Designation Of Stem-loop Structured Probes And Detection In Pathogenic Bacteria

After Tyagi and Kramer proposes the molecular beacon theoryin 1996, Bockisch provided a novel enzymatic conformationalswitch system based on the principle of molecular beacon forthe detection of bacterial nucleic acid. They utilized stem-loop structured oligodeoxynucleotide probes for the detectionof nucleic acid targets. This system, however, can display afalse-positive signal when the sequence of nucleic acidmismatches the loop segments of probe in a single position aswell as molecular beacon. Therefore, Using sequencecomparisons, we found out a segment of 16S rRNA gene sequencesof the bacteria, which is different from 16S rRNA gene sequencesof other highly homologous bacteria in more than two positions.As the loop of the stem-loop structured oligonucleotide probeis designed by this segment, its specificity has beenstrengthened and the system is able to successfully eliminatefalse-positive result that is mismatch in an oligonucleotide.The probes are immobilized on 96-well microtiter plates throughone terminus and carrying an affinity label at the other as theconformational switch. In the absence of a target, the labelis forced into close proximity to the surface of the solid support by the closed conformation of the probe so as toinaccessible to detector molecules. Upon target hybridization,exposure of the affinity label is able to be close to reportmolecules. This is might be due to the fact that the probe opensthe loop and turns to a linear conformation. Thus, this newprobe technology combining sequence comparisons is moresensitive than other conventional method at least one order ofmagnitude. The main researches included in this thesis arepresented as following:1) Based on Staphylococcus aureus 16S rRNA gene sequences,sequence comparisons have been applied to design a kind ofstem-loop structured oligonucleotide probes whose loopsequence mismatched other bacterial strains 16S rRNA genesequences in more than two positions with high specificity andsensitivity. According to the principle of molecular beacontechnology and enzyme linked immunosorbent assay, a method hasbeen evaluated using immobilized stem- loop structuredoligonucleotides probe as the conformation switch which isapplied to enzymatic detection of nucleic acid targets. As itsspecificity has been strengthened, the system is able tosuccessfully eliminate false-positive result that is mismatchin an oligonucleotide. 2) In order to confirm the possibility of this novelapproach in the detection of all bacterial nucleic acid targets,the Gram-positive bacterium (Staphylococcus aureus) and Gram-negative bacterium (Escherichia coli) are studied. As a result,they can be respectively detected.This thesis strongly implied that the novel approachcombining sequence comparisons and enzymatic conformationalswitch system could replace conventional techniques and detectmutations and single nucleotide polymorphism.