Food Safety Magazine, February/March 2012

FOOD SAFETY INSIDERNovel Developments inATP BioluminescenceSeveral industrial applications of ATP bioluminescence have been developedsince the 1970s. These have largely been non-specific applicationsused primarily for the direct objective assessment of cleaningverification and as a gross monitor of microbial biomass. Over the past 5years, significant developments have been made, leading to the first specifictest for the detection of low numbers of pathogenic and indicator organismsin 7 hours.The most widely used application of ATP bioluminescence is for surfacehygiene monitoring. This test is very rapid, giving results in seconds andproviding a direct objective test for cleaning verification. However, becausethis type of test looks for ATP, a universal energymolecule that is abundant in most organic materials,the test cannot differentiate between ATP from differentsources. Recently, Hygiena has developed anovel reagent technology to make an ATP test that isboth specific and sensitive to microorganisms, whilereducing the total test time to 7 hours or less. Thenew test platform is called MicroSnap.New Bioluminogenic AssayMicroSnap uses a novel reagent formulation where some of the essentialcofactors of the standard luciferase reaction are immobilized onto aIncubationLowesttimeinoculum(37 °C/hours) detectedTable 1: Detection Limit of MicroSnapwww.hygiena.net1 500,0002 100,0003 10,0004 1,0005 1006 107 1-5specific substrate that can only be released inthe presence of specific enzymes. Light generationwill only occur if the substrate is utilized.The enzyme activity and copy numbersare greater than microbial numbers, thus providingan early amplified signal such that oneorganism can be detected in 7 hours or less(Table 1).The test is dependent only on the specificenzyme-substrate reaction so there is little interferencefrom the sample, which has historicallylimited the applications of ATPbioluminescence for the detection of microorganisms. This allows for awide range of products to be tested.A Quick and Easy-to-Use TestThe MicroSnap system consists of two easy-to-use components:• Sample collection and an enrichment device• MicroSnap end detection deviceThe first two MicroSnap tests that have been launched and are expected tohave AOAC-RI approval in the second quarter of this year are for coliformsand E. coli. Tests for Listeria monocytogenes and total viable count are expectedto be launched by the end of 2012.The sample collection and enrichment device is fitted with a swab fortesting. Alternatively, a liquid sample (1 ml) can be added directly to theswab tube. The liquid can be a suspension of solid sample prepared in thesame way and with the same materials as traditional microbiological methods.After adding or collecting the sample, the device is activated by breakingthe patented Snap-Valve and squeezing the bulb to mix the samplewith the enrichment broth. The whole deviceis then incubated for up to 7 hours at37 °C. At the desired time points, analiquot (0.1 ml) of enrichment culture istransferred to the device. The device is activatedby breaking the valve and is incubatedfor 10 minutes at 37 °C; and thenplaced in the Hygiena hand-held luminometer.Results are generated in 15 secondsand are displayed in relative lightunits (RLUs). Pass/fail levels can be set for"go" or "no go" decisions or enumerationcan be done.The MicroSnap has been independentlyverified by a leading food safety andquality laboratory and shown to have asensitivity of 89 percent and a specificity of99 percent, which is better than alternativeculture method (Table 2).Testing methods for E. coli detectionDry media Dry media MicroSnapformat 1 format 2 E. coliSensitivity (%) 40 33 89Specificity (%) 91 79 100Table 2: Specificity and Sensitivity of MicroSnapFor low numbers of organisms (1-5),the detection time was confirmed as 7hours (Table 1) in both pure culture andinoculated foodstuffs, even where the targetorganism was outnumbered morethan 10,000:1 by competing natural microflora.The traditional method detected95 percent of samples, whereas MicroSnapdetected 99 percent of samples. Thespecification for many industrial applicationsfor coliforms and E .coli is lessthan10 per gram, which is effectively apresence/absence test for a 1 in 10 dilutionof a solid sample (