ATP bioluminescence and Lumac System

Dr. Denoj Sebastian Department of Life Sciences University of Calicut

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ATP bioluminescent assay Background

Food safety and quality management systems Proactive and preventative quality management Rely on end product testing and visual inspection Hazard Analysis Critical Control Point (HACCP) rapid methods for monitoring and verifying performance Monitoring usually relies on surveillance of physical or chemical parameters of a process such as time and temperature of heating or pH Index
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ATP bioluminescent assay Background

Validation of HACCP performance requires testing for the absence of specific pathogens
Salmonella Escherichia coli O157:H7 Campylobacter jejuni Listeria monocytogenes others

controversy over microbiological tests to monitor critical control points

length of time needed to generate results sampling strategy

ATP bioluminescence Assay - comes close to offering real-time results

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Principles of ATP bioluminescent assay

ATP bioluminescence technique for cell detection was first described in the 1960s by NASA scientists Makes use of the fact that all living cells contain ATP However, after cell death, the ATP content decreases sharply Intracellular ATP concentration serve as a measure of biomass and cell viability.

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Principles of ATP bioluminescent assay

An enzyme substrate complex, luciferase-luciferin, present in firefly tails converts the chemical energy associated with ATP into light by a stoichiometric reaction The amount of light emitted is proportional to the concentration of ATP present and can be quantified using light detecting devices. Because the quantum yield of this bioluminescent reaction is 0.88 almost 1 photon is emitted per reaction cycle. As instruments are available that can detect even single photons, the theoretical sensitivity of the bioluminescent ATP assay is extremely high (1 amol).

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Principles of ATP bioluminescent assay

The level of ATP in a cell remains relatively constant; thus, the light produced during the luciferase-luciferin catalyzed reaction is directly related to the number of metabolically active cells present in the assay. Assuming that the ATP content of an average bacterial cell is about 110 amol, even single cells should be detectable.

Sharpe et al. (1970) were the first to apply the method to the detection of microorganisms in food but the high level of ATP from nonmicrobial sources reduced the sensitivity substantially using this method

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Principles of ATP bioluminescent assay

In an attempt to improve the sensitivity of ATP bioluminescence, Squirrell and Murphy (1995) proposed the use of the enzyme adenylate kinase (AK) as a cell marker. AK is a key intracellular enzyme found in both prokaryotic and eukaryotic cells that equilibrates AMP, ADP and ATP by the reaction:

MgATP + AMP MgADP + ADP

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Principles of ATP bioluminescent assay

MgATP + AMP MgADP + ADP

When excess ADP is used as the substrate, ATP will be produced in an amount proportional to the concentration of AK present. This ATP can be assayed using the luciferin-luciferase system. From the turnover number and the cellular ratio of AK to ATP it was predicted that, with 1 minute incubation, 4050 times more ATP would be available for bioluminescent detection than in assays for ATP alone. This would correspond to a limit of detection between 20 and 25 bacterial cells. But This theoretical limit never achieved.
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Applications
Assay for testing the total bacterial count of food products The use of assays for particular foods
Raw milk Drinking water, brewing, beverage, fruit and fruit juice Poultry Meat

The use of assays for hygiene monitoring The use of assays to detect particular pathogens
Antibody-based bioluminescent methods for detection of bacterial pathogens Phage-based bioluminescent assays Phage-based biosorbents Bacterial luciferase (lux genes) incorporation

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Bacteriophage-based bioluminescent method for bacteria detection

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ATP bioluminescent reagent/instrument systems available for use as hygiene monitors and microbial contamination control in food industry
Type of system Bioluminometers and self-contained swab kits Manufacturer Bio trace Inc. Trade name and system description Clean-Trace sruface hygiene test and Aqua_Trace - water hygiene test with luminometers Uni-Lite or Uni-Lite XCEL Celsis-Lumac System SURE with Advance luminometer Pocket Swab surface hygiene kit with luminometers LUM-T or Firefly IDEXX Lightning MicroStar System for detection of microbial contamination in filterable samples ENLITEN* total ATP Rapid Biocontamination Detection Kit

Celsis Charm Sciences Biocontrol Systems Inc. CCD camera , filtering Millipore system&bioluminesce Corporation nt reagent kit Swab-based system Promega forhygiene monitoring Corporation

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Celsis Lumac

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Celsis Lumac System

Detecting the light emitted when ATP comes into contact with the bioluminescence reagent (luciferin-luciferase). Added a moment before cell lysis by an ATP releasing agent (or extractant) Bioluminescence measured using a luminometer

The luciferin is produced by chemical means. The luciferase is usually extracted from the tails of fireflies Celsis has produced a recombinant form of luciferase from bacterial fermentation

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Celsis Lumac Products

Lurainoraeters:
Celsis luminometers: Celsis Optocorap I and Celsis Optocorap II, Celsis Advance. The Lumac range of lumino meters, from manual and portable instruments to fully automated system.

ATP bioluminescence-based reagent systems kits

-Celsis kits; UHT milk and dessert sterility kits Fruit juice sterility kit Beer sterility kit Hygiene monitoring kits Assimilable organic carbon measurement kit to test the potential of water to support bacterial growth, 2 kits for resarch purposes: High sensitivity ATP bioluminescence kit, Low decay rate ATP bioluminescence kit

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Celsis Lumac Products

Lumac kits intended for use with Lumac luminometers:
Determination of microbial ATP in foods and beverages: Beer microbial kits Fruit juice test kit Dairy products sterility test kit Raw milk microbial kit Meat microbial kit

Determination of microbial and non-microbial ATP in water and aqueous solutions:

Aqueous solutions microbial kit Rinse water test kit Kit for determining microbial contamination in industrial samples. Kits for monitoring cleanliness of contact surface.

The Lumac/Kiwa Biofilm Monitor instrument

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Celsis Lumac Products

Handling and Filtration Systems for Testing Aqueous samples
The Celsis handling and filtration digital systems: MicroCount Digital method of quantitative microbial determination Celsis Colicount for the detection and enumeration of Coliforms The 2-place Lumac filtration system.

Antibiotic and Salmonella Test Kits Not Based on ATP Bioluminescence

Colorimetric test kit 200 -screening raw milk for antibiotic residues. Path-Stik kit -Salmonella using immunochromatography.

Tests for Personal Care Products and Consumer Products (NDITB)

Celsis Personal Care Products kit -for measuring microbial contamination of products such as shampoos, hair conditioners, creams, color cosmetics, cleansers and toothpastes Tests for control of consumer products: clinical nutrition kit, cough syrups and oral medicines

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