Our newly developed RNAssay^® technology enables highly specific molecular biology-based detection of salmonellae within 2 hours!

What is the RNAssay^® technology?

The RNAssay^® technology is based on the precise detection of unique nucleotide sequences present in molecules of ribosomal RNA (rRNA), by nucleic acid hybridisation. Just like a fingerprint, this sequence is characteristic for the respective microorganism. Furthermore, rRNA molecules are abundant in living cells, so there is no need for enzymatic amplification such as polymerase chain reaction. Because RNA is rapidly degraded in dead cells, only live bacteria will be detected.

An additional advantage is the enormous simplification of the working steps for the molecular biology-based detection. The assay can be performed in any laboratory, using a simple protocol without the need for special skills or availability of expensive equipment.

Fields of application

The RNAssay^® test system is perfectly suited for the rapid confirmation of the pathogenic microorganism directly from conventional selective enrichment media or from selective agar plates. Thereby, even the duration of standard detection methods can be considerably reduced.

Moreover, RNAssay^® is also ideal for the quick confirmation of reactive samples in rapid pathogen screening methods like impedance (SyLab BacTrac) or immunological tests (ELISA), and for the rapid identification of suspect colonies from chromogenic agar.

Even small numbers of samples can be tested very efficiently due to the specific test format.

RNAssay^® test principle

The ribosomal RNA of the target organism is released directly from the selective enrichment culture by a simple lysis reaction and detected using sandwich-hybridisation and subsequent enzyme-catalysed colour reaction on a test strip. An integrated reaction control enables monitoring of the reaction to exclude false negative test results due to user errors. The special test format allows for a strong reduction of the standard protocol for nucleic acid hybridisations and processing at ambient temperature (20-30° C). Therefore, in addition to the saving of time, the usual cumbersome incubation of hybridisation reactions in water baths or special incubators can be dispensed with. The only equipment required is an orbital shaker or a rocking platform.

Thus, RNAssay^® fulfills all the requirements of a simple and rapid, yet highly sensitive and specific detection assay.

 

Working steps (after pre-enrichment and selective enrichment)

Working step	duration
Preparation (lysis) of sample	5 minutes
Hybridisation	30 minutes
Washing and equilibration	approx. 25 Minuten
Colorimetric detection	≥ 45 minutes
In most cases, the result will be available within 2 hours after completion of the selective enrichment, with little hands-on time.

Test result:

Detected sequences (from left to right):
Salmonella-specific sequence (line A)
Integrated reaction control (line B)
Results:
1 = negative for Salmonella
2 = positive for Salmonella
3 = invalid result (no reaction control visible)

Procedure:

1. Pre-enrichment and selective enrichment (or plate culture) following standard protocols.

2. For plate cultures: Resuspend a single colony in 100µl of Lysis Buffer. For liquid cultures: Mix 200µl of liquid culture with 200µl of Lysis Buffer.

3. Put tests strips into incubation tray, mix 40µl of bacterial lysate with 20 µl of Denaturation Solution and incubate at ambient temperature for 5 minutes. Prepare hybridisation mix.

4. Add 0,5 ml of hybridisation mix per strip and hybridise 30 minutes at ambient temperature.

5. Wash 2 x 10 minutes using 2 ml each Washing Solution per strip.

6. Equilibrate strips for 1 minute in 1 ml of Substrate Buffer per strip

7. Colour development (45 minutes, up to a maximum of 2 hours).