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Laboratory Training - RTO Technical Services

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www.symbioalliance.com.au

18 September 2014

MINTRAC QA & MI CONFERENCE 2014

New technology in laboratories on and off plant

What is Driving New Technology in Labs?

Everyone wants this..... <GRAPHIC DEPICTING “FAST/SPEED”>

What is Driving New Technology in Labs?

Everyone also wants costs to go down ...

TEST

ING

CO

STS

TIME

TESTING COSTS

What is Driving New Technology in Labs?

But at the same time, nobody wants errors..... REALITY

TEST

RES

ULT

TRUE FALSE TR

UE CORRECT TYPE 1 ERROR

FALSE POSITIVE FA

LSE TYPE 2 ERROR

FALSE NEGATIVE CORRECT

e.g. E.coli O157 & Big 6 STEC

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

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)

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)

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)

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.

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

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

ATP Results - Overall

Mean of ATP Results

0

2000

4000

6000

8000

10000

12000

14000

All Personnel All Surfaces

RLU

ATP Results – All Surfaces

Pass <1000 100

Marginal 1000-3000 13

Fail >3000 7

Percentage Fail 5.8%

ATP Results – All Surfaces

Pass <100 50

Marginal 100-300 34

Fail >300 36

Percentage Fail 30%

ATP Results – All Personnel

Pass <1000 24 29%

Marginal 1000-3000 29 35%

Fail >3000 31 37%

ATP Results – All Personnel

Pass >3000 53 63%

Marginal 3000-5000 11 13%

Fail >5000 20 24%

Case Example – Immediate result to get cleaner PPE

Day 1 Day 2

Steel 872 133

Mesh Glove 43914 275

Hook (trimmers) 161265 41802

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

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

Non-traditional methods

Novel Methods - PCR

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

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

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

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.

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

What is Driving New Technology in Labs?

But at the same time, nobody wants errors..... REALITY

TEST

RES

ULT

TRUE FALSE TR

UE CORRECT TYPE 1 ERROR

FALSE POSITIVE FA

LSE TYPE 2 ERROR

FALSE NEGATIVE CORRECT

e.g. E.coli O157 & Big 6 STEC

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

THANKS FOR LISTENING