LAS approved laboratories: Information

Transcription

1 17 March 2010 Page 1 LAS approved laboratories: Information LAS approved laboratories: Information...1 Visit to ESR Forensic Biology Laboratory...1 Design and layout...2 Room design... 2 Air pressure and room design... 2 Staff clothing...3 Optimising use of reagents...3 Contamination...4 Cleaning...4 UV light use and environmental samples... 4 Cleaning chemicals and application... 4 Pipettors... 5 Room clean-up monthly cleanup... 5 Sample integrity...5 Listing of n60 bulk samplers...6 Visit to ESR Forensic Biology Laboratory On Tuesday 23 February 2010 a visit was made by the LAS Administrator to ESR Forensic Biology (ESR) at Mt Albert to view their set up for Polymerase Chain Reaction (PCR). Their laboratory layout, cleaning regimes and entry and egress of personnel were of interest. This document summarising the ESR visit, is just that, an information document. It does not replace any E. coli O157:H7 microbiological programme, LAS or IANZ requirements. As you might suspect there is a lot at stake if ESR has contamination or sample identification issues. Likewise there is lot at stake for the E. coli O157:H7 microbiological programme now GDS TM (Genetic Detection System) is being used. The purpose of this publication is to highlight some areas that ESR brought to the LAS Administrator s attention that E. coli O157:H7 laboratories might wish to focus on as they gain experience with GDS TM. As PCR requires very careful laboratory technique such that there is no risk of contamination all LAS approved laboratories will gain something from this publication; particularly the sections on Sample Integrity and Listing of n60 bulk samplers.

2 17 March 2010 Page 2 Design and layout Room design ESR started shoehorning the DNA laboratory in over 10 years ago and had used the dividing lines in the same room tactic. This proved to be unsatisfactory and now ESR have the sample preparation and casework (~Zone 2), reagent prep room (~Zone 1) and Instrumentation lab (~ Zone 3), along with other set up and research areas, each completely segregated. Air pressure and room design Air is a concern with PCR. One contamination event was deduced to be linked with air circulation between rooms. At ESR the hallway is neutral, the reagent preparation room is positive pressure of 5 Pa (~ Zone 1), and the sample preparation area is positive 10 Pa (~ Zone 2). Interestingly ESR laboratory has NEGATIVE PRESSURE in the instrumentation room (~ Zone 3). They have two air lock entries into the large instrumentation room. Each airlock entry has two doors that won t open at the same time. The reason for negative pressure and air locks is that the samples at Zone 3 stage are enclosed and amplification occurs in this space. The aim is to prevent anything going out of that room and being circulated into other areas. A cross contamination event 10 years ago led to this layout. I am not suggesting that there is a need for capital expenditure to install this air pressure and air lock arrangement, rather a neutral pressure for Zone 3 and that Zone 1 and Zone 3 rooms should at least be completely separate. By completely separate rooms I don t mean one room divided into two, but two separate rooms with separate access and egress as indicated below: Not suitable; these are just divided single rooms. Suitable; the rooms are completely segregated.

3 17 March 2010 Page 3 Zone 1 A discrete reagent dispensing room. Ideally a positive air pressure room for concentration reagent, wash solution, resuspension buffer, polymerase. Holding refrigerator, combitips and GDS TM repeat pipettors, TAQ polymerase and wells. Zone 2 Ideally a positive air pressure room dedicated for bulk meat sample weighing, suspension in enrichment broth (mtsb) and incubation, including the laminar flow cabinet with pickpen tips and filter tips for sample to well transfer. Zone 3 Ideally a negative pressure room with an airlock, next best would be a neutral room, housing the Laptop and Rotor Gene. Hallways connecting the rooms would be neutral pressure. Staff clothing At ESR they use gloves and lab coats, masks and hairnets in areas where the protection of the sample is important. No hairnets or masks are worn in the Instrumentation lab where contamination of the sample is unlikely. We need to use full lab coats, gloves, coats and booties because we are dealing with an enriched pathogen at the Zone 2 stage. Recommendation: Zone 1 Lab coat, hat, booties and gloves. Zone 2 Disposable lab coat over the top. Zone 3 A new set of gloves to be worn on entering Zone 3, plus an apron when emptying the GDS TM machine. It could be a challenge to ensure a discrete set of gloves are worn in Zone 3. If you choose to colour code things blue nitrile gloves are available. Optimising use of reagents In a word DON T! False economy! Kits have a 6 week expiry date. Attempts could be made to subdivide the polymerase solution tube into two lots, then adding 50% of the TAQ to one at a time in order for the kit to last 12 weeks instead of the usual 6 weeks. This can only lead to errors. The TAQ subdivided minute amount of 50µl plus attraction to surfaces will mean that there is none left to dispense. It would be impossible to determine if it had become contaminated. New consumables need to be verified. Each batch of consumables needs to be tested.

4 17 March 2010 Page 4 Contamination Operator contamination can be minimised by only conducting one case at a time. Records need to be taken of persons entering GDS TM Zones, particularly Zones 1 and 3 which are dedicated to GDS TM. A permanent record of the date and time of entry into Zones 1 and 3 should be taken. The use of blank, negative and positive controls is essential to monitor contamination and effective functioning of the PCR. Any contamination event needs to be monitored and all corrective actions, both effective and ineffective actions in response to the event, recorded. Corrective action procedures will be part of ISO17025, but it is essential issues such as contamination are documented and addressed immediately. Cleaning UV light use and environmental samples UV light denatures DNA and is used once a night by ESR. Environmental samples should be taken at least once a week. Environmental sampling needs to include pipettes, surfaces, rulers, doorhandles, hoods, all the equipment in hoods, the sides of benches. Determine where the shadow areas are in your rooms, for example the underside of benches and ledges where the UV light won t reach. Each time you take environmental samples ensure you include at least one from a shadow area. Cleaning chemicals and application Even though racks are only used to carry samples at ESR they are cleaned and soaked with Virkon, dishwashed in caustic powder and returned to the start of the process. Surfaces are cleaned with Virkon first and then wiped with 80% Ethanol. Bleach of 20% Janola to clean surfaces first, followed by 70% Ethanol is part of the GDS TM instructions. Trigene was not recommended by ESR. Trigene was reported to build up on surfaces leading to PCR inhibition.

5 17 March 2010 Page 5 Pipettors Ensure you are using aerosol resistant, filter pipette tips. Pull pipettors apart for cleaning at least once a month. Clean the outside of pipettors during weekly room clean-up. Room clean-up Clean all equipment, including equipment in hoods, and all surfaces including the sides of benches. Clean hoods. Everything in the rooms or passing through the rooms must be DNA free. This means DNA free, not just DNase and Rnase free. Once a month the air systems need to be turned off and given a maintenance check by trade professionals. 6 monthly clean-up At ESR every 6 months every nook and cranny from floor to ceiling, inside cupboards, inside and outside the refrigerator, the condensate trap, light fittings, hallways is cleaned. Everything everywhere is cleaned. ESR picked this idea up from one of the laboratories in Europe. Sample integrity Quality is a very big expense. At ESR 6 monthly Interlaboratory Comparison Programme (ILCP) samples are provided from the US. At ESR sample swapping is very rare and ESR has never had sample swapping errors in ILCP. Unfortunately this is a regular feature of LAS approved laboratories ILCP for microbiological testing, including E. coli O157 samples. LAS approved laboratories need to raise the bar! If sample swapping occurs with ILCP this does not give much confidence that it isn t happening with routine samples. At ESR procedures from the time of the receipt of the sample through to the completion of the analysis are carried out in a manner to preserve sample integrity. Precautions to prevent sample swapping include: A distinct bar code which is scanned. Where there is a transfer step of a supernatant to another tube, the tube is bar coded to ensure order.

6 17 March 2010 Page 6 If transfer is done manually then tubes must be moved up or down the racks diligently to ensure the sample integrity is maintained. All methods are posted in a step by step chart. Times are recorded, the time a sample went into the incubator and the time that particular sample came out. Tick boxes are used to know what stage one person finished one task related to that sample and another person commenced. Listing of n60 bulk samplers The current LAS includes a requirement for laboratory authorised representatives to notify the LAS Administrator of Certified and Associate Trainers sampling for the NMD microbiological sampling programme Listing of NMD Certified and Associate Trainers A list of Certified and Associate Trainers for the NMD programme will be published on the NZFSA web site and will be updated on a regular basis. Laboratory authorised representatives are responsible for informing the LAS Administrator of Certified and/or Associate Trainers currently undertaking NMD sampling on behalf of the laboratory NZFSA NMD administration has recently developed places in the Associate Trainers and Certified Trainers website listings to include n60 bovine and bobby calf bulk samplers. Bovine Bobby Calf Ovine Caprine Cervine Ostrich Porcine Poultry Ecoli Poultry Campy Buchanan, Trish Carcass Y Y Y N N N N N N N Alliance Mataura Primal Y Y Y N N Alliance Mataura(NMD), NZLabsCHCH(O157) NMD Bulk NMD Bulk (0157) Y Y Y N Y Y Poultry Caecal Note that O157 is an abbreviation used to represent the E. coli O157:H7 sampling programme; and LAS approved laboratories with official test 23.1 in their scope. Laboratory authorised

7 17 March 2010 Page 7 representatives of these laboratories need to review and record all their E. coli O157 programme n60 bulk samplers (Certified, Associate and Restricted) in addition to NMD bulk samplers. The LAS Administrator needs to be notified of n60 bulk Certified and Associate Trainers to list them. Information required by the LAS Administrator is the Trainers location (premises, or laboratory), species they sample; bobby calf and/or bovine. And for Associate Trainers the name of the Certified Trainer who trained them. Disclaimer: This publication is not a legal interpretation of the Animal Products Act or the Animal Products (Ancillary and Transitional Provisions) Act and is intended only as a guide. Contact for enquiries Gail Duncan LAS Administrator New Zealand Food Safety Authority Jervois Quay PO Box 2835 Wellington NEW ZEALAND Phone: Fax: March 2010