Padronização de PCR para detecção direta e diferenciação de espécies de Campylobacter em Queijo Minas Artesanal

Abstract

The Artisanal Minas Cheese (AMC) is one of the oldest and most traditional cheeses in Brazil and it contributes to the income generation of several rural producers. The production of artisanal minas cheese is characterized using raw milk freshly milked. Although very popular, artisanal minas cheese does not always present sufficient innocuousness, requiring intensifying the demand for more representative studies about microbiological safety of this product. Campylobacter spp. has been the most frequently pathogen associated with outbreaks of foodborne diseases worldwide in recent years, however it is a neglected pathogen in prevalence studies and in the microbiological standards for cheeses in the Brazilian legislation. The detection of Campylobacter spp. by conventional methods of identification is difficult to perform and may present significant variations in the results. Molecular methods, especially Polymerase Chain Reaction (PCR) are promising tools for the rapid and direct detection of Campylobacter spp. in food, due to its high specificity, sensitivity and good detection limit. The aim of this work was to develop a molecular methodology based on multiplex PCR for direct identification of Campylobacter spp. and differentiation of the species C. jejuni and C. coli in AMC from Serro. Multiplex PCR was developed using the primers 16S rRNA, hipO and ceuE, specific for the detection of Campylobacter spp., C. jejuni and C. coli, respectively. In order to develop the methodology, 5 reaction combinations were tested, with different concentrations of MgCl2 (1,0μL/2,0μ L), buffer (2,5μ L/3,5μ L), DNA(10μ L/5μ L), agarose gel (1,0%/1,5%), besides modifications in voltage of electrophoretic running (120V/80V) and anneling temperature (66°C/62°C). The detection limit (DL) was evaluated using the concentrations: 0,1, 10, 103 and 105 CFU/ml. Tests with restriction enzymes XhoI, HhaI and HindIII were performed to provide additional evidence about the selectivity of the method. The Campylobacter spp. presence in field samples was also evaluated. The multiplex PCR developed in this work has been shown to be a rapid and effective method for the detection of Campylobacter spp., C. jejuni and C. coli in artificially contaminated AMC samples. DL was estimated at 103 CFU/mL. Tests with the restriction enzymes confirmed the selectivity of the primers. When evaluating the presence of this pathogen in field samples, there was inconsistency in the results using multiplex PCR. The method was effective for the detection of Campylobacter spp. in the artificially contaminated samples and inconsistent for field samples, which can be explained by DL. As a future perspective, it was proposed to implement the selective enrichment step of the samples to improve the sensitivity of the method, as well as validation of the methodology to evaluate the PCR specificity.