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Nuclease-assisted suppression of human DNA background in sepsis.

Song Y, Giske CG, Gille-Johnson P, Emanuelsson O, Lundeberg J, Gyarmati P - PLoS ONE (2014)

Bottom Line: Our experiments show that human DNA concentration can be reduced approximately 100,000-fold without affecting the E. coli DNA concentration in a model system with similarly sized amplicons.With clinical samples, the human DNA background was decreased 100-fold, as bacterial genomes are approximately 1,000-fold smaller compared to the human genome.According to our results, background suppression can be a valuable tool to enrich rare DNA in clinical samples where a high amount of background DNA can be found.

View Article: PubMed Central - PubMed

Affiliation: Royal Institute of Technology, Science for Life Laboratory, Stockholm, Sweden.

ABSTRACT
Sepsis is a severe medical condition characterized by a systemic inflammatory response of the body caused by pathogenic microorganisms in the bloodstream. Blood or plasma is typically used for diagnosis, both containing large amount of human DNA, greatly exceeding the DNA of microbial origin. In order to enrich bacterial DNA, we applied the C0t effect to reduce human DNA background: a model system was set up with human and Escherichia coli (E. coli) DNA to mimic the conditions of bloodstream infections; and this system was adapted to plasma and blood samples from septic patients. As a consequence of the C0t effect, abundant DNA hybridizes faster than rare DNA. Following denaturation and re-hybridization, the amount of abundant DNA can be decreased with the application of double strand specific nucleases, leaving the non-hybridized rare DNA intact. Our experiments show that human DNA concentration can be reduced approximately 100,000-fold without affecting the E. coli DNA concentration in a model system with similarly sized amplicons. With clinical samples, the human DNA background was decreased 100-fold, as bacterial genomes are approximately 1,000-fold smaller compared to the human genome. According to our results, background suppression can be a valuable tool to enrich rare DNA in clinical samples where a high amount of background DNA can be found.

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Related in: MedlinePlus

Representative amplification curves of 10 experiments show background suppression on a septic blood sample, with blue lines representing the human DNA (β-actin), and pink lines show E. coli DNA amount.While the amount of human DNA has degraded, the E. coli DNA amount did not change.
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pone-0103610-g003: Representative amplification curves of 10 experiments show background suppression on a septic blood sample, with blue lines representing the human DNA (β-actin), and pink lines show E. coli DNA amount.While the amount of human DNA has degraded, the E. coli DNA amount did not change.

Mentions: The efficiency of background suppression of human DNA was evaluated in three different ways: 1) with amplicons resulting from PCRs targeting β-actin and E. coli in order to optimize the method, 2) with clinical plasma, and 3) blood samples from septic patients to prove the usability of the method (Figures S1–S2, Figure 3). In addition to the amplification curves, melting peaks specific for either β-actin or E. coli amplicons were used to identify positive signals (Figure S2b).


Nuclease-assisted suppression of human DNA background in sepsis.

Song Y, Giske CG, Gille-Johnson P, Emanuelsson O, Lundeberg J, Gyarmati P - PLoS ONE (2014)

Representative amplification curves of 10 experiments show background suppression on a septic blood sample, with blue lines representing the human DNA (β-actin), and pink lines show E. coli DNA amount.While the amount of human DNA has degraded, the E. coli DNA amount did not change.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4116218&req=5

pone-0103610-g003: Representative amplification curves of 10 experiments show background suppression on a septic blood sample, with blue lines representing the human DNA (β-actin), and pink lines show E. coli DNA amount.While the amount of human DNA has degraded, the E. coli DNA amount did not change.
Mentions: The efficiency of background suppression of human DNA was evaluated in three different ways: 1) with amplicons resulting from PCRs targeting β-actin and E. coli in order to optimize the method, 2) with clinical plasma, and 3) blood samples from septic patients to prove the usability of the method (Figures S1–S2, Figure 3). In addition to the amplification curves, melting peaks specific for either β-actin or E. coli amplicons were used to identify positive signals (Figure S2b).

Bottom Line: Our experiments show that human DNA concentration can be reduced approximately 100,000-fold without affecting the E. coli DNA concentration in a model system with similarly sized amplicons.With clinical samples, the human DNA background was decreased 100-fold, as bacterial genomes are approximately 1,000-fold smaller compared to the human genome.According to our results, background suppression can be a valuable tool to enrich rare DNA in clinical samples where a high amount of background DNA can be found.

View Article: PubMed Central - PubMed

Affiliation: Royal Institute of Technology, Science for Life Laboratory, Stockholm, Sweden.

ABSTRACT
Sepsis is a severe medical condition characterized by a systemic inflammatory response of the body caused by pathogenic microorganisms in the bloodstream. Blood or plasma is typically used for diagnosis, both containing large amount of human DNA, greatly exceeding the DNA of microbial origin. In order to enrich bacterial DNA, we applied the C0t effect to reduce human DNA background: a model system was set up with human and Escherichia coli (E. coli) DNA to mimic the conditions of bloodstream infections; and this system was adapted to plasma and blood samples from septic patients. As a consequence of the C0t effect, abundant DNA hybridizes faster than rare DNA. Following denaturation and re-hybridization, the amount of abundant DNA can be decreased with the application of double strand specific nucleases, leaving the non-hybridized rare DNA intact. Our experiments show that human DNA concentration can be reduced approximately 100,000-fold without affecting the E. coli DNA concentration in a model system with similarly sized amplicons. With clinical samples, the human DNA background was decreased 100-fold, as bacterial genomes are approximately 1,000-fold smaller compared to the human genome. According to our results, background suppression can be a valuable tool to enrich rare DNA in clinical samples where a high amount of background DNA can be found.

Show MeSH
Related in: MedlinePlus