HybriScan™ Rapid Test Systems: Rapid Detection, Identification and Quantification of Microorganisms in Beverages, Food and Water

By: Jvo Siegrist, AnalytiX Volume 8 Article 3

Rapid detection, identification and quantification of microorganisms in beverages, food and water

Jvo Siegrist, Product Manager Microbiology ivo.siegrist@sial.com

The new HybriScan Test System, which uses sandwich hybridization, provides fast, sensitive and reliable detection, identification and quantification of spoilage and pathogenic microorganisms in beverages, food and water. It is ideal for the comprehensive and reliable routine control of raw materials and concentrates in all production steps up to the quality check of finished goods. HybriScan is a simple, time-saving assay that can be performed with standard laboratory equipment.

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Benefits over conventional detection methods and PCR

HybriScan has significant time- and labour-saving benefits over traditional methods. It also has benefits over PCR and real-time PCR, which, although highly sensitive, are susceptible to experimental interferences, like template inhibition from insufficient purification, and lack quantification accuracy due to biases associated with PCR and reverse transcription reactions. In contrast, the HybriScan method is nearly independent of the influences of sample matrix and is able to distinguish between live and dead cells. It also permits the detection of non-culturable microbes. Table 1 compares the benefits and disadvantages of the various methods.

Table 1 Advantages of HybriScan over other detection techniques

Detection technology Advantage Disadvantage
HybriScan
  • differentiates live/dead cells
  • minimal interference by sample matrix
  • high specificity
  • low cross-reactivity
  • easy handling
  • cost-efficient read-out devices
  • quantitative and qualitative
  • high sample throughput (96-microwell plates)
  • detects non-culturable microbes
  • no differentiation of serotypes or subspecies
  • limited probe design (rRNA target)
PCR
  • high sample throughput
  • sensitive
  • quantitative
  • no live/dead-cell differentiation
  • sensitive to matrix interference
  • susceptible to polymerase inhibition
ELISA
  • differentiation of serotypes or subspecies
  • high sample throughput (96-microwell plates)
  • quantitative and qualitative
  • low sensitivity
  • low specificity, higher cross-reactivity
  • slow and expensive assay development
Conventional cultivationbased methods
  • relatively inexpensive
  • simple
  • specific
  • widely accepted method
  • time-consuming (up to 10 days)
  • no detection of nonculturable microbes 
  • low sample throughput
  • laborious

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Principles of the HybriScan method

The HybriScan method is based on the detection of rRNA via hybridization events and specific capture and detection probes (Figure 1). Specificity is achieved by targeting conserved or unique rRNA sequences. A labelled capture probe is used to immobilise the target sequence on a solid support plate (coated microtiter plate). A labelled detection probe provides an enzyme-linked optical signal read out. Detection results from application of antibody labelled enzyme. The bound complex is visualised by chromogenic substrate. Photometric data are measured at 450 nm and compared with standard solutions. The HybriScan software enables easy measurement and data analysis.


Figure 1. Principle of the HybriScan sandwich hybridization assay.

Figure 1. Principle of the HybriScan sandwich hybridization assay.

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Sensitivity, specificity, flexibility and applicability of HybriScan technology

Sandwich hybridization is very sensitive, detecting attomoles of the respective target rRNA molecules.[1] 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.

By using specific probes, HybriScan allows flexible group- and species-specific detection. It is applicable to many analytical fields, including monitoring the microbial content of beer, wine, nonalcoholic beverages, drinking water, a wide variety of foods and wastewater. HybriScan rapidly and accurately identifies, detects and quantifies many important pathogenic species, including Salmonella, Campylobacter, Listeria and Legionella including the most relevant species L. pneumophila.[2,3]

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HybriScan Listeria monocytogenes: Rapid and innovative test system

One of the most important foodborne pathogens is Listeria monocytogenes (Figure 2), which poses a health threat in foods that have long, refrigerated shelf lives. Listeriosis, caused by ingestion of foods contaminated with Listeria monocytogenes, has increased dramatically in recent years, causing a great deal of distress and even death. Milk, cheese, ice cream and meat contaminated with this pathogen have led to recent outbreaks of listeriosis. L. monocytogenes proliferates at refrigeration temperatures and is able to grow over a wide pH range from 4.39 to 9.40.

 

Figure 2 HybriScan Listeria mono Confirmatory Agar Fluka 92302 In front Listeria moncytogenes.

Figure 2 HybriScan Listeria mono Confirmatory Agar 92302 In front Listeria moncytogenes. Listeria monocytogenes permits rapid identification of suspect colonies within one hour.

Conventional culture-based methods to detect L. monocytogenes generally involve selective enrichment followed by culturing on selective medium, isolation and biochemical identification. This laborious and time-consuming approach often takes several days to show results. Also, compared to molecular biological and immunological methods, culture-based methods often give false negatives.

HybriScan Listeria monocytogenes is an excellent alternative to lengthy culture-based methods. It is as reliable and comprehensive as classical methods, but permits rapid detection and quantification with results available within 48 hours. The species-specific probe permits direct detection of L. monocytogenes, thereby eliminating false positives caused by other Listeria species. Even more compelling, suspected single colonies can be identified within one hour using the HybriScanI 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 the culture-based method according to § 64 LFGB. Five different food categories were tested. Results of validation showed a relative accuracy of 99.2%, relative specificity of 98.5% and relative sensitivity of 99.6%.

 

Figure 3 Validation of HybriScan Listeria monocytogenes.

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 are the number of L. monocytogenes positive food samples in each category. Validation was according to ISO 16140:2003 (ASU L00.00-22).

Two versions are available. HybriScanI Listeria monoytogenes is used for the extremely rapid, sensitive and economical identification of suspect colonies of L. monocytogenes. HybriScanD Listeria monocytogenes is used for the detection, identification and quantification of L. monocytogenes in different food matrixes.

HybriScan kits are the result of a joint project between Sigma- Aldrich and Scanbec GmbH. For more details please visit us at www.sigma-aldrich.com/hybriscan

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Materials

     

References

  1. Tenhunen, J.; Eloranta, J.; Kallio, A.; Soderlund, H. A solution hybridization method for quantification of mRNAs: determining the amount and stability of oncogene mRNA. Genet. Anal. Tech. Appl. 1990, 7, 228–233.
  2. Huhtamella, S.; Leinonen, M.; Nieminen, T.; Fahnert, B.; Myllykoski, L.; Breitenstein, A.; Neubauer, P. RNA-based sandwich hybridisation method for detection of lactic acid bacteria in brewery samples. J. Microbiol. Methods 2007, 68(3), 543–53.
  3. Leskela, T.; Tilsala-Timisjarvi, A.; Kusnetsov, J.; Neubauer, P.; Breitenstein, A. Sensitive genus-specific detection of Legionella by a 16S rRNA based sandwich hybridization assay. J. Microbiol. Methods 2005, 62(2), 167–79.

 

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