Object's details: Zastosowanie chemiluminescencji do immunoenzymatycznego oznaczania produktów PCR w diagnostyce molekularnej Bacillus anthracis

Provider:Repozytorium Uniwersytetu Łódzkiego

Description

Title:
Zastosowanie chemiluminescencji do immunoenzymatycznego oznaczania produktów PCR w diagnostyce molekularnej Bacillus anthracis
Creator:
Michalski, Aleksander
Contributor:
Ośrodek Diagnostyki i Zwalczania Zagrożeń Biologicznych w Puławach, Wojskowy Instytyt Higieny i Epidemiologii im. generała Karola Kaczkowskiego w Warszawie;
Uniwersytet Łódzki, Wydział Biologii i Ochrony Środowiska
Description:
Bacillus anthracis is one of the most relevant bacteria considered as biological agent of weapon of mass destruction, which may be used in bioterroristic attacks and biological weapon production. Under these circumstances, it is necessary to develop and possess inexpensive, rapid, specific and sensitive method of biological agents detection and identification, which may be applied in stationary and field laboratories. Among various detection and identification methods, the molecular biology based ones are very valuable tools, that can detect genetic material with very high level of sensitivity. The aim of the study was application of PCR and chemiluminescence based technique for identification of Bacillus anthracis. The original assay setup PCR-CLEIA (ChemiLuminescence Enzyme ImmunoAssay) was designed for this purpose. Initially the specific amplification of Bacillus anthracis genes fragments and digoxigenin-dUTP labeling of resulting product during PCR were applied. Then the denatured amplicon hybridized with biotin labeled probe, and thanks to biotin interaction the complexes were immobilized on streptavidin coated microbeads. The amplicons were detected by affinity interaction of enzyme labeled antibodies against digoxigenin and chemiluminescent substrates. The small, filed, portable microluminometer Profile 1 model 4560 (New Horizons Diagnostic Corp., USA) was utilized for this purpose. The device, which is used in sanitary control, for bacterial contamination assessment and spores detection by bioluminescent detection of ATP, is optimized for signal readout around 550-560 nm, emitted by luciferine/luciferase complex. Triplex PCR was applied in this study. It allowed for simultaneous detection of three sequences of Bacillus anthracis from chromosome and plasmids: pXO1 and pXO2. Available sequences: pag from pXO1, cap from pXO2 and chromosomal sequences were analyzed and selected. The triplex PCR technique was optimized in terms of magnesium ions concentration and annealing temperature as well as its sensitivity and specificity were assessed with PCR products analysis in agarose gels. Four chemiluminescent substrates for horseradish peroxidase POD (PS-Atto, TMA-6, SuperSignal ELISA Femto and CPS-1) and five for alkaline phosphate AP (APS, CSPD with sapphire and emerald enhancer and CDP-Star with sapphire and emerald enhancer) were tested in following stages of the study in terms of reaction kinetics and emission spectra. Subsequently utilization of substrates were optimized in terms of its concentration and time of readout. Based on obtained results two substrates CPS-1 for POD labeled antibodies and CDP-Star with emerald enhancer for AP labeled antibodies were selected and used in the experiments. Following parameters of CLEIA assay were optimized: concentration of probes and microbeads (M-280 Streptavidin, Dynal), time and temperature of hybridization and immobilization of complexes, time of binding and concentration of enzyme labeled antibodies. The necessity of utilization of substances preventing unspecific binding of digoxygenin-dUTP or/and unspecific amplicons and/or enzyme labeled antibodies was demonstrated. Intensity of signal level and test specificity, influenced by probe labeling sites (3’ or 5’ end) and usage of the oligonucleotide arm (TTT TTT) was assessed during PCR-CLEIA method optimization. For the most of probes 5’ end labeling or usage of six-nucleotides arms were favorable for assay results. The utilization of AP or POD labeled antibodies and selected substrates in optimized conditions resulted in comparable readouts in terms of specificity and sensitivity. The application of chemiluminescence based technique in immunoenzymatic detection of PCR products in molecular diagnostic of Bacillus anthracis enabled for very sensitive (10 fg template DNA) and specific (against of wide panel of Bacillus cereus group strains) detection of genetic material of Bacillus anthracis. The developed method allows for identification of pathogenic and non-pathogenic strains, as well as plasmid-bearing and plasmidless ones. It was the first noted application of PCR-CLEIA method for molecular detection of Bacillus anthracis by portable microluminometer Profile 1 with high sensitivity and specificity. The innovative PCR-CLEIA method developed in this study is one hundred times more sensitive when compared to classical PCR with agarose gel electrophoresis.;
Badania były częściowo wykonane w ramach realizacji prac badawczo-rozwojowych pt. „Postępowanie diagnostyczne, lecznicze i profilaktyczne oraz doskonalenie systemów monitorowania epidemiologicznego w warunkach stacjonarnych i polowych w przypadkach zagrożenia bronią biologiczną.” 2007/MON/IWSZdr/119, finansowanych przez Inspektorat Wojskowej Służby Zdrowia MON
Object availability:
free access
Rights:
Aleksander Michalski
Date:
2016.01.29;
2016
Type:
PhD/Doctoral Dissertation
Language:
Polish
Format:
application/pdf
Subject:
PCR-CLEIA;
Bacillus anthracis;
laseczka wąglika;
chemiluminescencja
Identifier: http://hdl.handle.net/11089/16810
Data provider:
Repozytorium Uniwersytetu Łódzkiego

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Object's details: Zastosowanie chemiluminescencji do immunoenzymatycznego oznaczania produktów PCR w diagnostyce molekularnej Bacillus anthracis

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