Development and optimisation of a protocol for the extraction of microbial DNA from clinical pulmonary samples

Abstract

Ventilator-associated pneumonia (VAP) is the most common nosocomial infection in patients admitted to the ICU and a leading cause of mortality in critically ill patients. The rise in antimicrobial resistance threatens current treatment methods, and if not addressed will lead to a significant increase in mortality. Rapid and accurate diagnostic methodologies are needed to combat this issue. To develop and test the potential of these DNA based technologies, we focus here on Staphylococcus aureus as it is a common cause of antimicrobial resistant VAP.

The first step in the application of any DNA-based test is the reliable extraction of the bacterial DNA. In this project three DNA extraction methods were compared for accuracy and sensitivity: a commercially available DNA extraction kit, and two methods which use traditional phenol/chloroform methods for DNA purification but differ in their lysis technique; one using physical bead-beating and the other using MetaPolyzyme, a multi-lytic enzyme mix.

To test each method’s ability to extract S. aureus, extracted DNA was amplified using qPCR with generic 16S rRNA gene primers and S. aureus specific nuc gene primers to determine the relative quantity of S. aureus within a sample. On pure broth grown cultures the bead-beating method was determined as the most effective and was subsequently developed and optimised for use on pleural fluids provided to us by the NHS. With these more complex materials the use of phase lock gel (PLG) reduced the variability observed when using phenol/chloroform in standard laboratory tubes.

Having established the optimal extraction methodology, and alongside control plasmids, the limit of detection was determined for two sets of PCR primers, a generic bacterial 16S set and a S. aureus specific nuc set. The limit of detection of spiked pleural fluids was ~1x104 CFU/ml using the 16S primer set and ~1x103 CFU/ml for the nuc set. As this work is developed further its use in a clinical diagnostic setting will be evaluated to provide rapid diagnoses of pleural infections.

Date of Award	29 Sept 2020
Original language	English
Awarding Institution	University of Bristol
Supervisor	Ruth C Massey (Supervisor)