Evaluation of a multiplex PCR assay as an alternative method for Listeria monocytogenes serotyping

被引:107
作者
Kerouanton, Annaelle [1 ]
Marault, Muriel [1 ]
Petit, Laetitia [1 ]
Grout, Joel [1 ]
Dao, Trinh Tam [1 ]
Brisabois, Anne [1 ]
机构
[1] LERQAP, Agence Francaise Securite Sanit Aliments, F-94706 Maisons Alfort, France
关键词
Listeria monocytogenes; Molecular serogroup; Serotyping; Subtyping; INTERNATIONAL STANDARD; TYPING METHODS; FOOD; IDENTIFICATION; STRAINS;
D O I
10.1016/j.mimet.2009.11.008
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Listeria monocytogenes serotyping is commonly used as the first level of characterisation in the epidemiological surveillance of food and clinical isolates and is therefore widely accepted. The aim of this study was to define a scheme for multiplex molecular serotyping of L monocytogenes based on a previously described PCR assay and then to evaluate and compare this new procedure with conventional serotyping by agglutination. The study included 1204 Listeria strains collected from food products in France, from March 2005 to October 2006. Two multiplex PCR assays were designed to cluster L monocytogenes strains into five molecular serogroups: IIa, IIb, IIc, IVa, IVb in agreement with the most commonly encountered serotypes. Amplification of the prfA gene was added to the multiplex PCR to check for L monocytogenes species; forty-eight (4%) of the isolates tested belonged to the genus Listeria but were not L monocytogenes. Using this first multiplex PCR, the concordance between conventional and molecular methods was 90.6%, 97.8%, 100% and 100%, for 1/2a, 1/2c, 1/2b and 4b serotypes respectively. False results were observed for some atypical 1/2a, 3a and 1/2c strains. Therefore, this lack of specificity was resolved by using an additional PCR assay based on amplification of the flaA gene, a specific target of 1/2a and 3a strains. When applying the second PCR assay to IIa and IIc molecular serogroup strains, total agreement was obtained between molecular and conventional serotyping methods with a lower level of discrimination for the molecular one. This study proposes to define a strategy for molecular serotyping using both PCR assays: a multiplex and the flaA PCR in order to assign the atypical 1/2a, 3a and 1/2c strains. Moreover, prs gene detection was added for Listeria genus recognition as a positive control in association with flaA detection. Indeed, this molecular serotyping scheme could be considered as a useful and rapid method for first-level characterisation of the most frequently encountered L monocytogenes serotypes. (C) 2009 Elsevier B.V. All rights reserved.
引用
收藏
页码:134 / 137
页数:4
相关论文
共 20 条
[1]   Listeria monocytogenes serotype identification by PCR [J].
Borucki, MK ;
Call, DR .
JOURNAL OF CLINICAL MICROBIOLOGY, 2003, 41 (12) :5537-5540
[2]   A validated PCR-based method to detect Listeria monocytogenes using raw milk as a food model -: Towards an international standard [J].
D'Agostino, M ;
Wagner, M ;
Vazquez-Boland, JA ;
Kuchta, T ;
Karpiskova, R ;
Hoorfar, J ;
Novella, S ;
Scortti, M ;
Ellison, J ;
Murray, A ;
Fernandes, I ;
Kuhn, M ;
Pazlarova, J ;
Heuvelink, A ;
Cook, N .
JOURNAL OF FOOD PROTECTION, 2004, 67 (08) :1646-1655
[3]   Multiplex PCR for the identification and Serotyping of L-monocytogenes isolated from sheep cheese-processing plants [J].
De Santis, E. P. L. ;
Pilo, A. L. ;
Cosseddu, A. M. ;
Canu, N. A. ;
Scarano, C. ;
Marongiu, P. .
VETERINARY RESEARCH COMMUNICATIONS, 2007, 31 (Suppl 1) :359-363
[4]  
DENNY J, 2008, EUROSURVEILL, V13
[5]   Multicenter validation of a multiplex PCR assay for differentiating the major Listeria monocytogenes Serovars 1/2a, 1/2b, 1/2c, and 4b:: Toward an international standard [J].
Doumith, M ;
Jacquet, C ;
Gerner-Smidt, P ;
Graves, LM ;
Loncarevic, S ;
Mathisen, T ;
Morvan, A ;
Salcedo, C ;
Torpdahl, M ;
Vazquez, JA ;
Martin, P .
JOURNAL OF FOOD PROTECTION, 2005, 68 (12) :2648-2650
[6]   Differentiation of the major Listeria monocytogenes serovars by multiplex PCR [J].
Doumith, M ;
Buchrieser, C ;
Glaser, P ;
Jacquet, C ;
Martin, P .
JOURNAL OF CLINICAL MICROBIOLOGY, 2004, 42 (08) :3819-3822
[7]   LISTERIA-MONOCYTOGENES, A FOOD-BORNE PATHOGEN [J].
FARBER, JM ;
PETERKIN, PI .
MICROBIOLOGICAL REVIEWS, 1991, 55 (03) :476-511
[8]   Increasing incidence of listeriosis in France and other European countries [J].
Goulet, Veronique ;
Hedberg, Craig ;
Le Monnier, Alban ;
de Valk, Henriette .
EMERGING INFECTIOUS DISEASES, 2008, 14 (05) :734-740
[9]   PulseNet standardized protocol for subtyping Listeria monocytogenes by macrorestriction and pulsed-field gel electrophoresis [J].
Graves, LM ;
Swaminathan, B .
INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY, 2001, 65 (1-2) :55-62
[10]  
GRAVES LM, 2007, ISOPOL 16 MARCH 20 2