PFGE Updates. Re-evaluation of existing protocols. Update on protocols in development. SDS in the plug agarose. Cell suspension concentration

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1 PFGE Update Kara Cooper, Ph.D. Centers for Disease Control and Prevention CCID/NCZVED/EDLRB PulseNet Methods Development and Reference Laboratory April 17 th 2006

2 PFGE Updates Re-evaluation of existing protocols SDS in the plug agarose Is it necessary for cell lysis? Cell suspension concentration Is more always better? Update on protocols in development

3 Proposed Revisions to the Existing PulseNet Standardized PFGE Protocols for E. coli O157, Salmonella, and Shigella Revision #1: Removal of the SDS from the plug agarose. Revision #2: Reduction of cell suspension concentration. Spectrophotometer: 610 nm wavelength, absorbance (Optical Density) of 1.00 Dade Microscan Turbidity Meter: (measured in Falcon 2054 tubes) (measured in Falcon 2057 tubes) biomérieux Vitek colorimeter: 17-18% transmittance (measured in Falcon 2054 tubes)

4 Validation of Proposed Revisions Internal Validation Tested by several laboratories within CDC External Validation 21 PulseNet participating laboratories involved Tested 2 E. coli O157:H7, 2 Shigella, and 3 Salmonella isolates with the protocol with revised conditions

5 Removal of SDS from Plug Agarose Internal Validation (CDC) No difference observed between plugs made with and without SDS No detrimental effect was observed with E. coli, Salmonella, and Shigella plugs made without SDS over the past year at CDC External Validation (21 PulseNet Laboratories) No indication of incomplete lysis was observed (i.e. high background, smearing, or incomplete restriction) A number of laboratories indicated that they had removed SDS from their plug preparation procedures prior to this study Final Recommendation Remove SDS from plug agarose

6 Proposed Revision of Cell Suspension Concentration Internal Validation (CDC) Fewer cells resulted in more efficient lysis Reduced background and sharper bands Increased resolution of closely migrating bands External Validation (21 PulseNet Laboratories) Band intensities were sufficient for analysis of all images submitted Final Recommendations Optimal cell suspension concentration may differ from lab to lab due to differences in equipment, reagents, and overall cell lysis efficiency Proposed revisions represent a more appropriate starting point, therefore we recommend accepting revised values

7 Validation Results XbaI BlnI Gels were of good to excellent quality High level of pattern similarity Difference that did exist were in difficult to resolve bands

8 Validation Recommendations Recommend accept proposed revisions Detailed description of proposed revisions available in your notebooks A sheet is provided for any comments you have on the proposed protocol Please turn in with your meeting survey

9 Worksheet Summary Purpose: To identify variations in the performance of PFGE in different laboratories that may influence gel/image quality Outcome: Several recommendations were generated to assist in improving gel/image quality and reproducibility among participating laboratories Final report and recommendations is provided in your notebooks

10 V. cholerea Update Database On-line: 256 patterns Certification Program is now in place Announcement will be posted to the PulseNet WebBoard Please submit request for certification to the PFGE Inbox with the phrase Vcholerae Certification in the subject line

11 Vibrio parahaemolyticus US External Validation External US validation by SHD: AL, HI, NY Accepted for publication in this summers edition of Foodborne Pathogens and Disease International Protocol Evaluation and Validation Collaborative effort with PulseNet Asia-Pacific to validate a PFGE protocol suitable for strains of V. parahaemolyticus isolated worldwide Summary of results presented during the poster session

12 Vibrio parahaemolyticus SfiI Plug Preparation Identical to Vibrio cholerae Electrophoresis conditions Initial Switch Time : 10s Final Switch Time: 35s NotI

13 Progress Report Plug Preparation Restriction Enzymes Electrophoresis Conditions Internal Validation External Validation V. vulnificus Non-O157 STEC Y. enterocolitica

14 Acknowledgments PulseNet Methods Development and Validation Laboratory Mary Ann Fair Efrain Ribot Nancy Garrett Eija Hyytia-Trees Jessica Halpin Michele Bird

PulseNet PFGE Protocol Development A Historical Perspective

PulseNet PFGE Protocol Development A Historical Perspective Mary Ann Fair Division of Foodborne, Diarrheal and Mycotic Diseases PulseNet Laboratory, CDC PAST PFGE - Major Developments In 1980s, several