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Microbiology

HybriScan®


- Introduction
- Benefits over conventional detection methods and PCR
- Table with advantages over other detection techniques
- Principles of the HybriScan method
- Sensitivity, specificity, flexibility and applicability
- Example: HybriScan Listeria monocytogenes
- Product list of HybriScan Kits
- References
- HybriScan Waste Water Calculator




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

 

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 (see Figure 1).

Work flow diagram of HybriScan Rapid Test System test method

Figure 1: Work flow of HybriScan test method


 



Benefits over conventional detection methods and PCR

 


HybriScan has significant time- and labor-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 [1], and lack quantification accuracy due to biases associated with PCR and reverse transcription reactions (a general accepted error connected to these methods). In contrast, the HybriScan method is nearly independent of the influences of sample matrix and detects only living cells. It also permits the detection of non-culturable microbes. Table 1 compares the benefits and disadvantages of the various methods.

 

Advantages of HybriScan over other detection techniques

Detection technology Advantage Disadvantage
HybriScan
  • detects only living 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 of 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 (high extraction effort)
  • 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 cultivation-based methods
  • relatively inexpensive
  • simple
  • specific
  • widely accepted method
  • time-consuming (up to 10 days)
  • no detection of non-culturable microbes
  • low sample throughput
  • laborious


 




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 2). Sandwich hybridization assays from crude cell samples or in connection to PCR have been extensively used in clinical diagnostics for detection of nucleic acids from bacteria [3, 9, 10] and viruses [11]. Specificity is achieved by targeting conserved or unique rRNA sequences. A labeled capture probe is used to immobilize the target sequence on a solid support plate (coated microtiter plate). A labeled detection probe provides an enzyme-linked optical signal read out. Detection results from application of antibody labeled enzyme. The bound complex is visualized by chromogenic substrates. Photometric data are measured at 450 nm and compared with standard solutions. The HybriScan software enables easy measurement and data analysis.

Image showing detection of rRNA via HybriScan sandwich hybridization assay.

Figure 2:
Principle of the HybriScan sandwich hybridization assay.



Sensitivity, specificity, flexibility and applicability of HybriScan technology

 


Sandwich hybridization is very sensitive, detecting attomoles of the respective target rRNA molecules. [2] 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, non-alcoholic 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. [3, 4, 5]




HybriScan Listeria monocytogenes: Rapid and innovative test system

 

One of the most important foodborne pathogens is Listeria monocytogenes (Figure 3), which poses a health threat in foods that have long, refrigerated shelf lives. [6] 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. [7] L. monocytogenes proliferates at refrigeration temperatures and is able to grow over a wide pH range from 4.39 to 9.40. These are important characteristics particularly with regard to food safety [12].

Image of Listeria monocytogenes colonies grown on PALCAM agar

Figure 3: Listeria monocytogenes colonies grown on PALCAM agar. HybriScan 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. [8] 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 4 shows the validation results of HybriScan Listeria monocytogenes. Food samples were analyzed 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%. 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.


Graph showing validation of Hybridscan Listeria monocytogenes vs. cultured base methods.

Figure 4: Validation of HybriScan Listeria monocytogenes. 355 food samples were analyzed and compared to culture-based method according to § 64 LFGB. The blue values are the number of L. monocytogenes positive tested food samples in each category. Validation was according to ISO 16140:2003 (ASU L00.00-22).



Product list of HybriScan Kits

 


Cat. no. Description old
Cat. No.
old Name Reactions Add to Cart
Detection Kits
62533 HybriScanD Beer FS G 01 FastScan Beer 96
56917 HybriScanD Campylobacter - - - - 96
68301 HybriScanD Drinks FS G 02 FastScan Drinks 96
96343 HybriScanD E. coli FS F 05 FastScan E. coli 96
12838 HybriScanD Enterobacter sakazakii - - - - 96
59744 HybriScanD Lactobac FS G 04 FastScan Lactobac 96
16593 HybriScanD Legionella FS W 01 FastScan Legionella 96
07190 HybriScanD Legionella pneumophila FS W 02 FastScan Legionella pneumophila 96
55661 HybriScanD Listeria FS F 03 FastScan Listeria 96
49699 HybriScanD Listeria monocytogenes FS F 04 FastScan Listeria monocytogenes 96
55662 HybriScanD Salmonella FS F 01 FastScan Salmonella 96
02349 HybriScanD Total Bacterial Count FS B 01 FastScan Total Bacterial Count 96
04447 HybriScanD Waste Water Microthrix parvicella FS W 04 FastScan Waste Water Microthrix parvicella 96
78436 HybriScanD Waste Water Total Bacterial Count FS W 03 FastScan Waste Water Total Bacterial Count 96
61397 HybriScanD Yeast FS G 03 FastScan Yeast 96
Identification Kits
79742 HybriScanI Brettanomyces FS I 09 FastScan Brettanomyces 48
19503 HybriScanI Candida albicans FS I 08 FastScan Candida albicans 48
76545 HybriScanI E. coli FS I 04 FastScan E. coli 48
75724 HybriScanI Lactobacillus brevis FS I 10 FastScan Lactobacillus brevis 48
80065 HybriScanI Lactobacillus buchneri FS I 12 FastScan Lactobacillus buchneri 48
86827 HybriScanI Lactobacillus lindneri FS I 11 FastScan Lactobacillus lindneri 48
49417 HybriScanI Legionella pneumophila FS I 06 FastScan Legionella pneumophila 48
77007 HybriScanI Leuconostoc FS I 13 FastScan Leuconostoc 48
49712 HybriScanI Listeria monocytogenes FS I 01 FastScan Listeria monocytogenes 48
42875 HybriScanI Megasphaera FS I 17 FastScan Megasphaera 48
33018 HybriScanI Pectinatus cerevisiiphilus FS I 16 FastScan Pectinatus cerevisiiphilus 48
73582 HybriScanI Pectinatus frisingensis FS I 15 FastScan Pectinatus frisingensis 48
67289 HybriScanI Pediococcus damnosus FS I 14 FastScan Pediococcus damnosus 48

 


Table 2:
HybriScan products (HybriScanD = detection kit; HybriScanI = identification kit)

 




References

 

  1. Bustin, S.A. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 2000, 25, 169-193.
  2. 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.
  3. 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.
  4. 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.
  5. Rautio, J.; Barken, KB.; Lahdenpera, J.; Breitenstein, A.; Molin, S.; Neubauer, P. Sandwich hybridisation assay for quantitative detection of yeast RNAs in crude cell lysates. Microb. Cell Fact. 2003, 2(1), 4-12.
  6. Mellefont, L.A.; McMeekin, T.A.; Ross, T. Effect of relative inoculum concentration on Listeria monocytogenes growth in co-culture. Int. J. Food Microbiol. 2008, 121, 157-168.
  7. McLauchlin, J. The relationship between Listeria and listeriosis. Food Control 1996, 7, 187-193.
  8. Donnelly, C.W. Detection and isolation of Listeria monocytogenes from food samples: implications of sublethal injury, J. AOAC Int. 2002, 85, 495-500.
  9. Casademont I, Bizet C, Chevrier D and Guesdon JL Rapid detection of Campylobacter fetus by polymerase chain reaction combined with non-radioactive hybridization using an oligonucleotide covalently bound to microwells. Mol Cell Probes 2000, 14, 233-240
  10. Chevrier D, Popoff MY, Dion MP, Hermant D and Guesdon JL Rapid detection of Salmonella subspecies I by PCR combined with non-radioactive hybridisation using covalently immobilised oligonucleotide on a microplate. FEMS Immunol Med Microbiol. 1995, 10(3-4), 245–501.
  11. Albretsen C, Kalland KH, Haukanes BI, Havarstein LS and Kleppe K Applications of magnetic beads with covalently attached oligonucleotides in hybridization: isolation and detection of specific measles virus mRNA from a crude cell lysate. Anal. Biochem. 1990, 189, 40-50
  12. Uyttendaele, M., De Troy, P., Debevere, J. Incidence of Salmonella, Campylobacter jejuni, Campylobacter coli, and Listeria monocytogenes in poultry carcasses and different types of poultry products for sale on the Belgian retail market. Journal of Food Protection 1999, 62, 735–740.

 




HybriScan Waste Water Calculator

 

For the Waste Water Kits (04447 HybriScanD Waste Water Microthrix parvicella, 78436 HybriScanD Waste Water Total Bacterial Count ) we provide an excel sheet for calculation of total bacterial count, Microthrix count and ratio of Microthrix : Total Bacterial Count.

For download the file please click on the links below.


Hybriscan Waste Water Calculator English

Excel Sheet 28KB

Waste Water Calculator English
Hybriscan Waste Water Calculator Deutsch

Excel Sheet 56KB

Waste Water Calculator Deutsch