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Orthopaedic Proceedings
Vol. 98-B, Issue SUPP_23 | Pages 32 - 32
1 Dec 2016
APPLICATION OF NEXT GENERATION SEQUENCING FOR THE DIAGNOSIS OF ORTHOPAEDIC INFECTION: AN EVALUATION OF FOUR DNA EXTRACTION TECHNIQUES
Cleaver L Gorton R Gandy M Palanivel S Mack D Warren S
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Aim

Diagnosing Orthopaedic infection is limited by the sensitivity of culture methods. Next generation sequencing (NGS) offers an alternative approach for detection of microorganisms from clinical specimens. However, the low ratio of pathogen DNA to human DNA often inhibits detection of microorganisms from specimens. Depletion of human DNA may enhance the detection of microbial DNA1. Our aim was to compare four DNA extraction methods for the recovery of microbial DNA from orthopaedic samples for NGS.

Method

Simulated samples; pooled culture negative sample matrix was spiked with known concentrations of microorganisms, each panel consisting of 7 samples. Broth culture was performed on simulated samples for comparison with NGS*.

DNA Extraction; total nucleic acid extraction was performed on an automated extraction platform** using the viral NA assay. Modifications included: (1) mechanical lysis (glass beads), (2) lysis of human cells (saponin 0.025%), turbo DNase treatment and (3) mechanical lysis and addition of MspJI enzyme post-extraction for methylated DNA digestion.

Detection of human and microbial DNA; human endogenous (HE) gene rtPCR*** was utilised following manufacturer's recommendations. Microbial DNA was detected using SYBR green 16s ribosomal RNA rtPCR with high resolution melt-curve analysis****.