Optimization of PCR Sensitivity for Detection of Bacterial Species in Blood of Patients with Suspected Sepsis
2015 (English)Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE credits
Student thesis
Abstract [en]
Sepsis is commonly caused by bacteria, fungi or both present in the blood stream during inflammation. In response, inflammatory cascades are released in multiple organ systems which if prolonged causes sepsis and can eventually lead to organ failure and death. The major diagnostic technique of sepsis is blood culturing. However, the technique is time consuming and lacks sensitivity; especially in patients under antimicrobial therapy. Molecular techniques particularly PCR could possibly become implemented in sepsis diagnostics in the future. The aim of the thesis was to compare the effect on PCR sensitivity by different PCR kits, with optimized PCR conditions to find an ideal Real-time PCR applicable for direct detection of rRNA or rDNA in whole blood, using the 16S rDNA gene. The study also surveyed the overall background flora of bacterial species circulating in the blood. During the optimization Haemophillus influenzae and Streptococcus pneumoniae were added to whole blood, rRNA or rDNA was isolated and extracted and subsequently processed by Real-time PCR. Four commercially available PCR kits were compared. Attempts using rRNA did not significantly increase the PCR sensitivity. LightCycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics) used for rDNA, generated low cp-values, the cleanest sequences and the finest separation between amplification curves. Twenty whole blood and pre-cultured patient samples were processed by the optimized PCR. The effect on PCR sensitivity by pre-culturing patient blood samples was studied and no statistical difference was noted. Increased PCR sensitivity is essential for implementation of PCR techniques in sepsis diagnostics.
Place, publisher, year, edition, pages
2015. , p. 35
Keywords [en]
Sepsis, PCR, Microbiology, Diagnostics, Molecular Biology
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:his:diva-12246OAI: oai:DiVA.org:his-12246DiVA, id: diva2:928005
External cooperation
Department of laboratory medicine, clinical microbiology/molecular diagnostics R&D at the University Hospital in Örebro Sweden
Subject / course
Biology
Educational program
Infection Biology - Master’s Programme
Supervisors
Examiners
2016-05-232016-05-132016-05-23Bibliographically approved