Entwicklung von Rekombinase-Polymerase-Amplifikations-Nachweisverfahren für virale Erreger von Atemwegsinfektionen

Development of a panel of recombinase polymerase amplification assays for detection of respiratory viruses

by Kai Ilmo Ehnts
Doctoral thesis
Date of Examination:2013-08-06
Date of issue:2013-07-19
Advisor:PD Dr. Manfred Weidmann
Referee:Prof. Dr. Frank Torsten Hufert
Referee:Prof. Dr. Iris Bartels
Referee:Prof. Dr. Martin Oppermann
Persistent Address: http://dx.doi.org/10.53846/goediss-3943

 

 

Files in this item

Name:Dissertation Ehnts.pdf
Size:2.06Mb
Format:PDF

The following license files are associated with this item:

Abstract

English

Objective: Acute respiratory infections (ARI), ranging from the common cold to life threatening Influenza, are frequent causes for medical consultations. Point-of-Care (PoC) diagnosis of Influenza facilitates early antiviral treatment and can lead to a reduction in antibiotic use. We designed three assays for the fast and sensitive isothermal PoC detection of Influenza A, Influenza B and Adenovirus using Recombinase-Polymerase-Amplification. Methods: Following existing PCR amplicons, we aligned several random Genbank sequences and designed specific primer and probe sets for Influenza A, Influenza B and Adenoviruses. We developed quantitative standards of synthetic viral RNA and DNA respectively, which where calibrated using real-time PCR. For human Adenoviruses three standard preparations of serotype 1, 4 and 7 were developed to cover those serotypes causing severe respiratory tract infections. Sensitivity of each assay was assessed in 8 similar runs in a ESEQuant Tube Scanner instrument for 20 minutes at 42°C. The obtained data was statistically analysed via Probit analysis. Results: The number of molecules detected in 95% of test runs in a 50 µl volume were as per Probit analysis: Influenza-A 218 copies, Influenza-B 131 copies, HAdV-1 12 copies, HAdV-4 131 copies, HAdV-7 16 copies. The runtime until positive results was for Influenza-A not exceeding 9 minutes, for Influenza-B not exceeding 8 minutes and for Adenoviruses not exceeding 14 minutes. Conclusion: Recombinase-Polymerase-Amplification provides a very fast, sensitive and specific way of isothermal detection of respiratory viruses. These assays can be easily adapted to microfluidic Point-of-Care devices.
Keywords: RPA; recombinase polymerase amplification; Influenza; Adenovirus; PoC; point-of-care diagnostic; detection of nucleic acid; PCR; Nucleic Acid Amplification Test; NAAT; Flu; Acute respiratory infection
Schlagwörter: RPA; Rekombinase-Polymerase-Amplifikation; Influenza; Adenovirus; Schnelltest; Nukleinsäurenachweisverfahren; Grippe; Atemwegsinfektion
 
Statistik