Jensen, Peter Lüttge

Larsen, Lone Heimann

Schønheyder, Henrik Carl

Thomsen, Trine Rolighed

English

    Aalborg UniversitetDetection and destruction of risidual dna on surgical steelJensen, Peter Lüttge; Larsen, Lone Heimann; Thomsen, Trine Rolighed; Schønheyder,Henrik CarlPublication date:2012Document VersionEarly version, also known as pre-printLink to publication from Aalborg UniversityCitation for published version (APA):Jensen, P. L., Larsen, L. H., Thomsen, T. R., & Schønheyder, H. C. (2012). Detection and destruction of risidualdna on surgical steel. Poster presented at Symposium of The Danish Microbiological Society, Copenhagen,Denmark.General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.            ? Users may download and print one copy of any publication from the public portal for the purpose of private study or research.            ? You may not further distribute the material or use it for any profit-making activity or commercial gain            ? You may freely distribute the URL identifying the publication in the public portal ?Take down policyIf you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access tothe work immediately and investigate your claim.Downloaded from vbn.aau.dk on: December 26, 2020PRIS DETECTION AND DESTRUCTION OF RESIDUAL DNA ON SURGICAL STEEL  Peter Lüttge Jensen1, Lone Heimann Larsen2, Trine Rolighed Thomsen1, Henrik Carl Schønheyder3  1: Section for Medical Biotechnology, Danish Technological Institute  2: Section for Biotechnology, Institute for Chemistry and Biotechnology, Aalborg University  3: Department of Clinical Microbiology, Aalborg Hospital, Aarhus University Hospital,  WHY? HOW? RESULTS CONCLUSION Polymerase chain reaction (PCR) is being used increasingly in the field of clinical microbiology. One of the methods’ advantages is the extreme sensitivity, but this can also be a problem, because even trace contamination with DNA can lead to erroneous diagnosis. This has been the case previously where carry-over of residual microorganisms REFS Kaul K, et al. Amplification of residual DNA sequences in sterile bronchoscopes leading to false-positive PCR results. J Clin Microbiol. 1996;34(8):1949-51.    Gefrides LA, et al. UV irradiation and autoclave treatment for elimination of contaminating DNA from laboratory consumables.  Forensic Sci Int Genet. 2010;4(2):89-94.   Patel R, et al. Death from inappropriate therapy for Lyme disease. Clin Infect Dis. 2000;31(4):1107-9.   from improperly cleansed bronchoscopes lead to false-positive PCR results. Current autoclave procedures are known to be adequate for killing of microorganisms, but it is not known whether the routines are sufficient for removal/inactivation of DNA. With molecular biology-based methods for diagnosis it could proove necessary to combine the autoclave step with a DNA removal/ inactivation method. Unfortunately, current strategies for removal/destruction of DNA from surfaces are hazardous, corrosive or expensive.   The aim of the current study was therefore to: 1) Develop a non-corrosive, non-hazardous, inexpensive method for making residual DNA non-amplifiable,  2) Devise a protocol for detection of residual DNA on surgical steel instruments 3) Determine whether residual DNA was present after sterilization routines at Aalborg University Hospital.  Gefrides 2009: ”2 h of autoclave treatment will eliminate nanogram  quantities of DNA from laboratory consumables” S. aureus P. aeruginosa Autoclave step a) Duration: 0 & 60 & 120 min b) Temperature: 125 & 139 °C DNA isolation and quantification Sonication  soil kit TaqMAN qPCR P. ae S. au IN VITRO TEST Wash  100 min max temp 100°C CLIN. SIMUL. Autoclave a) 4 min,137°C b) 120 min,134°C P. aeruginosa S. aureus The simulation at the CSR documented that the current disinfection routines are adequate. If more rigorous DNA-destruction is needed, prolonged autoclaving at 137°C for 120 min can be implemented to provide >10 mio fold reduction in the amount of residual amplifiable DNA. K. pneumoniae (thermotolerant) 110100100010000100000100000010000000A B C D A B C D A B C DK. pneumoniae (16S rRNA) S. aureus (femA 63 bp) P. Aeruginosa (ecfX 142 bp) Approximately 105 cells/scalpel A) Control (no wash, no autoclave) B) Wash (100 min, max temp 100°C) C) Wash & autoclave (4 min, 137°C) D) Wash & autoclave (120 min, 134°C) Dense inoculum:  109 cells/mL # respective genes 11,0001,000,0001,000,000,0001H Wet2H Wet1H Dry2H Dry139 °C125 °C11,0001,000,0001HWet2HWet1H Dry2H Dry

Detection and destruction of risidual dna on surgical steel

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Detection and destruction of risidual dna on surgical steel

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