ACCURACY OF A MULTIPLEX PCR PROTOCOL FOR Staphylococcus aureus, Streptococcus agalactiae AND Escherichia coli DETECTION IN BULK TANKS
Palavras-chave:
Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli, microorganisms, , molecular diagnosis, dairy, bulk tank, milkResumo
The objective of the present study was to evaluate the accuracy of a multiplex PCR (mPCR) protocol for Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli detection in bovine milk samples collected from bulk tanks. Twenty dairy farms in mid-west of Sao Paulo State, Brazil were enrolled. Milk samples collected from each bulk tank were evaluated by mPCR and microbiological culture (gold standard test). S. aureus, S. agalactiae and E. coli were isolated by culture in 30%, 10% and 40% respectively, and detected by mPCR in 0%, 10% and 35% bulk tank milk samples respectively. mPCR has demonstrated 93.2% specificity and 37.5% sensitivity values, with 78.3% accuracy. It was possible to conclude that this mPCR protocol was efficient for S. agalactiae and E. coli detection in the evaluated bulk tank milk samples, but failed to detect S. aureus. Despite the high specificity values obtained by mPCR, the sensitivity levels were low, indicating that this molecular method should only be recommended as a complementary diagnosis test for milk quality monitoring in bulk tanks.
Referências
Brasil 2002. Instrução Normativa 51, de 20 de setembro de 2002. Aprova os regulamentos técnicos de produção, identidade e qualidade do leite tipo... Diário Oficial da União, Seção 1:13.
Brasil 2011. Instrução Normativa 62, de 29 de dezembro de 2011. Alterar o caput, excluir o parágrafo único e inserir os §§ 1º ao 3º, todos do art. 1º, da Instrução Normativa MAPA nº 51, de 18 de setembro de 2002... Diário Oficial da União, Seção 1.
Gillespie BE & Oliver SP. 2005. Simultaneous detection of mastitis pathogens, Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae by Multiplex Real-time Polymerase Chain Reaction. J. Dairy Sci. 88:3510-3518.
Phuektes P, Mansell PD & Browning GF. 2001. Multiplex polymerase chain reaction assay for simultaneous detection of Staphylococcus aureus and Streptococcal causes of bovine mastitis. J. Dairy Sci. 84:1140-1148.
Phuektes P, Browning GF, Anderson G & Mansell PD. 2003. Multiplex polymerase chain reaction as a mastitis screening test for Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis in bulk milk samples. J. Dairy Res. 70:149-155.
Silva MA. 2008. Utilização de PCR Multiplex para o diagnóstico etiológico da mastite bovina. Dissertação de Mestrado, Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Hoizonte.32p.
Maxwell ML, Gillespie BE & Oliver SP. 2008. Real-time polymerase chain reaction for simultaneous detection of mastitis pathogens directly from milk. Proc. NMC Annual Meeting, New Orleans, Louisiana - USA, p.212-213.
Carneiro DMVF. 2009. Detecção microbiológica e molecular de Staphylococcus aureus em amostras de leite bovino obtidas de tanques de expansão – correlação com resíduos de antibióticos. Tese de Doutorado, Faculdade de Medicina Veterinária e Zootecnia, Universidade Estadual Paulista, Botucatu-SP, Brasil.
Quinn PJ, Markey B, Carter ME, Donnelly WJ & Leonard FC. 2005. Microbiologia Veterinária e Doenças Infecciosas. Artmed. Porto Alegre. 512p.
Chótar M,Vidova B & Godany A. 2006.A development of specific and rapid detection of bacterial pathogens in dairy products by PCR. Folia Microbiol. 51:639-646.
Mackinnon A. 2000. A spreadsheet for the calculation of comprehensive statistics for the assessment of diagnostic tests and inter-rater agreement. Comput. Biol. Med.30:127-134.
Moussa IM, Ashgan MH, Alwathnani HA, Mohamed KHF & Al-doss AA. 2010. Multiplex polymerase chain reaction for detection and characterization of shiga toxigenic Escherichia coli (STEC). African J. Biotech.9:4356-4363.
Cremonesi P, Castiglioni B, Malferrari G, Biunno I, Vimercati C, Moroni P, Morando S. & Luzzana M. 2006. Technical Note: improved method for rapid DNA extraction of mastitis pathogens directly from milk. J. Dairy Sci. 89:163-169.
Chapaval L, Moon DH, Gomes JE, Duarte FR, Tsai SM. 2008. An alternative method for Staphylococcus aureus DNA isolation. Arq. Bras. Med. Vet. Zootec., 60(2):299-306.
Zocche F, Bastos CP & Silva WP. 2010. Detecção de genes do cluster egc em Staphylococcus aureus isolados de alimentos de origem animal. Ciência Rural, 40:1134-1140.
Barkema HW, Schukken YH, Zadoks, RN. 2006. Invited Review: The Role of Cow, Pathogen, and Treatment Regimen in the Therapeutic Success of Bovine Staphylococcus aureus Mastitis J. Dairy Sci. 89:1877-1895.
Yamagishi N, Jinkawa Y, Omoe K, Makino S & Oboshi K. 2007. Sensitive test for screening for Staphylococcus aureus in bovine mastitis by broth cultivation and PCR. Vet. Rec.161:381-383.
Faccioli PY. 2010. Detecção molecular de Staphylococcus aureus e de suas toxinas no leite de tanque de rebanhos bovinos, em condições de refrigeração e sob temperatura ambiente. Tese de Doutorado, Faculdade de Medicina Veterinária e Zootecnia, Universidade Estadual Paulista, Botucatu, SP. 132p.
Riffon R, Sayasith K, Khalil H, Dubreuil P, Drolet M & Lagacé J. 2001. Development of a rapid and sensitive test for identification of major pathogens in bovine mastitis by PCR. J. Clin. Microbiol. 39:2584-2589.
Silvennoinen M, Holopainen J & Koskinen MT. 2010. Pathoproof mastitis PCR assay and sensitivity validation of a new test kit for rapid identification of Staphylococcus aureus, Streptococcus agalactiae and Mycoplasma bovis in bovine milk. Proc. Annual Meeting of the National Mastitis Council, Albuquerque, New Mexico, p.240-241.
Ramesh A, Padmapriya BP, Chrashekar A & Varadaraj MC. 2002. Application of a convenient DNA extraction method and multiplex PCR for the direct detection of Staphylococcus aureus and Yersinia enterocolitica in milk samples. Mol. Cell. Probes, 16:307-314.
Downloads
Publicado
Como Citar
Edição
Seção
Licença
Este obra está licenciado com uma Licença Creative Commons Atribuição-NãoComercial 4.0 Internacional.
