HybriScan^® Rapid Microbial Test System

Rapid Microbiological Method: Sandwich Hybridization

The method simply needs a centrifuge, a thermal mixer and an optional a microplate reader. The complete analysis can be achieved in 2 hours for the HybriScan^®D kit (quantitative kits) and about 1 hour for HybriScan^®I kit (identification kits). Qualitative results can be determined by eye or, for quantitative measurement, use with a microplate reader.

Figure 1. HybriScan^® system screening workflow with analysis time of approx. 2–2.5 hours

Sensitivity, Specificity and Flexibility of Sandwich Hybridization

Sandwich hybridization is very sensitive and can detect attomoles of the target rRNA molecules in yeast or bacteria.^1-3 These types of cells contain a large number of rRNA-containing ribosomes; a single cell therefore contains several thousand copies of rRNA but only one DNA copy. Sandwich hybridization provides sensitivity in crude biological samples because it is not susceptible to matrix interference. By using specific probes, the HybriScan^® system allows both genus or species-specific detection and it is therefore 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.

• Detects viable microorganisms only—minimizes false-positive results from dead bacteria
• Not sensitive to sample matrix
• Genus- and species-specific detection
• Results in just 2 hours after enrichment
• Broad application range: beer, wine, non-alcoholic beverages, drinking water, food and wastewater

Advantages of HybriScan^® technology over other microbial detection techniques

Detection Technology	Advantage	Disadvantage
HybriScan® system	Detects only living cells  Minimal interference by sample matrix High specificity Low cross-reactivity Easy handling Cost-efficient read-out 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

Figure 2. HybriScan^® technology

HybriScan^® Waste Water Bacterial Count Calculator

For our Wastewater Kits (04447 HybriScan^® D Waste Water Microthrix parvicella, 78436 HybriScan^® D Waste Water Total Bacterial Count), we provide an excel sheet (below) for calculation of total bacterial count, Microthrix count, and ratio of Microthrix: Total Bacterial Count.

• Waste Water Calculator (English)
• Waste Water Calculator (Deutsch)

References

1. Huhtamella S, Leinonen M, Nieminen T, Fahnert B, Myllykoski L, Breitenstein A, Neubauer P. 2007. RNA-based sandwich hybridisation method for detection of lactic acid bacteria in brewery samples. Journal of Microbiological Methods. 68(3):543-553. https://doi.org/10.1016/j.mimet.2006.10.009

2. Leskelä T, Tilsala-Timisjärvi A, Kusnetsov J, Neubauer P, Breitenstein A. 2005. Sensitive genus-specific detection of Legionella by a 16S rRNA based sandwich hybridization assay. Journal of Microbiological Methods. 62(2):167-179. https://doi.org/10.1016/j.mimet.2005.02.008

3. Rautio J, Barken K, Lahdenperä J, Breitenstein A, Molin S, Neubauer P. 2003. Microb Cell Fact. 2(1):4. https://doi.org/10.1186/1475-2859-2-4