Evaluation of the performances of the GeneDisc ID Listeria real-time PCR
assay for the rapid identification of Listeria species
Nicolas DESROCHE(1), Patrice CHABLAIN(2) , Jean GUZZO(1) and Patrice ARBAULT(1)
(1)
Nexidia SAS, AgroSup Dijon, 26 Boulevard du Docteur Petitjean, BP 87999, 21079 DIJON Cedex,
(2)GeneSystems,
centre d’affaires CICEA, 1, rue du courtil, 35170 Bruz, France;
INTRODUCTION
The Listeria genus is composed of 6 different species (L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. grayi and L. welshimeri) but only L. monocytogenes and L. ivanovii are pathogenic, respectively to humans and animals,
especially ruminants. L. monocytogenes is a rare cause of food-borne disease resulting in dramatic failures such as encephalitis, abortion and septicemia. Therefore it is imperative to have rapid and sensitive assays for the detection
and identification of Listeria sp. in foodstuff and clinical specimens. For foodstuff, the conventional methods for the detection and enumeration of Listera sp. (ISO, BAM, USDA) require biochemical tests to differentiate amongst the 6
species of Listeria. These biochemical tests are performed from isolated colonies and rely on catalase activity, hemolytic characteristic and sugar fermentation. Rapid biochemical kits such as API® Listeria, Microbact™ Listeria are
available for carrying out these biochemical test panels. More recently, new techniques were developed for rapid and accurate speciation of Listeria isolates: classical PCR, real-time PCR, RAPD, FITR, etc…
A new automated real-time PCR systems has been developed for the rapid identification of Listeria species (GeneDisc ID Listeria) within less than 1 hour. In our study, we evaluated the performances of the GeneDisc ID Listeria
and compared it to the commercialized biochemical kits, API® Listeria and Microbact™ Listeria.
BACTERIAL STRAINS
RESULTS
166 strains of Listeria species were isolated on TSA plates (Trypton Soy Agar) at 35°C.
Species
Serotype
L. monocytogenes 1/2 (a, b and c)
L. innocua
L. ivanovii
Species
Origin
Collection
Food or clinical
3
4b
Others
3
15
6
5
6a, 6b and 4ab
2
60
ND
8
L. monocytogenes
28
L. innocua
L. ivanovii
L. grayi
3
Number of
strain tested
API® Listeria
Microbact ™ Listeria 12L
% of correct identification
GeneDisc® Listeria
60
100%(a)
100%(a)
100%
62
100%
100%(b)
100%
11
73%(c)
73%
100%
11
91%(d)
91%
100%
90%
100%
L. grayi
ND
8
3
L. seeligeri
10
90%(d)
L. seeligeri
L. welshimeri
ND
ND
1
0
9
12
L. welshimeri
12
100%
100%
100%
161
161
166
Total strains correctly identified
METHODS
• API® Listeria and Microbact™ 12L Listeria galleries were purchased from BioMérieux (Marcy l’Etoile, France) and
Oxoid (Dardilly, France). GeneDisc Listeria ID was supplied by Pall/GeneSystems (Bruz, France).
Few colonies (equivalent to 1 Mc
Farland) in suspension medium
(a)
One strain of L. monocytogenes was first identified as L. innocua but the biochemical profiles were correct after a second test.
One strain of L. innocua was first identified as L. welshimeri but the biochemical profile was correct after a second Microbact™ 12L test.
(c) One strain of L. ivanovii was identified as L. seeligeri after a first API® Listeria test but the biochemical profiles was correct after a second test.
(d) One strain was misidentified after a first API® Listeria test but the second test allowed a correct identification.
(b)
16- 24 hours
API Listeria
• For the Listeria species monocytogenes, innocua and welshimeri, both biochemical galleries and the realtime PCR assay showed perfect matching.
One colony in suspending medium
16- 24 hours
Microbact 12L
Streaking onto TSA
16 -24 hours – 35°C
• The GeneDisc ID Listeria gave correct identifications for all the Listeria strains.
One colony (DNA extract
by boiling in Chelex)
0.5 - 1 hour
• Both biochemical galleries showed erroneous results for the identification of 3 out of 11 L. ivanovii
strains: API and Microbact yielded a biochemical profile for L. seeligeri. These 3 L. ivanovii collection
strains were clearly confirmed as L. ivanovii by the 16S rDNA sequencing method, in agreement with the
GeneDisc ID Listeria.
CONCLUSIONS
The GeneDisc ID Listeria allowed a highly accurate identification of Listeria species. The time to result was
significantly lower with the GeneDisc ID Listeria (approximately 1 hour) compared to the biochemical
galleries (approximately 24 hours).
GeneDisc ID Listeria
• For the strains which gave different identifications between biochemical tests and molecular test, a fragment of 550 bp of the
16S rDNA sequence was amplified with specific primers and sequenced. Sequences were aligned with the nucleotide banks.
The GeneDisc ID Listeria is an ideal tool for the routine rapid identification of Listeria strains in food and
clinical laboratories.