Problem: The detection of pathogenic or spoiling microorganisms has always been an important topic in the food and beverage industry. Adequate microbial examination can be time-consuming, and yet short shelf life dates and the costs related to storing freshly prepared food for extended periods demand that products be shipped in a timely manner. Upsetting this balance can lead to costly recall campaigns: For example in 2011, 15 people died and 84 fell ill, from a Listeriosis outbreak after consuming contaminated cantaloupe grown by Jensen Farms in Colorado, USA. More recently, in January 2012, the Centers for Disease Control and Prevention (Georgia, USA) reported 19 individuals were sickened by a “multi-drug resistant strain of Salmonella Typhimurium” after consuming tainted beef sold by Hannaford grocery stores.

One of today’s major problems is the changing behaviour of food consumption coupled with an increasing demand for a longer shelf life. For a growing variety of food products, the trend towards “ready-toeat” and “ready-to-cook” products addresses this issue by prolonged storage at low temperatures (4 – 8 °C). In addition, emerging preparation technologies to extend shelf life, such as “Cook & Chill” and “sous vide,” lead to increasing microorganism contamination.

Solution: Rapid methods for detection of microorganisms are in demand. HybriScan is a recent example of a rapid method that saves up to 10 days compared to traditional cultivation methods. The HybriScan test system is capable of detecting groups of organisms (e.g. all bacteria, Listeria ssp.) and specific species, for example Listeria monocytogenes. No PCR is required, and the result is based on genetic information; the target molecule is the ribosomal RNA (rRNA).

Figure 1: The capture probe is used to immobilize the target sequence on a solid support and the detection probe is labelled with a detectable marker.

This test method is designed on a simple 96-well microplate, with 12 strips of 8 wells, and is a sandwich hybridization assay. The principle of the analysis is based on the detection of hybridization events between two specific oligonucleotide probes and target nucleic acids. The capture probe is used to immobilize the target sequence on a solid support and the detection probe is labelled with a detectable marker (Figure 1). The test is not sensitive enough to sample matrices and detects only living cells; therefore, no special equipment is needed and the test is complete in approximately 2 to 2.5 hours. A positive result is visible to the naked eye, but it is also possible, by a standard microplate reader, to quantify the number of cells at 450 nm (based on the fact that the average number of ribosomes is similar from cell to cell).

Also, the sandwich hybridization is relatively sensitive and can be performed with crude biological samples. These types of assays from crude cell samples, or in connection to PCR, have been extensively used in clinical diagnostics for detection of nucleic acids from bacteria and viruses. The sandwich hybridization method is ideal for an identification of specific rRNAs in bacterial cells and yeasts. Although a direct detection of the ribosomal RNA does not match the sensitivity of a PCR-based DNA assay, it offers advantages like quantification, live/ dead-discrimination, and no additional amplification steps; plus, simple assay protocols with a standard laboratory equipment. The sensitivity reaches up to 1 CFU per 1 ml or 25 g with a preenrichment step (24 - 30 hours). For the assay itself, 1000-2000 bacteria or about 100 yeasts are needed. The available kits were validated according the official methods in Germany (§ 64 LFGB).

For more information, visit www.sigma-aldrich.com/hybriscan