Construction, application and analysis of the oligonucleotide database, VirOligo.

摘要

Scope and method of study. 5738 virus species are known as of October 31, 2002. The number of known viruses has been exponentially increasing. However, current methods do not allow simultaneous detection of such a large number of viruses. Virus Signature Amplification (ViSA) card and Virus Signature Hybridization (ViSH) chip methods have been created previously to detect multiple viruses simultaneously. Such universal virus detection systems can be used for early detection of outbreaks to protect us from bioterrorism and biowarfare, and may resolve causes of chronic diseases. For further development, these methods need a large number of oligonucleotides. Thus, an oligonucleotide database called VirOligo was created. Then, the influenza virus specific oligonucleotides from VirOligo were tested as probes in the ViSH chip. Oligonucleotide sequences and PCR conditions were also analyzed to provide oligonucleotide design and PCR optimization methods for those suggested methods.; Findings and conclusions. VirOligo consists of Common data and Oligo data tables. The Oligo data table contains PCR primers and hybridization probes used for detection of viral nucleic acids and the Common data table contains the experimental conditions used in their detection. Each entry has links to PubMed, GenBank, NCBI Taxonomy databases and BLAST. VirOligo currently holds 4318 virus specific oligonucleotides for more than 100 virus species. Oligonucleotide sequences obtained from VirOligo were applied to the ViSH chip to compare to oligonucleotides designed by primer design software. The hybridization intensities for both groups of probes were scattered, and a superiority of one method over another could not be determined. Oligonucleotides tested in the ViSH chip were designed as PCR primers. Since ViSH chip used these oligonucleotides as hybridization probes, the results may be caused by differences in the requirements of primer and probe design. Using the data from VirOligo, the range of each condition used most frequently was analyzed, and primer design and PCR optimization methods were suggested. One of the suggested equations in the analysis was for estimation of maximum annealing temperatures in PCR cycles. The equation was tested in ten actual PCR experiments and the validity of the equation was demonstrated.