Mastitisli İnek Sütlerinde Önemli Patojenlerin Direkt Tespiti İçin Bir Multipleks PCR Yönteminin Geliştirilmesi

Bu çalışmanın amacı, inek sütlerinde major mastitis patojenlerinin (Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli ve Mycoplasma bovis) saptanabilmesi için hızlı, basit ve spesifik bir DNA ekstraksiyon ve multipleks PCR yöntemi geliştirmektir. Kaliforniya mastitis testi (CMT) pozitif 200 süt örneğinin kültürü sonrasında S. aureus, S. agalactiae ve E. coli varlığı sırasıyla 45 (%22.5), 21 (%10.5) ve 11 (%5.5) örnekte saptandı. Çalışmada optimizasyonu yapılan DNA ekstraksiyon metodu ile elde edilen DNA örneklerinin mPCR analizi neticesinde S. aureus, S. agalactiae ve E. coli varlığı sırasıyla %26.5 (53/200), %12 (24/200) ve %6 (12/200) olarak belirlendi. Ticari DNA izolasyon kitiyle elde edilen DNA örneklerinde de yukarıdaki etkenlerin varlığı benzer oranda bulundu. Öte yandan süt örneklerinin hiçbirinde kültür veya mPCR ile M. bovis tespit edilmedi. Kültür ve mPCR sonuçları arasındaki fark istatistiki olarak önemli bulundu (P

Development of A Multiplex PCR Method for Direct Detection of Common Mastitis Pathogens in Bovine Milk Samples

The aim of this study was to evaluate a simple and rapid DNA extraction method combined with a multiplex polymerase chain reaction (mPCR) for the identification of the major mastitis pathogens (Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli and Mycoplasma bovis) from milk samples. Of the 200 California Mastitis Test (CMT) positive milk samples, 45 (22.5%), 21 (10.5%) and 11 (5.5%) were detected as positive for the presence of S. aureus, S. agalactiae and E. coli by culture, respectively. In mPCR by DNA isolation method optimised here, S. aureus, S. agalactiae and E. coli were detected in 26.5% (53/200), 12% (24/200) and 6% (12/200) of the milk samples, respectively. The abovementioned agents were observed in similar proportions when the samples were analysed by a commercial DNA isolation kit. On the other hand, M. bovis was not detected in any of the milk samples by either culture or mPCR methods. A significant difference was determined between the results of culture and mPCRs (P<0.001). Diagnostic sensitivity and specificity of the optimised mPCR were calculated as 100% and 89.2% respectively, when culture results were considered as reference. The results suggest that the mPCR assay employed in this study could be used as an alternative routine diagnostic method for rapid, sensitive, and speciŞc simultaneous detection of major mastitis agents in bovine milk samples.

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Kafkas Üniversitesi Veteriner Fakültesi Dergisi-Cover
  • ISSN: 1300-6045
  • Yayın Aralığı: Yılda 6 Sayı
  • Başlangıç: 1995
  • Yayıncı: Kafkas Üniv. Veteriner Fak.
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