mdh.sePublications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Utveckling av analysmetoder för en tillämpning inom beredskapsdiagnostik
Mälardalen University, School of Sustainable Development of Society and Technology.
2007 (English)Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesisAlternative title
Development of diagnostic PCR-based methods for an application in biopreparedness (English)
Abstract [en]

Bacillus anthracis is a risk class III organism and needs to be handled inside a biosafety level 3-laboratory. A major problem when working with airborne, spore-forming bacteria like B. anthracis are the hazardous aerosols created when using an automated DNA-extraction method to prepare samples suspected to contain the organism. This study has therefore evaluated the possibility of enclosing a DNA-extraction-robot inside an air tight container (glove box). A prototype of a class III safety cabinet (also known as a glove box) was designed and built to enclose a BioRobot EZ1 from Qiagen. The purpose of this prototype was to evaluate the measurements needed to and also the feasibility of working with the robot inside the cabinet. During the manual DNA-extractions, there was some contamination found on the glove box gloves, probably due to the significantly lowered dexterity that was seen with the thick gloves. The enclosing of the robot revealed no obstacles as the machine was very easy to operate. In addition, protocols have been created for the operation of a transportable class III safety cabinet from Germfree available at SVA. The protocols include the different pressure tests that needed before every experiment take place and also decontamination steps before and after each run. Bacillus cereus was used as a model organism for different DNA-extractions, i.e. automated and manual extractions. The extracted DNA was analysed by real-time polymerase chain reaction (PCR). DNA was also extracted and analysed from B. cereus-spores. When using a manual DNA-extraction kit, B. cereus-DNA was detected at the femtogram level, i.e. 10-15 g DNA / PCR. When using the automated BioRobot EZ1, detection level was found to be at 10-16 g DNA / PCR. The PCR-efficiency for the manual kit was 89-90 % for all samples, whereas with the EZ1, efficiency was 99 %, showing the strengths of the magnetic bead separation used by the machine. A novel PCR-machine, the AlphaHelix QuanTyper™, was evaluated and compared to an ABI 7500 with regards to efficiency, speed and consistency. The QuanTyper™ was found to be superior in ramping speeds, performing a 40-cycle real-time PCR-run with melting point analysis in only 14 minutes. The fastest run accomplished on the ABI 7500 took 1 h 40 min. A ready made master mix for PCR was used for most tests (Platinum® SYBR® Green qPCR SuperMix-UDG), but faster and more robust enzymes are available and further studies need to be performed on the QuanTyper™ to fully evaluate the platform. Three target genes in Bacillus anthracis-DNA were analysed in only 38 minutes with efficiencies between 96-104 % for the virulence plasmids and detection at femtogram amount of DNA. This master thesis has addressed rapid pathogen-detection with automated DNA-extraction and novel PCR-technology, coupled with a strong biosafety aspect. The thesis will hopefully contribute to the surprisingly small area of biosafety and safety cabinet research.

Place, publisher, year, edition, pages
2007. , 47 p.
Keyword [en]
bacillus, anthracis, antrax, BSL-3, glovebox, biosafety, PCR
National Category
Chemical Engineering
Identifiers
URN: urn:nbn:se:mdh:diva-1064OAI: oai:DiVA.org:mdh-1064DiVA: diva2:25218
Presentation
(English)
Uppsok
bio-/geovetenskap
Supervisors
Examiners
Available from: 2009-04-28 Created: 2008-09-23 Last updated: 2009-04-28Bibliographically approved

Open Access in DiVA

fulltext(1408 kB)290 downloads
File information
File name FULLTEXT01.pdfFile size 1408 kBChecksum SHA-512
c3f1f352b4016d780cdac31a31a878d58077f69ebbf0e6f27aba2dc8d9468053c56dc4baf6c77ae934dfe2d159a787d9d7c7a9e0202d9502fe99b465db46193d
Type fulltextMimetype application/pdf

By organisation
School of Sustainable Development of Society and Technology
Chemical Engineering

Search outside of DiVA

GoogleGoogle Scholar
Total: 290 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Total: 191 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf