Entwicklung von Rekombinase-Polymerase-Amplifikations-Verfahren zum schnellen Nachweis von hochpathogenen Erregern

Development of a panel of recombinase polymerase amplification assays for rapid detection of highly pathogenic agents

by Anna Milena Euler
Doctoral thesis
Date of Examination:2015-07-07
Date of issue:2015-06-26
Advisor:PD Dr. Manfred Weidmann
Referee:Prof. Dr. Frank T. Hufert
Referee:Prof. Dr. Peter Burfeind
Referee:Prof. Dr. Martin Oppermann
Persistent Address: http://dx.doi.org/10.53846/goediss-5157

 

 

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Abstract

English

Zoonoses are infectious diseases transmissible from vertebrate animals to humans and vice versa. Some zoonotic diseases caused by highly pathogenic agents represent a public health risk. Early diagnosis is essential to improve treatment and to prevent the further spread of agents.  To test the performance of isothermal recombinase polymerase amplification (RPA) assays, we developed a panel of RPAs for 8 highly pathogenic zoonotic agents: DNA-RPA-assays were developed for detection of Sudan Ebola Virus (SUDV), Zaire Ebola Virus (EBOV), Marburg Virus (MARV) and Rift Valley Fever Virus (RVFV). Reverse transcriptase (RT) RPAs were developed for detection of Variola Virus (VARV), Francisella tularensis (Ftu), Yersinia pestis (Ype) and Bacillus anthracis (BA), composed of RNA genome. An additional assay was designed for Sigma-Virus (negative-strand-RNA-Virus, Rhabdoviridae) as internal positive control. Target genomes were selected, specific primer and probe sets were designed for each amplicon and quantitative standards were developed. For each assay 8 runs were performed with ESEQuant Tube Scanner instrument. Their analytical sensitivities ranged from 16 to 21 molecules detected for the majority of RPA and RT-RPA assays, analysed via probit analysis. RPA showed comparable sensitivities in these extracts to real-time-PCR assays. Comparable results were also shown by RPA and RT-PCR testing inactivated whole organisms spiked into plasma (RVFV, SIGV, BA und Ype). The run times of the assays ranged at 42°C from 6 to 10 min, and they showed no cross-detection of any of the target genomes of the panel nor of the human genome.  Recombinase polymerase amplification is a highly sensitive and specific method for rapid detection of pathogenic zoonotic agents composed of RNA- or DNA-genome. The assays seem suitable for implementation in Point-of-Care-devices.
Keywords: RPA; recombinase polymerase amplification; Point-of-Care diagnostic; detection of nucleic acid; RT-PCR; Nucleic Acid Amplification Test; isothermal
Schlagwörter: RPA; Rekombinase-Polymerase-Amplifikation; Molekulare Nachweisverfahren; Isothermale Amplifikationsverfahren; Echtzeit-PCR
 
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