Limit of detection of genomic DNA by conventional PCR for estimating the load of Staphylococcus aureus and Escherichia coli associated with bovine mastitis
Jazyk angličtina Země Spojené státy americké Médium print-electronic
Typ dokumentu hodnotící studie, časopisecké články, práce podpořená grantem
PubMed
25773783
DOI
10.1007/s12223-015-0384-0
PII: 10.1007/s12223-015-0384-0
Knihovny.cz E-zdroje
- MeSH
- bakteriální proteiny genetika MeSH
- DNA bakterií genetika MeSH
- Escherichia coli genetika izolace a purifikace MeSH
- infekce vyvolané Escherichia coli diagnóza mikrobiologie veterinární MeSH
- limita detekce MeSH
- mastitida skotu diagnóza mikrobiologie MeSH
- polymerázová řetězová reakce metody MeSH
- skot MeSH
- stafylokokové infekce diagnóza mikrobiologie veterinární MeSH
- Staphylococcus aureus genetika izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny MeSH
- DNA bakterií MeSH
Detection of mastitis-associated bacteria can be accomplished by culturing or by molecular techniques. On the other hand, rapid and inexpensive methods to enumerate bacterial load without culturing can be better achieved by molecular methods. Staphylococcus aureus and Escherichia coli are the predominant bacterial pathogens associated with bovine mastitis. Here, we describe the application of conventional PCR for the limit of detection (LOD) of genomic DNA of S. aureus and E. coli based on single-copy genes. The selected genes were thermonuclease (nuc), aureolysin (aur), and staphopain A (scpA) for S. aureus and β-D-glucuronidase A (uidA), cytochrome d oxidase (cyd), and rodA (a gene affecting cell shape and methicillin sensitivity) for E. coli. The LOD was 5.3, 15.9, and 143 pg for aur, nuc, and scpA genes, corresponding to S. aureus genomic copies of 1.75 × 10(3), 5.16 × 10(3), and 4.71 × 10(4), respectively. The LOD was 0.45, 12.3 and 109 pg for uidA, rodA and cyd genes, corresponding to E. coli genome copies of 8.91 × 10(1), 2.43 × 10(3), and 2.16 × 10(4), respectively. Application of uidA and aur PCRs to field strains revealed that as low as approximately 100 genome copies of E. coli and 1000-10,000 copies of S. aureus could be detected. This study is the first to report LOD of genomic DNA using conventional PCR for aur and scpA genes of S. aureus, and rodA and cyd genes of E. coli. The results should be useful for developing assays to assess bacterial load in milk and to determine the load that contributes to subclinical or clinical mastitis.
Ella Foundation Genome Valley Turkapally Shameerpet Mandal Hyderabad 500078 India
Institute of Wildlife Veterinary Research Kudige Kodagu 571232 India
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