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Extraction method comparison for PCR analysis of human samples
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry.
2023 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesisAlternative title
Utvärdering av extraktionsmetoder inför PCR analys av humana prov (Swedish)
Abstract [sv]

Den här studien genomfördes för att jämföra två instrumentuppsättningar, EasyMag och Kingfisher Flex, med avseende på effektivitet i förmåga att extrahera DNA och RNA inför diagnostiska tester med PCR. Studien utfördes SYNLAB Sverige AB i samband med avslutandet av allmän screening för Covid-19 och övergång till IVDR godkända instrument. Extraktionerna analyserades i diagnostiska test av olika ursprung (Sars-CoV-2 RNA, dermatofyt DNA och human genotypning av laktosintolerans LCT-13910 variant). Projektet inkluderar testning och utvärdering av instrumentens extraktionsförmåga samt diskussion kring när respektive instrument lämpar sig mer. Analyserna visade ingen signifikant skillnad mellan kvalitén i något av de extraherade materialen, men det fanns en antydan till att EasyMag extraherade dermatofyter effektivare (spädning serie-experimentet p=0,00006, n=15; övergripande analys p=0,057, n=35, parat t-test), i synnerhet då koncentrationerna var låga. Från ett handhavande perspektiv var EasyMag enklare att variera mellan de olika extraktions metoderna, medan Kingfisher var behändigare när man arbetade med fler prover. Därför är slutsatserna att Kingfisher är lämpligare vid höga provvolymer låg variation av metoder medan EasyMag är lämpligare med mindre provvolymer och många olika extraktions metoder.

Abstract [en]

In this study, a comparison between the capabilities of two different systems for extraction of DNA and RNA, EasyMag and Kingfisher Flex, for downstream use in real-time RT PCR-based diagnostic tests was performed. The project was completed at SYNLAB Sverige AB in conjunction with the discontinuation of public screening of Covid-19 and transitioning into IVDR-approved instruments. The extractions were evaluated in diagnostic tests of different type of samples (Sars-CoV-2 RNA, dermatophytes DNA, and human DNA genotyping of lactose intolerance genetic variants LCT-13910). This report describes the testing and evaluation of the extraction capabilities and discussion around the choice of instruments. The analysis proved no statistically significant difference between the quality of the extracted sample for neither analysis, but there was an indication that EasyMag extracted low concentration of dermatophytes more efficiently, especially at lower concentrations (dilution series p=0.00006, n=15; overall analysis p=0,057, n=35). In the aspect of working with the instruments, EasyMag was more manageable to swap between extraction methods, while Kingfisher excelled at higher sample volumes. Thus, Kingfisher is more appropriate with a larger number of samples with lower extraction method variance and EasyMag with fewer samples with more variation in extraction methods.

Place, publisher, year, edition, pages
2023.
Series
TRITA-CBH-GRU ; 2023:038
Keywords [sv]
PCR, RNA, DNA, diagnostik, Sars-Cov-2, PCR, RNA, DNA, diagnostics, SARS-Cov-2
National Category
Biochemistry Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-324235OAI: oai:DiVA.org:kth-324235DiVA, id: diva2:1739048
External cooperation
SYNLAB Sverige AB
Subject / course
Chemical Science and Engineering
Educational program
Degree of Master - Molecular Science and Engineering
Examiners
Available from: 2023-02-23 Created: 2023-02-23 Last updated: 2025-02-20

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