A non-organic and non-enzymatic extraction method gives higher yields of genomic DNA from whole-blood samples than do nine other methods tested

Molecular methods are important tools in the diagnosis of bloodstream bacterial infections, in particular in patients treated with antimicrobial therapy, due to their quick turn-around time. Here we describe a new broad-range real-time PCR targeting the 23S rDNA gene and capable to detect as low as 10 plasmid copies per reaction of targeted bacterial 23S rDNA gene. Two commercially available DNA extraction kits were evaluated to assess their efficiency for the extraction of plasma and whole blood samples spiked with different amount of eitherStaphylococcus aureusorEscherichia coli, in order to find the optimal extraction method to be used. Manual QIAmp extraction method with enzyme pre-treatment resulted the most sensitive for detection of bacterial load. Sensitivity of this novel assay ranged between 10 and 103 CFU per PCR reaction forE. coliandS. aureusin human whole blood samples depending on the extraction methods used. Analysis of plasma samples showed a 10- to 100-fold reduction of bacterial 23S rDNA in comparison to the corresponding whole blood specimens, thus indicating that whole blood is the preferential sample type to be used in this real-time PCR protocol. Our results thus show that the 23S rDNA gene represents an optimal target for bacteria quantification in human whole blood.

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Comparison of genomic DNA extraction techniques from whole blood samples: a time, cost and quality evaluation study

Molecular Biology Reports ◽

10.1007/s11033-011-1408-8 ◽

2012 ◽

Vol 39 (5) ◽

pp. 5961-5966 ◽

Cited By ~ 26

Author(s):

Diego Chacon-Cortes ◽

Larisa M. Haupt ◽

Rod A. Lea ◽

Lyn R. Griffiths

Keyword(s):

Dna Extraction ◽

Whole Blood ◽

Genomic Dna ◽

Quality Evaluation ◽

Evaluation Study ◽

Time Cost ◽

Extraction Techniques ◽

Blood Samples ◽

Genomic Dna Extraction ◽

Whole Blood Samples

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Methods for extracting genomic DNA from whole blood samples: current perspectives

Journal of Biorepository Science for Applied Medicine ◽

10.2147/bsam.s46573 ◽

2014 ◽

pp. 1 ◽

Cited By ~ 14

Author(s):

Lyn Griffiths ◽

Diego Chacon-Cortes

Keyword(s):

Whole Blood ◽

Genomic Dna ◽

Blood Samples ◽

Whole Blood Samples

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Comparison of commercial DNA extraction kits for the detection of bacterial genomic DNA from whole-blood samples using a broad-range PCR

Critical Care ◽

10.1186/cc8069 ◽

2009 ◽

Vol 13 (Suppl 4) ◽

pp. P13

Author(s):

B Krulova ◽

E Nemcova ◽

B Zaloudikova ◽

P Nemec ◽

T Freiberger

Keyword(s):

Dna Extraction ◽

Whole Blood ◽

Genomic Dna ◽

Blood Samples ◽

Whole Blood Samples

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An efficient and novel technology for the extraction of parasite genomic DNA from whole blood or culture

BioTechniques ◽

10.2144/btn-2019-0086 ◽

2020 ◽

Vol 68 (2) ◽

pp. 79-84 ◽

Cited By ~ 1

Author(s):

David J Clark ◽

Catherine M Moore ◽

Marc Flanagan ◽

Katrien Van Bocxlaer ◽

Evangelia-Theophano Piperaki ◽

...

Keyword(s):

Whole Blood ◽

Genomic Dna ◽

Leishmania Donovani ◽

Small Volume ◽

Small Sample ◽

Blood Samples ◽

Novel Technology ◽

Spin Column ◽

Whole Blood Samples ◽

Pathogen Dna

The aim of this study was to assess pathogen DNA extraction with a new spin column-based method (DNA-XT). DNA from either whole-blood samples spiked with Plasmodium falciparum or Leishmania donovani amastigote culture was extracted with DNA-XT and compared with that produced by a commercial extraction kit (DNeasy®). Eluates from large and small sample volumes were assessed by PCR and spectroscopy. Using a small volume (5 μl) of blood, the DNA-XT and DNeasy methods produced eluates with similar DNA concentrations (0.63 vs 1.06 ng/μl, respectively). The DNA-XT method produced DNA with lower PCR inhibition than DNeasy. The new technique was also twice as fast and required fewer plastics and manipulations but had reduced total recovered DNA compared with DNeasy.

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