On the specificity of PCR detection of Listeria monocytogenes in food: a comparison of published primers.
A total of nine pairs of primers, seven previously published and two newly developed, have been assayed for PCR detection of Listeria monocytogenes in food. They have been tested for specificity on a total of 72 strains including reference and food isolates belonging to L. monocytogenes and other species in the genus. First of all, a polyphasic approach has been carried out in order to establish a reference strain collection. They were biochemically and genetically characterized by API-Lis and randomly amplified polymorphic DNA PCR (RAPD-PCR), respectively. Random amplification of DNA was performed with M13, T7 and T3 universal primers and a data bank was created to compile the RAPD patterns of all the analyzed strains. The UPGMA cluster analysis of RAPD profiles with primer M13 showed eight clusters at 72.3% similarity. Clusters 2 and 7 corresponded to L. monocytogenes. Clusters 1 and 6 grouped L. ivanovii strains. Clusters 3, 4, 5 and 8 corresponded to L. grayi, L. innocua, L. welshimeri and L. seeligeri, respectively. Pattern analysis revealed the existence of miss-identified reference strains which was confirmed by 16S rDNA sequence analysis. RAPD-PCR is a rapid genetic test which helped to confirm strain identity. On the basis of PCR specificity results, primers LM1-LM2 were the best combination for the detection of L. monocytogenes since they only amplified the specific fragment in strains that had been genetically and biochemically assessed as belonging to the species. Specificity of other assayed primers is discussed.
Aznar
R
R
Departamento de Microbiología y Ecología, Universitat de València, Spain. rosa.aznar@uv.es
Alarcón
B
B
D017931:DNA Primers; D005516:Food Microbiology; D008089:Listeria monocytogenes / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D019105:Random Amplified Polymorphic DNA Technique / Q000379:methods* / Q000639:trends; D012680:Sensitivity and Specificity
D017931:DNA Primers; D012336:RNA, Ribosomal, 16S