The application of tmRNA as a marker molecule in bacterial diagnostics using microarray and biosensor technology
Date
2012-05-15
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Abstract
Bakterite uurimiseks on traditsiooniliselt kasutatud erinevaid kultuuris kasvatamise meetodeid. Kuigi mainitud meetodid on töökindlad ja täpsed, on nad samas ka küllaltki töö- ja ajamahukad ning ei võimalda kõikide bakterite uurimist. Seetõttu kasutatakse tänapäeval tihti nende uurimiseks erinevaid molekulaarbioloogilisi meetodeid, mis põhinevad spetsiifiliste nukleiinhappe järjestuste tuvastamisel ja kirjeldamisel. Käesolevas töös tutvustatakse erinevaid tehnoloogiaid, mida kasutatakse nukleiinahappe põhises bakteriaalses diagnostikas. Põhirõhk on erinevatel nukleiinhapete paljundamise meetoditel ning hübridisatsiooni-põhistel detektsiooni tehnoloogiatel. Käsitletud on erinevate mikrokiibi ja biosensor tehnoloogiate põhimõtteid ning nende võimalikke kasutusviise bakterite tuvastamisel. Lisaks antakse ülevaade DNA ja RNA järjestustest, mida saab kasutada markerjärjestusena erinevate bakterite tuvastamisel ja üksteisest eristamisel. Pikemalt tutvustatakse tmRNA molekule, mida kasutatakse markerjärjestusena käesoleva doktoritöö raames välja töötatud diagnostiliste meetodite puhul. tmRNA on kõikides bakterites leiduv, keskmiselt 300-400 nukleotiidi pikkune spetsiifiline RNA molekul, mis abistab rakus valgusünteesi mehhanismi, ning mille molekuli järjestuse põhjal on võimalik tuvastada ning eristada erinevaid bakteriliike ja ka kõrgemaid taksonoomilisi üksusi. Töö praktilises osas kirjeldatakse kahte erinevat meetodit, kus tmRNA detektsiooni kaudu tuvastatakse erinevaid baktereid. Nendest esimene põhineb tmRNA molekulide spetsiifilisel paljundamisel NASBA tehnoloogia abil, millele järgneb märgistatud tmRNA molekulide tuvastatamine ja täpne identifitseerimine mikrokiibi tehnoloogia abil. Teise puhul toimub tmRNA-de detektsioon märkevaba reaal-ajas toimiva biosensor süsteemi abil, mis põhineb optilisel mikroring resonaator tehnoloogial. Kuigi mõlema meetodi puhul kasutati testsüsteemina erinevaid hingamisteede haigusi põhjustavaid baktereid ning nende vastavaid liigispetsiifilisi tmRNA molekule, on kirjeldatud tehnoloogiad lihtsasti rakendatavad ka teiste RNA järjestuste ning erinevate bakteri-liikide korral.
There is a growing need for faster and more reliable approaches for microorganism detection and identification that could complement or replace conventional rather time- and labor-consuming culture-based technologies. A common tactics nowadays is to analyze the nucleic acid component of analyte solution and determine the bacterial composition according to specific nucleic acid profiles that are detected and identified. Theoretically every bacterial species and strain contain unique characteristic target regions that can be used for their specific identification. In the first part of current thesis a literature overview is given about the different technologies that are used for nucleic acid-based bacterial detection. Main focus is on nucleic acid amplification and hybridization-based detection methods with emphasis on microarray and biosensor technologies, and their practical application in bacterial diagnostics. In second part of the literature overview, a description of different DNA and RNA molecules that have been targeted for bacterial detection and identification is reviewed. Longer explanation is given about the trans-translation mediating RNA molecule called tmRNA that is used as a target marker molecule in the current thesis. The research section describes two different methods that apply tmRNA for bacterial detection and identification. Firstly, a microarray-based technology is described where target tmRNA molecules are amplified using Nucleic Acid Sequence Based Amplification (NASBA) and labeled fluorescently prior the hybridization experiment. The developed method was applied for tmRNA detection from bacterial total RNA samples. In second part of the research tmRNA molecules are specifically targeted using real-time label-free biosensing platform that is based on the optical microring resonator technology. Potential quantitative nature and sensitivity of the biosensor is demonstrated using in vitro synthesized tmRNA molecules.
There is a growing need for faster and more reliable approaches for microorganism detection and identification that could complement or replace conventional rather time- and labor-consuming culture-based technologies. A common tactics nowadays is to analyze the nucleic acid component of analyte solution and determine the bacterial composition according to specific nucleic acid profiles that are detected and identified. Theoretically every bacterial species and strain contain unique characteristic target regions that can be used for their specific identification. In the first part of current thesis a literature overview is given about the different technologies that are used for nucleic acid-based bacterial detection. Main focus is on nucleic acid amplification and hybridization-based detection methods with emphasis on microarray and biosensor technologies, and their practical application in bacterial diagnostics. In second part of the literature overview, a description of different DNA and RNA molecules that have been targeted for bacterial detection and identification is reviewed. Longer explanation is given about the trans-translation mediating RNA molecule called tmRNA that is used as a target marker molecule in the current thesis. The research section describes two different methods that apply tmRNA for bacterial detection and identification. Firstly, a microarray-based technology is described where target tmRNA molecules are amplified using Nucleic Acid Sequence Based Amplification (NASBA) and labeled fluorescently prior the hybridization experiment. The developed method was applied for tmRNA detection from bacterial total RNA samples. In second part of the research tmRNA molecules are specifically targeted using real-time label-free biosensing platform that is based on the optical microring resonator technology. Potential quantitative nature and sensitivity of the biosensor is demonstrated using in vitro synthesized tmRNA molecules.
Description
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Keywords
mikroobid, bakterid, määramismeetodid, nukleiinhapped, biomarkerid, biokiibid, microbes, bacteria, fixed methods, nucleic acids, biomarkers