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

ABSTRACT: 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.

SUBMITTER: Clark DJ

PROVIDER: S-EPMC7115988 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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

Clark David J DJ Moore Catherine M CM Flanagan Marc M Bocxlaer Katrien Van KV Piperaki Evangelia-Theophano ET Yardley Vanessa V Croft Simon L SL Tyson John J Whitehouse Sam P SP O'Halloran Jonathan J Krishna Sanjeev S Staines Henry M HM

BioTechniques 20191127 2

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 produc ...[more]

PMID: 31774301

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Project description:PURPOSE:Speed of bloodstream infection diagnosis is vital to reduce morbidity and mortality. Whole genome sequencing (WGS) performed directly from liquid blood culture could provide single-assay species and antibiotic susceptibility prediction; however, high inhibitor and human cell/DNA concentrations limit pathogen recovery. We develop a method for the preparation of bacterial DNA for WGS-based diagnostics direct from liquid blood culture. METHODOLOGY:We evaluate three commercial DNA extraction kits: BiOstic Bacteraemia, Amplex Hyplex and MolYsis Plus. Differential centrifugation, filtration, selective lysis and solid-phase reversible immobilization bead clean-up are tested to improve human cells/DNA and inhibitor removal. Using WGS (Illumina/MinION), we assess human DNA removal, pathogen recovery, and predict species and antibiotic susceptibility inpositive blood cultures of 44 Gram-negative and 54 Staphylococcus species.Results/Key findings. BiOstic kit extractions yield the greatest mean DNA concentration, 94-301 ng µl-1, versus 0-2.5 ng µl-1 using Amplex and MolYsis kits. However, we note higher levels of inhibition (260/280 ratio 0.9-2.1) and human DNA (0.0-4.4×106 copies) in BiOstic extracts. Differential centrifugation (2000 g, 1 min) prior to BiOstic extraction reduces human DNA by 63-89 % with selective lysis minimizing by a further 62 %. Post-extraction bead clean-up lowers inhibition. Overall, 67 % of sequenced samples (Illumina MiSeq) contain <10 % human DNA, with >93 % concordance between WGS-based species and susceptibility predictions and clinical diagnosis. If >60 % of sequencing reads are human (7/98 samples) susceptibility prediction becomes compromised. Novel MinION-based WGS (n=9) currently gives rapid species identification but not susceptibility prediction. CONCLUSION:Our method for DNA preparation allows WGS-based diagnosis direct from blood culture bottles, providing species and antibiotic susceptibility prediction in a single assay.

Dataset Information

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

Publications

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

Similar Datasets

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