Agence Française de Sécurité Sanitaire des Aliments, LERQAP, 94706 Maisons-Alfort Cedex, France.
J Microbiol Methods. 2010 Feb;80(2):134-7. doi: 10.1016/j.mimet.2009.11.008. Epub 2009 Dec 1.
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.
李斯特菌血清分型通常作为食品和临床分离株流行病学监测的第一级特征，因此被广泛接受。本研究的目的是基于先前描述的 PCR 检测方法，定义一种李斯特菌多重分子血清分型方案，然后评估并比较该新方法与传统凝集血清分型。本研究包括 2005 年 3 月至 2006 年 10 月期间从法国食品中收集的 1204 株李斯特菌菌株。设计了两个多重 PCR 检测方法，将李斯特菌菌株聚类为五个分子血清群：IIa、IIb、IIc、IVa 和 IVb，与最常见的血清型一致。prfA 基因的扩增被添加到多重 PCR 中，以检查李斯特菌物种；48 株（4%）被测试的分离物属于李斯特菌属，但不是李斯特菌。使用该第一个多重 PCR，传统和分子方法之间的一致性分别为 90.6%、97.8%、100%和 100%，对于 1/2a、1/2c、1/2b 和 4b 血清型。一些非典型 1/2a、3a 和 1/2c 菌株出现假阳性结果。因此，通过使用基于 flaA 基因扩增的特异性靶标 1/2a 和 3a 菌株的额外 PCR 检测，解决了这种特异性缺乏的问题。当将第二个 PCR 检测应用于 IIa 和 IIc 分子血清群菌株时，分子和传统血清分型方法之间完全一致，分子方法的分辨率较低。本研究提出了一种使用两种 PCR 检测方法进行分子血清分型的策略：多重和 flaA PCR，以分配非典型 1/2a、3a 和 1/2c 菌株。此外，prs 基因检测被添加到李斯特菌属识别中，作为与 flaA 检测相关的阳性对照。实际上，这种分子血清分型方案可以被认为是一种有用且快速的方法，用于对最常见的李斯特菌血清型进行第一级特征描述。
