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Utveckling av analysmetoder för en tillämpning inom beredskapsdiagnostik / Development of diagnostic PCR-based methods for an application in biopreparednessÅgren, Joakim January 2007 (has links)
<p>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.</p>
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Utveckling av analysmetoder för en tillämpning inom beredskapsdiagnostik / Development of diagnostic PCR-based methods for an application in biopreparednessÅgren, Joakim January 2007 (has links)
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.
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Sekuritizace biologie: Biologické hrozby a připravenost státu v kontextu pandemie / Securitising biology: Biological threats and state preparedness in the wake of a pandemicArtico, Chiara January 2021 (has links)
64053643 SECURITISING BIOLOGY: BIOLOGICAL THREATS AND STATE PREPAREDNESS IN THE WAKE OF A PANDEMIC ABSTRACT The management of infectious diseases in the realm of public health has shown increasingly overlapping areas with biological warfare preparedness. While the acknowledgement of these common elements is not only frequent but also codified in an international treaty and subject to distinct regulations, research into how these two fields connect is scarce. Potential deliberate use of biological weapons typically leads to intense political mobilisation and ensuing dedication of financial resources. Contrarily, the management of health crises over the last decades has been severely flawed, and no country in the world is considered fully prepared to a pandemic, according to the Global Health Security Index. The current COVID-19 pandemic has recently been further proof of the inadequacy of state-level prevention and preparedness capabilities. This dissertation aims at bridging the existing conceptual gap and policy divide between biological warfare and infectious disease preparedness, and to analyse elements that can be mutually applicable and potentially beneficial. It will do so by establishing analytical equivalence between the securitisation of an artificial biothreat and a nature-borne infectious disease...
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