Singlepath® Listeria

EMD Cat. No. 1.04142.0001
25 tests


GLISA-Rapid Test (Gold Labelled ImmunoSorbent Assay) for the qualitative detection of Listeria spp. in food.


Introduction
 Listeria are gram-positive, non-spore-forming, rod-shaped bacteria. Of the six known species of the Listeria genus, Listeria monocytogenes deserves particular mention as a human and animal pathogen, while L. ivanovii is pathogenic only in animals and L. innocua, L. seeligeri, L. grayi, and L. welshimeri are considered harmless environmental bacteria.

Listeriosis, the disease caused by L. monocytogenes, manifests itself not only as sepsis, but also and primarily as meningitis or even as encephalitis. Since L. monocytogenes is capable of crossing the placenta barrier, an infection of the pregnant mother with listeria constitutes a special risk for the fetus or result in the infection of the newborn child. As a result of the ubiquitous distribution of listeria and their capability to grow at refrigerator temperatures (+4°C to + 8°C), foods constitute one of the main sources of infection.

In the conduct of risk-related quality controls in foods and in the context of state-of-the-art hygiene-status monitoring procedures, tests should be run not only for L. monocytogenes, but also for the Listeria genus in general. The presence of listeria - in particular of L. innocua - is an indicator for critical hygienic conditions in the production process.

The drastic increase in the incidence of food infection caused by Listeria demands reliable and rapid methods of detection. Apart from traditional culture methods, immunological techniques are becoming more and more popular with users due to their better specificity and sensitivity.

Singlepath® Listeria is an immunological screening test based on the immune flow principle and is designed in such a way that time-consuming and personnel intensive working steps are avoided. Singlepath® Listeria is specific to all species within the Listeria genus.

Principle
 Singlepath® Listeria ( 1.04142) is an immunochromatographic rapid test based on gold-labelled antibodies. The test device has a circular sample port, and an oval shaped test (T) and control (C) window.
 1. The sample is applied to the chromatography paper via the circular sample port
 2. The sample is absorbed through the pad to the reaction zone containing colloidal, gold-labelled antibodies specific to Listeria.
 3. Any Listeria antigen present complexes with the gold-labelled antibody and migrates through the port until it encounters a binding zone in the test (T) area.
 4. The binding zone (T) contains another anti- Listeria Antibody, which immobilises any Listeria -antibody complex present. Due to the gold-labelling, a distinct red line is then formed.
 5. The rest of the sample continues to migrate to a second binding reagent zone within the control (C) zone, and also forms a second distinct red line (positive control). Regardless of whether any Listeria is present or not, this distinct red line is always formed in the control (C) zone, thus ensuring the test is working correctly.

Storage/Stability
 Singlepath® Listeria is stable until the expiry date printed on the box, when stored at +2 to +8°C.

Materials required for the test
 Package contents
 25 test devices (individually pouched in aluminium foil)
 Additionally required materials and instrumentation
 1. Enrichment media, e.g. 1.09628 Listeria enrichment broth, buffered (Base) acc. to FDA/BAM 1995 (bLEB), 1.11781 Listeria selective enrichment supplement acc. to FDA/BAM 1995, 1.10³98 FRASER Listeria selective enrichment broth (base), 1.10³99 FRASER Listeria Supplement
 2. Stomacher / Stomacher bags
 3. Incubators +35°C and +37°C
 4. Distilled or deionized water
 5. Autoclave
 6. Waterbath for boiling of samples
 7. Disposable Polypropylene tubes for boiling of samples
 8. Disposable plastic transfer pipettes and/or appropriate micro pipettes and disposable tips for dispensing 1-2 ml (sample for boiling) and 160 µl (application of oiled sample onto tests)

Sample material / sample enrichment
 Mix 25 g solid sample or 25 ml liquid sample with 225 ml of half-concentrated FRASER broth and homogenise with a Stomacher if necessary.
Incubate for 18-24 h at +30°C.
 Transfer 0,1 ml in 9.9 ml buffered LEB
 Incubate for 18-24 h at +30°C.

Test procedure
 Sample Preparation
 1. Transfer approx. 1-2 ml of enriched culture to an appropriate (polypropylene) tube. Cover with a loose-fitting cap.
 2. Incubate tubes at 80°C for 20 min.
3. Allow cooling to room temperature ( 18 - 26°C ), prior to use.
 Procedure
 1. Remove the foil pouches from the required number of Singlepath® Listeria devices. Place the test device(s) on a flat surface and label with appropriate sample identification. (Note: Perform the tests within a period of 2 hours after opening!)
 2. Using  a micro pipette and disposable pipette tip, draw up 160µl from the boiled, cooled enrichment. Dispense 160µl of the sample into the circular sample port on the test device.
Alternatively using a disposable transfer pipette, squeeze the pipette bulb, insert the stem into the boiled sample and release pressure on bulb. This will draw sample up into the pipette. Dispense five (5) free falling drops (about 150-160µl) into the circular sample port on the test device.
3. Observe the test result 20 minutes after applying the sample to the device.

Interpretation of results
 The test can be regarded as working correctly if a distinct red line appears in the control zone (C) within 20 minutes.
 A sample can be considered POSITIVE if at or prior to 20 minutes, red lines appear on both test (T) and control (C) zones.
 A sample can be considered NEGATIVE if no red line appears in the test (T) zone but does appear distinctly in the control (C) zone 20 minutes after application of sample to the device.

Technical specifications
 Detection limit
 Depending on serogroup, a range of approx. 104 106 bacteria/ml can be regarded as being the lower detection limit.
 Interferences
 Results obtained to date on numerous food samples indicate that there is no interference of Singlepath® Listeria with food ingredients.

The test has been developed based on using LEB and Palcam selective enrichment broth from MERCK KGaA. Interference from other types of selective enrichment broths and other brands cannot be excluded. In particular use of broth of red-brown color could potentially mask weak signals due to background coloration of the test zone.

Trouble-shooting
Problem
 		Measures
No line appears in either zone after 20 minutes test period.
Re-run sample.




Singlepath® Listeria



© 2002 Merck KGaA, Darmstadt, Germany