J P Rasmussen1, P H Barbez, L A Burgoyne, C P Saint. 1. Cooperative Research Centre for Water Quality and Treatment, Australian Water Quality Centre, South Australian Water Corporation, Salisbury, SA, Australia. paul.rasmussen@uwi.com.au
Abstract
AIMS: To develop a rapid preparation method for real-time PCR analysis of cyanobacteria from cultures or field samples. METHODS AND RESULTS: Field samples and cultures containing Anabaena circinalis, Cylindrospermopsis raciborskii or Microcystis aeruginosa were subjected to three cell disruption treatments: (i) heating during thermocycling, (ii) microwave irradiation in the presence of detergent and (iii) probe sonication. Treated samples were directly added to the PCR reaction and analysed on two different real-time devices. A statistically significant difference was evident in the cycle thresholds for each of the treatments in all but one culture and one environmental sample, sonication and microwave treatments performing better than direct addition. The microwave treatment was also compared to the Qiagen DNA Mini kit and performance was equivalent when treated samples were analysed as above. CONCLUSIONS: Whilst microwave treatment was slightly less effective than probe sonication across all samples, it was more amenable to processing multiple samples and significantly better than heat treating the sample during thermocycling. SIGNIFICANCE AND IMPACT OF THE STUDY: The microwave method described here is a simple, rapid and effective preparation method for cyanobacterial DNA that can be easily deployed in the field, making the most of the speed and flexibility offered by fixed and portable real-time PCR devices.
AIMS: To develop a rapid preparation method for real-time PCR analysis of cyanobacteria from cultures or field samples. METHODS AND RESULTS: Field samples and cultures containing Anabaena circinalis, Cylindrospermopsis raciborskii or Microcystis aeruginosa were subjected to three cell disruption treatments: (i) heating during thermocycling, (ii) microwave irradiation in the presence of detergent and (iii) probe sonication. Treated samples were directly added to the PCR reaction and analysed on two different real-time devices. A statistically significant difference was evident in the cycle thresholds for each of the treatments in all but one culture and one environmental sample, sonication and microwave treatments performing better than direct addition. The microwave treatment was also compared to the Qiagen DNA Mini kit and performance was equivalent when treated samples were analysed as above. CONCLUSIONS: Whilst microwave treatment was slightly less effective than probe sonication across all samples, it was more amenable to processing multiple samples and significantly better than heat treating the sample during thermocycling. SIGNIFICANCE AND IMPACT OF THE STUDY: The microwave method described here is a simple, rapid and effective preparation method for cyanobacterial DNA that can be easily deployed in the field, making the most of the speed and flexibility offered by fixed and portable real-time PCR devices.
Authors: Johan H Melendez; Tonya M Santaus; Gregory Brinsley; Daniel Kiang; Buddha Mali; Justin Hardick; Charlotte A Gaydos; Chris D Geddes Journal: Anal Biochem Date: 2016-06-17 Impact factor: 3.365