Standardization of a rapid quadruplex PCR method for the simultaneous detection of bovine, buffalo, Salmonella spp., and Listeria monocytogenes DNA in milk
ABSTRACT The objective of the present study was to Standardize a Polymerase Chain Reaction (PCR) protocol for the authentication of bovine and buffalo milk, and to detect the presence of Salmonella spp. and Listeria monocytogenes. For this, the target DNA was extracted, mixed, and subjected to a PCR assay. Milk samples were defrauded and experimentally contaminated with microorganisms to assess the detection of target DNA at different times of cultivation, bacterial titers, and concentration of genetic material. In addition, the protocol was tested with DNA extracted directly from food, without a pre-enrichment step. The proposed quadruplex PCR showed good accuracy in identifying target DNA sequences. It was possible to simultaneously identify all DNA sequences at the time of inoculation (0h), when the samples were contaminated with 2 CFU/250mL and with 6h of culture when the initial inoculum was 1 CFU/250mL. It was also possible to directly detect DNA sequences from the food when it was inoculated with 3 CFU/mL bacteria. Thus, the proposed methodology showed satisfactory performance, optimization of the analysis time, and a potential for the detection of microorganisms at low titers, which can be used for the detection of fraud and contamination.
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Universidade Federal de Minas Gerais, Escola de Veterinária
2021
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oai:scielo:S0102-093520210004007812021-08-20Standardization of a rapid quadruplex PCR method for the simultaneous detection of bovine, buffalo, Salmonella spp., and Listeria monocytogenes DNA in milkLima,J.S.Sampaio,A.P.P.O.Dufossé,M.C.S.Rosa,A.M.B.P.Sousa,P.F.M.Silva,J.B.Cardoso,G.V.F.Moraes,C.M.Roos,T.B. fraud food safety molecular techniques food pathogens ABSTRACT The objective of the present study was to Standardize a Polymerase Chain Reaction (PCR) protocol for the authentication of bovine and buffalo milk, and to detect the presence of Salmonella spp. and Listeria monocytogenes. For this, the target DNA was extracted, mixed, and subjected to a PCR assay. Milk samples were defrauded and experimentally contaminated with microorganisms to assess the detection of target DNA at different times of cultivation, bacterial titers, and concentration of genetic material. In addition, the protocol was tested with DNA extracted directly from food, without a pre-enrichment step. The proposed quadruplex PCR showed good accuracy in identifying target DNA sequences. It was possible to simultaneously identify all DNA sequences at the time of inoculation (0h), when the samples were contaminated with 2 CFU/250mL and with 6h of culture when the initial inoculum was 1 CFU/250mL. It was also possible to directly detect DNA sequences from the food when it was inoculated with 3 CFU/mL bacteria. Thus, the proposed methodology showed satisfactory performance, optimization of the analysis time, and a potential for the detection of microorganisms at low titers, which can be used for the detection of fraud and contamination.info:eu-repo/semantics/openAccessUniversidade Federal de Minas Gerais, Escola de VeterináriaArquivo Brasileiro de Medicina Veterinária e Zootecnia v.73 n.4 20212021-08-01info:eu-repo/semantics/articletext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S0102-09352021000400781en10.1590/1678-4162-12218 |
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Lima,J.S. Sampaio,A.P.P.O. Dufossé,M.C.S. Rosa,A.M.B.P. Sousa,P.F.M. Silva,J.B. Cardoso,G.V.F. Moraes,C.M. Roos,T.B. |
spellingShingle |
Lima,J.S. Sampaio,A.P.P.O. Dufossé,M.C.S. Rosa,A.M.B.P. Sousa,P.F.M. Silva,J.B. Cardoso,G.V.F. Moraes,C.M. Roos,T.B. Standardization of a rapid quadruplex PCR method for the simultaneous detection of bovine, buffalo, Salmonella spp., and Listeria monocytogenes DNA in milk |
author_facet |
Lima,J.S. Sampaio,A.P.P.O. Dufossé,M.C.S. Rosa,A.M.B.P. Sousa,P.F.M. Silva,J.B. Cardoso,G.V.F. Moraes,C.M. Roos,T.B. |
author_sort |
Lima,J.S. |
title |
Standardization of a rapid quadruplex PCR method for the simultaneous detection of bovine, buffalo, Salmonella spp., and Listeria monocytogenes DNA in milk |
title_short |
Standardization of a rapid quadruplex PCR method for the simultaneous detection of bovine, buffalo, Salmonella spp., and Listeria monocytogenes DNA in milk |
title_full |
Standardization of a rapid quadruplex PCR method for the simultaneous detection of bovine, buffalo, Salmonella spp., and Listeria monocytogenes DNA in milk |
title_fullStr |
Standardization of a rapid quadruplex PCR method for the simultaneous detection of bovine, buffalo, Salmonella spp., and Listeria monocytogenes DNA in milk |
title_full_unstemmed |
Standardization of a rapid quadruplex PCR method for the simultaneous detection of bovine, buffalo, Salmonella spp., and Listeria monocytogenes DNA in milk |
title_sort |
standardization of a rapid quadruplex pcr method for the simultaneous detection of bovine, buffalo, salmonella spp., and listeria monocytogenes dna in milk |
description |
ABSTRACT The objective of the present study was to Standardize a Polymerase Chain Reaction (PCR) protocol for the authentication of bovine and buffalo milk, and to detect the presence of Salmonella spp. and Listeria monocytogenes. For this, the target DNA was extracted, mixed, and subjected to a PCR assay. Milk samples were defrauded and experimentally contaminated with microorganisms to assess the detection of target DNA at different times of cultivation, bacterial titers, and concentration of genetic material. In addition, the protocol was tested with DNA extracted directly from food, without a pre-enrichment step. The proposed quadruplex PCR showed good accuracy in identifying target DNA sequences. It was possible to simultaneously identify all DNA sequences at the time of inoculation (0h), when the samples were contaminated with 2 CFU/250mL and with 6h of culture when the initial inoculum was 1 CFU/250mL. It was also possible to directly detect DNA sequences from the food when it was inoculated with 3 CFU/mL bacteria. Thus, the proposed methodology showed satisfactory performance, optimization of the analysis time, and a potential for the detection of microorganisms at low titers, which can be used for the detection of fraud and contamination. |
publisher |
Universidade Federal de Minas Gerais, Escola de Veterinária |
publishDate |
2021 |
url |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0102-09352021000400781 |
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