarray hybridization. These methods include the dendrimer-based system NAIMA [27], biotin-streptavidin binding assisted labeling [28] and aminoreactive dye coupling to aminoallyl-UTP (aa-UTP) molecules in NASBA products [29]. In this report we present a complete technological solution for detection of low amounts of bacterial tmRNA molecules. We describe a new software program, SLICSel, for designing specific oligonucleotide probes for microbial diagnostics using nearest-neighbor thermodynamic modeling and evaluate SLICSel by testing the specificity of the designed tmRNA specific probes. Finally we demonstrate the sensitivity of these probes using a molecular diagnostics method that combines tmRNA amplification by NASBA with microarray-based detection [29]. Using this approach we were able to specifically detect S.pneumoniae tmRNA in the amount that corresponds to a single bacterium or less in the presence of 4000-fold excess of other bacterial tmRNA. Methods SLICSel program for probe design The nearest-neighbor thermodynamic (NN) modeling of probe hybridization strength with target (specific hybridization) and control (nonspecific hybridization) nucleotide sequences at exact annealing temperature is used as design criterion of the SLICSel program. The previously published empirical formula was used to adjust the calculated thermodynamic values to the actual annealing temperature and salt concentration [15]. No surface and positional effects were added to the model to keep it universal and not bound to specific technology. We also expect that NN parameters on surface, although slightly different, are in correlation with the ones in solution [19]. Bacterial strains Streptococcu (...truncated)