Symbio Alliance. Laboratory Training - RTO Technical Services.

Transcription

1 Symbio Alliance Laboratory Training - RTO Technical Services Brisbane: Brandl St, Eight Mile Plains, Qld, Melbourne: Unit 15, Geelong Rd, Brooklyn, Vic, Sydney: 1 Braidwood St, Enfield, NSW, Wagga Wagga: Unit 5, Koorignal Rd, Wagga Wagga, NSW, Rockhampton: St Christophers Chapel Rd, NERIMBERA, Qld September 2014

2 MINTRAC QA & MI CONFERENCE 2014 New technology in laboratories on and off plant

3 What is Driving New Technology in Labs? Everyone wants this... <GRAPHIC DEPICTING FAST/SPEED >

4 What is Driving New Technology in Labs? Everyone also wants costs to go down... TESTING COSTS TESTING COSTS TIME

5 What is Driving New Technology in Labs? But at the same time, nobody wants errors... REALITY TRUE FALSE TEST RESULT TRUE FALSE CORRECT TYPE 2 ERROR FALSE NEGATIVE TYPE 1 ERROR FALSE POSITIVE CORRECT e.g. E.coli O157 & Big 6 STEC

6 What is Driving New Technology in Labs?... and.. everyone assumes they get the most ACCURATE & PRECISE numbers e.g. E.coli, Coliform, Staphyloccus, EB, LAB, Listeria counts

7 Developments in Laboratory Technology 1. Testing methods Traditional vs Novel Methods Screening vs Confirmation Testing 2. New Technologies for On-plant Labs Keep the simple and cheap, outsource the complex and costly Rapid screen tests with easy interpretation of results e.g. Rapid Micro kits, Allergen Kits, Immunoassay, ELISA kits Alternative/rapid hygiene monitoring e.g. ATP 3. New Technologies in Independent/commercial lab technologies Novel methods Automation & labour saving devices Robotics (saves labour cost, adds capital cost, saves a little time, but not necessarily TAT which is method dependent) E-everything (CoC s, Results Access, Trend Analysis)

8 On plant Hygiene Monitoring ATP vs Traditional Micro Adenosine Tri-Phosphate (ATP) is the energy store in cells It is in ALL living cells (microbes, plants, animals, bodily fluids), and gradually fades away after death The phosphates break off to release energy ATP machine uses fire-fly Luciferase to capture the energy Therefore more Cells/Organisms > More ATP > More Light Light is measured as Relative Light Units (RLU)

9 ATP vs Visual vs Micro? Visual - rapid but naked eye not sensitive beyond very dirty Micro testing - objective, but not in real time and only get a result for microbes, not biofilm ATP - rapid, picks up everything that is giving off energy from ATP (cells of all types, incl micro)

10 Idea of using ATP.. Using food residue (measured by ATP) as an indicator of cleanliness is a similar idea to use of generic E. coli as an indicator of faecal contamination.

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17 ATP Machines & RLUs Manufacturer of ATP machine determines their own value for a Light Unit and all their measurements are Relative to that The machines all have inherent variance Very important to use a machine that has been validated and that you can identify what the known variance is and how repeatable the result is If the result is not consistent, you might re-clean when you don t need to or not re-cleaning when you should Recommend getting detailed info on this aspect from the manufacturer To use this technology in the meat industry and have in an Approved Arrangement is likely to require manufacturers or plants to do a little validation study in order to set proper limits

18 Potential ATP use in pre-op? Identify what needs to be clean Direct contact surfaces Indirect contact surfaces Personnel PPE, knives, steels, pouches Set limits Pass, Fail, Marginal (e.g. m = 100 M = 300; OR m = 1000 M = 1500) Base on your relative performance Use the Results Today s results available in real time Track over time, build database, use trend analysis Experiments different methods of cleaning; before vs after; type of equipment

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20 ATP Results - Overall Mean of ATP Results RLU All Personnel All Surfaces

21 ATP Results All Surfaces Pass < Marginal Fail > Percentage Fail 5.8%

22 ATP Results All Surfaces Pass < Marginal Fail > Percentage Fail 30%

23 ATP Results All Personnel Pass < % Marginal % Fail > %

24 ATP Results All Personnel Pass > % Marginal % Fail > %

25 Case Example Immediate result to get cleaner PPE Day 1 Day 2 Steel Mesh Glove Hook (trimmers)

26 New Technologies - testing methods Traditional vs Novel Methods Cultural grow bugs and count them Other reactions e.g. Immunoassays Molecular e.g PCR & move to more multiplexes Proteins using MALDI-TOF Mass Spec Screening vs Confirmation Testing Reference Methods still mostly traditional

27 Non-cultural tests More instrument based & automated detection systems for pathogens Enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) used widely in food e.g. Listeria and Salmonella, was used for rapid E.coli O157 Range from simple lateral flow tests to fully automated (e.g. VIDAS by biomérieux and Tecra Unique Plus from 3M) PCR based tests amplification phase is automated, new systems coming which automate the prep stages e.g. Bax, GDS Further developments in robotics for repetitive manual tasks highly likely

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29 Novel Methods - PCR

30 How PCR Works Amplifies (replicates) specific fragments of bacterial DNA unique to the target organism DNA from the sample + polymerase, nucleotides and primers Apply heating and cooling cycles Heating denatures the DNA, separating it into single strands During cooling, primers find and stick to the target The polymerase and primers create a copy of the DNA fragment The process of copying (doubling each time) gives an exponential increase in the number of target DNA fragments So you get a detectable number of copies in rapid time rather than have to wait for days while the bugs grow If you know the DNA of your targets, you can build primers to copy it using PCR Combining multiple DNA targets into one assay = multiplexing

31 Issues with Rapid Methods Qualitative (D/ND) vs Quantitative (Number) E.g. Listeria present vs Listeria <100 cfu/g Sensitivity and Limits of Detection Cross reactivity

32 Very New - MALDI-TOF MS Looking for Microbes using the Chem Lab equipment Matrix-Assisted Laser Desorption Ionization (MALDI) Ion generation and desorption of analytes embedded in a solid matrix by using Laser irradiation Ions are all given the same energy hit and the time of flight (TOF) depends on the mass of the analyte. MALDI-TOF mass spectrometry is typically used to determine molecular mass of whole proteins (intact) and/or the mass of peptide/s in a digest Key point - if you have reference standards for the protein and peptides, you can apply this approach to numerous targets in one run = very quick Pathogenic microbes, Allergens, all have proteins so big potential Quick, very sensitive, no false positives a good confirmation method

33 Use of MALDI-TOF MS - NeoSEEK US based company, Neogen, platform to detect the presence and identity of STEC from a sample Uses MALDI-TOF Mass Spectrometry-based multiplexing at the back end. Platform has three steps: 1. PCR amplification 2. Primer extension to get DNA products of different masses; and 3. Chip-based mass spectrometry for analysis of the extension products. Has ~70 independent targets at present Gathers enough evidence to make an identification of O-group AND other virulence markers without the need for single colony isolation Aim is for a method that could be applied to both an isolated organism (colony), but also to detect and identify STECs in enrichment broth (i.e. with lots of other background in it. Claiming it works well further work to be done, but exciting.

34 Automation Time and cost savings but better for larger laboratories Reduces potential for human error Can give more reliable and accurate results controlled process Food labs slower adopters than human pathology labs Targets efficiency gains in the traditional laboratory process sample preparation e.g. gravimetric dilutor - balance linked to diluent dispensing pump, multiple diluents, liquid handling robots labelling e.g. bar codes and scanners media preparation and dispensing - automated plate pourers plating out of sample dilutions serial dilutions, spiral plater, TEMPO MPN plate reading and colony counting - e.g. new imaging technology, using colours and light sources to improve the images

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37 What is Driving New Technology in Labs? But at the same time, nobody wants errors... REALITY TRUE FALSE TEST RESULT TRUE FALSE CORRECT TYPE 2 ERROR FALSE NEGATIVE TYPE 1 ERROR FALSE POSITIVE CORRECT e.g. E.coli O157 & Big 6 STEC

38 What is Driving New Technology in Labs?... and.. everyone assumes they get the most ACCURATE & PRECISE numbers e.g. E.coli, Coliform, Staphyloccus, EB, LAB, Listeria counts

39 THANKS FOR LISTENING