1105. Maximum Release of DNA from Difficult-to-lyse Bacteria for Nucleic Acid Testing Using a Novel Chemical Lysis Reagent
Session: Poster Abstract Session: Molecular and Nucleic Acid Testing Diagnostics
Friday, October 9, 2015
Room: Poster Hall
Posters
  • IDweek poster final.pdf (2.8 MB)
  • Background: Identification of microorganisms by nucleic acid testing (NAT) is a critical tool for the rapid diagnosis of infectious diseases and identification of bio-threat agents. The sensitivity and clinical utility of any NAT strategy relies on the availability of nucleic acids in a sample. Hardy bacteria such as Bacillus spores and Mycobacterium tuberculosis (MTB) are notoriously difficult to break open, and as such, the total nucleic acid available for NAT may limit the effectiveness of the assays. The development of a new chemical lysis method called spore•LYSE now enables larger amounts of high quality nucleic acids to be released in a safe manner from difficult-to-lyse microbes. 

    Methods: Extraction of DNA by spore•LYSE was compared to bead-beating in phosphate-buffered saline or SDS buffer for release of DNA from in vitro­-grown microbes, and MTB spiked into sputum. spore•LYSE was evaluated by comparing the amount of DNA released from bacteria to the amount of DNA present inside the bacteria prior to lysis treatment. This was done using acid-hydrolysis/HPLC, and by quantification of extracted DNA using qPCR. 

    Results: spore•LYSE was able to release approximately 85% of DNA from Mycobacterium smegmatis and greater than 90% of DNA from surrogates of Category A bioterrorism agents, including Bacillus spores. Overall, qPCR results support that spore•LYSE releases an equal or greater amount of DNA than bead-beating from both gram positive and negative bacteria. Lysis of Bacillus thuringiensis spores with spore•LYSE resulted in qPCR Ct values of 18.4 +/- 0.1, whereas Ct values of bead-beating extracts were 19.3 +/- 0.1. spore•LYSE was also compared to standard-of-care for DNA extraction from MTB in sputum. When 7 x 104 cells of MTB were spiked into sputum, the Cvalue of samples extracted with spore•LYSE was 24.3 +/- 0.5 compared to 25.7 +/- 0.9 for bead-beating. 

    Conclusion: spore•LYSE employs an easy-to-use liquid lysis reagent that only requires incubation for 5-20 min at 70°C for release of large amounts of high quality DNA from a variety of bacteria, including MTB and Bacillus spores. spore•LYSE simplifies DNA extraction protocols and eliminates the need for bead-beating, and can decrease the time/cost associated with diagnosis and identification of low-abundance or difficult-to-lyse bacteria.

    Olle De Bruin, PhD, Jacques Niles, MSc, Bitapi Ray, MSc and Cassandra Kelly-Cirino, PhD, DNA Genotek Inc., Ottawa, ON, Canada

    Disclosures:

    O. De Bruin, DNA Genotek Inc.: Employee , Salary

    J. Niles, DNA Genotek Inc.: Employee , Salary

    B. Ray, DNA Genotek Inc.: Employee , Salary

    C. Kelly-Cirino, DNA Genotek Inc.: Employee , Salary

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