I want to optimize direct PCR detection of S. aureus. Does somebody have a good protocol or kit? Experience with comparison of efficiency of some methods or kits for isolating staphylococcal DNA from sputum?
I have experience with bacterial DNA extraction from sputum. Basically, procedure was take specimen and centrifuge, decant supernatant and resuspend in PBS. Spin and decant. Use chelex (10% w/v) extraction. Mix well and spin. Use supernatant for PCR. Hope this is some help.
Refer to Assam Assam JP et al 2012 Genetic analyses revealed by Mycobacterium tuberculosis as the causative agent of TB in the southern ecological zones of Cameroon.
There is a recent review of the molecular detection of CF lung pathogens in the Journal of Cystic Fibrosis 12 (2013) 194-205. It considers automated detection systems. We used a method published in Current Protocols in Molecular Biology (1990) Eds.: Ausubel et al, author: Wilson K. Preparation of genomic DNA from bacteria. It worked on sticky CF sputum samples.
You'll need a procedure involving bead beating otherwise you won't 'crack' the tough cell walls of S. aureus particularly in a mixed ecosystem. I have used QBiogene soil kit for sputum previously and it works well.
- Badges
- Science topic
- Similar topics
- Medicine
- Disease
- Infectious Diseases