title: Using nested PCR to detect the hlyA gene of Listeria monocytogenes in Minas Frescal cow's milk cheese.
abstract: To evaluate a nested PCR protocol for Listeria monocytogenes detection, Minas Frescal cow's milk cheeses were produced, artificially inoculated with this pathogen at concentrations ranging from 1 to 1,000 CFU/g, and stored at 4°C for 10 days. The International Organization  for Standardization (ISO) standardized method 11290-1/A1 was used to detect L. monocytogenes in the inoculated samples, and DNA was extracted from aliquots (1, 5, and 10 ml) of 1:10 dilution, followed by a nested PCR protocol for the hlyA gene. The ISO standardized reference  method and nested PCR both detected L. monocytogenes at all concentrations and during all storage periods; McNemar's test showed no significant difference (P > 0.05). The results indicated that the nested PCR protocol can be used as a screening test to detect L. monocytogenes  in Minas Frescal cheese, allowing earlier detection of the pathogen that can later be confirmed by the ISO standardized reference method.
authors.affiliation: Faculdade de Agronomia e Veterinária, Universidade de Brasília, Campus Darcy Ribeiro, 70910-900, Brasília, Distrito Federal, Brazil.
authors.forename: Stefania Márcia de Oliveira
authors.initials: SM
authors.lastname: Souza
authors.forename: Isabela Sarmento
authors.initials: IS
authors.lastname: Brasileiro
authors.forename: Antonio Fernandes
authors.initials: AF
authors.lastname: de Carvalho
authors.forename: Abelardo
authors.initials: A
authors.lastname: da Silva
authors.forename: Luís Augusto
authors.initials: LA
authors.lastname: Nero
authors.forename: Márcia de Aguiar
authors.initials: Mde A
authors.lastname: Ferreira
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics*; D001938:Brazil; D002417:Cattle; D002611:Cheese / Q000382:microbiology*; D015169:Colony Count, Microbial / Q000379:methods; D003257:Consumer Product Safety; D004269:DNA, Bacterial / Q000032:analysis*; D005506:Food Contamination / Q000032:analysis*; D006360:Heat-Shock Proteins / Q000235:genetics*; D006460:Hemolysin Proteins / Q000235:genetics*; D006801:Humans; D008089:Listeria monocytogenes / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D001427:Bacterial Toxins; D004269:DNA, Bacterial; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; C038705:hlyA protein, Listeria monocytogenes