Digital amplification for pathogen detection in bloodstream infections from whole blood

Sensitive and specific detection of bacteria is essential for control and treatment of bloodstream infections. Sepsis, a diagnostic challenge of blood stream infections, causes thousands of deaths each year. The gold standard for detection and identification of microorganisms is blood culture, which can take up to 5 days to yield a diagnosis or a negative result. Molecular diagnostic methods and nucleic acid assays developed to detect pathogens often require enrichment through blood culture prior to DNA extraction. Nonetheless, state of the art diagnostic tests that detect pathogens directly from blood are available with limitations. Increased amount of sample processing and purification is required before detection of pathogens and turnaround time to results can be up to 8 hours. Moreover, the sensitivity and specificity of such assays are broad in range. Thus, there is a need for a rapid detection method that can identify pathogens from whole blood within 1-2 hours, with minimal sample processing and high sensitivity and specificity. In this work, we demonstrate detection of bacterial pathogens from whole blood through loop mediated isothermal amplification without the steps of DNA purification and extraction. Whole blood with pathogens is dried to tubes, creating separated phases of the cellular debris of blood from the amplification reaction and allowing for separation of background human DNA from target DNA as well as better detection of amplicons that are not masked by red blood cells. We show that this method is robust, having a detection limit of 1 cfu of pathogens in 4uL of whole blood. In preparation of processing higher blood volumes in a digital amplification method, we also show characterization experiments and preliminary results of amplification of pathogens from whole blood on chip.

Name thesis:degree_name

M.S.

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Bioengineering

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2020

Author dc:creator

• Mostafa, Ariana

Contributors dc:contributor

• Bashir, Rashid

• Amplification, Pathogen Detection, Sepsis, Whole Blood, Digital, Chip

Statement dc:rights

• Copyright 2019 Ariana Mostafa

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/106369

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/106369