HybriScan® Rapid Test SystemRapid, qualitative and quantitative detection of micro- organisms by means of nucleic acid (rRNA) based methods in food, beverages and water.

• Time save of 5 to 7 days• High flexibility• Easy handling• Analysis in 2,5 hours• Only living cells are detected

(rRNA is fast decomposed in a dead cell)

Sensitive (up to 1 CFU/ml with enrichment step)High specificity of the probes (low cross reaction)Robust system, not sensitive sample matrixCost efficient analyses (96-well microplate format)

Principles of the HybriScan method

The HybriScan method is based on the detection of rRNA via hybridization events and specific capture and detection probes. Sandwich hybridization is very sensitive, detecting attomoles of the respective target rRNA molecules. The ideal hybridization target for bacteria and yeast is rRNA. These cells contain a large number of rRNA-containing ribosomes; a single cell therefore contains several thousand copies of rRNA but only one DNA. Sandwich hybridization also provides sensitivity in crude biological samples because it is not susceptible to matrix interference.

substrate product

3. - labeling with enzyme - washing - detection

CCCCCCCCCCCC

DDDDDDDDDDDD

binding molecule

labeled cature probe

labeled detection probe

enzym with binding molecule

target rRNA

hybridisation

1.

2.

microtiter plate

capture step

Specificity is achieved by targeting conserved or unique rRNA sequences. A biotin-labeled capture probe is used to immobilize the target sequence on a solid support plate (streptavidin-coated microtiter plate). A digoxigenin-labeled detection probe provides an enzyme-linked optical signal read out. Detection results from application of anti-DIG-horseradish peroxidase Fab fragments. The bound complex is visualized by horseradish peroxidase substrate TMB (3,3’,5,5’-tetramethylbenzidine). Photometric data are measured at 450 nm and compared with standard solutions. The HybriScan software enables easy measurement and data analysis.

How it works

More Information about HybriScan®E-mail: Eurtechserv@sial.comInternet: www.sigma-aldrich.com/hybriscan

Detection Kits for:

Beer spoiling organismsCampylobacterCronobacter spp.Beverage spoiling organismsE. coliLactobacilliLegionellaLegionella pneumophilaListeriaListeria monocytogenesSalmonellaTotal Bacterial CountWaste Water Microthrix parvicellaWaste Water Total Bacteria CountYeasts

analysis time: approx. 2 – 2.5 hours

Identification Kits for:

BrettanomycesCandida albicansE. coliLactobacillus brevisLactobacillus buchneriLactobacillus lindneriLegionella pneumophilaLeuconostocListeria monocytogenesMegasphaeraPectinatus cerevisiiphilusPectinatus frisingensisPediococcus damnosus

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HybriScan® �Rapid Test Systemsfor the Detection and Identification of Microorganisms

Rapid, sensitiveand reliable,without PCR

Save up to 10 daysin comparison tocultivation-basedassays

Live/dead-discrimination

Easy handlingwith standard labequipment, like96-wellplate reader

Quantifiable bystandards

The technologyThe HybriScan® technology is based on the detection of target molecules,specific for the microorganism of interest, with capture and detection probes bymeans of a so-called sandwich hybridisation. The signal read-out is triggeredoptically by an enzymatically generated colour change.

Easy sample preparation:

Our Innovation, Your Research – Shaping the Future of Life Science

17

HybriScan® Rapid Test SystemsRapid detection, identification and quantification of microorganisms

The new HybriScan® Test System, which uses sandwich hybridisation, providesfast, sensitive and reliable detection, identification and quantification of spoil-age and pathogenic microorganisms. It is ideal for the comprehensive and reli-able analysis of microorganism and pathogens. HybriScan® is a simple, time-saving assay that can be performed with standard laboratory equipment.

Benefits over conventional detection methods and PCRHybriScan® has significant time- and labour-saving benefits over traditionalmethods. It also has benefits over PCR and real-time PCR, which, althoughhighly sensitive, are susceptible to experimental interferences, like templateinhibition from insufficient purification [1], and lack quantification accuracy dueto biases associated with PCR and reverse transcription reactions (a generalaccepted error connected to these methods). In contrast, the HybriScan®

method is nearly independent of the influences of sample matrix and is able todistinguish between live and dead cells. It also permits the detection of non-culturable microbes. Table 1 compares the benefits and disadvantages of thevarious methods.

Principles of the HybriScan® methodThe HybriScan® method is based on the detection of rRNA via hybridisationevents and specific capture and detection probes (Figure 1). Sandwich hybridi-sation assays from crude cell samples or in connection to PCR have been exten-sively used in clinical diagnostics for detection of nucleic acids from bacteria [3,9, 10] and viruses [11]. Specificity is achieved by targeting conserved or uniquerRNA sequences. A labelled capture probe is used to immobilise the targetsequence on a solid support plate (coated microtiter plate). A labelled detectionprobe provides an enzyme-linked optical signal read-out. Detection resultsfrom application of antibody labelled enzyme. The bound complex is visualisedby chromogenic substrate. Photometric data are measured at 450 nm andcompared with standard solutions. The HybriScan® software enables easy mea-surement and data analysis.

By using specific probes, HybriScan® allows flexiblegroup- and species-specific detection. It is applicable tomany analytical fields, including monitoring the micro-bial content of beer, wine, non-alcoholic beverages,drinking water, a wide variety of foods and wastewater.HybriScan® rapidly and accurately identifies, detectsand quantifies many important pathogenic species,including Salmonella, Campylobacter, Listeria andLegionella including the most relevant species L. pneu-mophila. [3, 4, 5]

HybriScan® Listeria monocytogenes: Rapidand innovative test systemOne of the most important foodborne pathogens isListeria monocytogenes (Figure 2), which poses ahealth threat in foods that have long, refrigerated shelflives. [6] Listeriosis, caused by ingestion of foods con-taminated with Listeria monocytogenes, has increaseddramatically in recent years, causing a great deal of dis-tress and even death. Milk, cheese, ice cream and meatcontaminated with this pathogen have led to recentoutbreaks of listeriosis. [7] L. monocytogenes prolifer-ates at refrigeration temperatures and is able to growover a wide pH range from 4.39 to 9.40. These areimportant characteristics particularly with regard tofood safety.

Figure 1: Principle of the HybriScan® sandwich hybridisation assay.

Figure 2: Listeria mono Confirmatory Agar (Fluka 92302; infront Listeria moncytogenes)

2

1

Sensitivity, specificity, flexibility and applicability ofHybriScan® technologySandwich hybridisation is very sensitive, detecting attomoles of the respectivetarget rRNA molecules. [2] The ideal hybridisation target for bacteria and yeastis rRNA. These cells contain a large number of rRNA-containing ribosomes; asingle cell therefore contains several thousand copies of rRNA but only oneDNA. Sandwich hybridisation also provides sensitivity in crude biological sam-ples because it is not susceptible to matrix interference.

Our Innovation, Your Research – Shaping the Future of Life Science

ReferencesBustin, S.A. Absolute quantification of mRNA using real-1]time reverse transcription polymerase chain reactionassays. J. Mol. Endocrinol. (2000), 25, 169–93.

Tenhunen, J., Eloranta, J., Kallio, A., Soderlund, H. A2]solution hybridisation method for quantification ofmRNAs: determining the amount and stability of oncogenemRNA. Genet. Anal. Tech. Appl. (1990), 7, 228–233.

Huhtamella, S., Leinonen, M., Nieminen, T., Fahnert, B.,3]Myllykoski, L., Breitenstein, A., Neubauer, P. RNA-basedsandwich hybridisation method for detection of lactic acidbacteria in brewery samples. J. Microbiol. Methods (2007),68(3), 543–53.

Leskela, T., Tilsala-Timisjarvi, A., Kusnetsov, J., Neubauer,4]P., Breitenstein, A. Sensitive genus-specific detection ofLegionella by a 16S rRNA based sandwich hybridisationassay. J. Microbiol. Methods (2005), 62(2), 167–79.

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Figure 3: Validation of HybriScan® Listeria monocytogenes.355 food samples were analysed and compared to culture-based method according to § 64 LFGB. The blue values arethe number of L. monocytogenes positive-tested analysedfood samples in each category. Validation was according toISO 16140:2003 (ASU L00.00-22).

Table 1: Advantages of HybriScan® over other detection techniques.

Table 2: HybriScan® products (HybriScan® D = detection kit;HybriScan® I = identification kit)

Detectiontechnology

Advantage Disadvantage

HybriScan® differentiates live/dead cells,•minimal interference bysample matrix, high specificitylow cross-reactivity•easy handling•cost-efficient read-out devices•quantitative and qualitative•high sample throughput•(96-microwell plates)detects of non-culturable•microbes

no differentiation of serotypes or•subspecieslimited probe design (rRNA target)•

PCR high sample throughput•sensitive•quantitative•

no live/dead cell differentiation•sensitive to matrix interference•(high extraction effort)susceptible to polymerase•inhibition

ELISA differentiation of serotypes or•subspecieshigh sample throughput•(96-microwell plates)quantitative and qualitative•

low sensitivity•low specificity, higher cross-•reactivityslow and expensive assay•development

Conventionalcultivation-basedmethods

relatively inexpensive•simple•specific•widely accepted method•

time consuming (up to 10 days)•no detection of non-culturable•microbeslow sample throughput•laborious•

Conventional culture-based methods to detect L. monocytogenes generallyinvolve selective enrichment followed by culturing on selective medium, isola-tion and biochemical identification. [8] This laborious and time-consumingapproach often takes several days to show results. Also, compared to molecularbiological and immunological methods, culture-based methods often give falsenegatives.

HybriScan® Listeria monocytogenes is an excellent alternative to lengthy cul-ture-based methods. It is as reliable and comprehensive as classical methods,but permits rapid detection and quantification with results available within 48hours. The species-specific probe permits direct detection of L. monocytogenes,thereby eliminating false positives caused by other Listeria species. Even morecompelling, suspected single colonies can be identified within one hour, usingthe HybriScan® I identification kit without need for further cultivation.

Figure 3 shows the validation results of HybriScan® Listeria monocytogenes.Food samples were analysed with the HybriScan® method and compared to theculture-based method according to § 64 LFGB. Five different food categorieswere tested. Results of validation showed a relative accuracy of 99.2%, relativespecificity of 98.5% and relative sensitivity of 99.6%.

Two versions are available. HybriScan® I Listeria monoy-togenes is used for the extremely rapid, sensitive andeconomical identification of suspect colonies of L.monocytogenes. HybriScan® D Listeria monocytogenesis used for the detection, identification and quantifica-tion of L. monocytogenes in different food matrixes.

HybriScan® kits are the result of a joint project betweenSigma-Aldrich and Scanbec GmbH. For more detailsplease visit us at www.sigma-aldrich.com/hybriscan

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Prod. no. Description Reactions

96343 HybriScan® D E. coli 96

59744 HybriScan® D Lactobac 96

16593 HybriScan® D Legionella 96

07190 HybriScan® D Legionellapneumophila

96

55661 HybriScan® D Listeria 96

49699 HybriScan® D Listeriamonocytogenes

96

55662 HybriScan® D Salmonella 96

02349 HybriScan® D Total BacterialCount

96

61397 HybriScan® D Yeast 96

19503 HybriScan® I Candida albicans 48

76545 HybriScan® I E. coli 48

49417 HybriScan® I Legionellapneumophila

48

49712 HybriScan® I Listeriamonocytogenes

48

89384 HybriScan® I Pectinatuscerevisiiphilus

48

73582 HybriScan® I Pectinatusfrisingensis

48

67289 HybriScan® I Pediococcusdamnosus

48

44492 HybriScan® Software

Rautio, J., Barken, KB., Lahdenpera, J., Breitenstein, A.; Molin, S., Neubauer, P. Sandwich5]hybridisation assay for quantitative detection of yeast RNAs in crude cell lysates. Microb.Cell Fact. (2003), 2(1), 4–12.

Mellefont, L.A., McMeekin, T.A., Ross, T. Effect of relative inoculum concentration6]on Listeria monocytogenes growth in co-culture. Int. J. Food Microbiol. (2008), 121,157–68.

McLauchlin, J. The relationship between Listeria and listeriosis. Food Control (1996),7]7, 187–93.

Tasara, T. et al. Incorporation of Reporter Molecule-labeled Nucleotides by8]DNA Polymerases. I. Chemical Synthesis of Various Reporter Group-labeled2’-deoxyribonucleoside-5’-triphosphates. Nucleic Acid Research, Vol. 31, No. 10(2003), 2630–5.

Giller, G. et al. Incorporation of Reporter Molecule-labeled Nucleotides by DNA9]polymerases. II. High-density labelling of natural DNA. Nucleic Acids Research, Vol.31, No. 31 (2003), 2636–46.

Buschmann, V. et al. Spectroscopic Study and Evaluation of Red-Absorbing10]Fluorescent Dyes. Bioconjugate Chem. 14 (2003), 195–204.

Hinze, K., et al. Triple-Colour Coincidence Analysis: One Step Further in Following Higher11]Order Molecular Complex Formation. Biophysical Journal, Vol. 86 (2004), 506–16.