title: Limit of detection of genomic DNA by conventional PCR for estimating the load of Staphylococcus aureus and Escherichia coli associated with bovine mastitis.
abstract: 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.
authors.affiliation: Department of Microbiology, Karnataka Veterinary Animal and Fisheries Sciences University, Hebbal, Bengaluru, 560024, India.
authors.forename: K M
authors.initials: KM
authors.lastname: Chandrashekhar
authors.affiliation: Department of Microbiology, Karnataka Veterinary Animal and Fisheries Sciences University, Hebbal, Bengaluru, 560024, India.
authors.forename: Shrikrishna
authors.initials: S
authors.lastname: Isloor
authors.affiliation: Department of Microbiology, Karnataka Veterinary Animal and Fisheries Sciences University, Hebbal, Bengaluru, 560024, India.
authors.forename: B H
authors.initials: BH
authors.lastname: Veeresh
authors.affiliation: Institute of Animal Health and Veterinary Biologicals, Karnataka Veterinary Animal and Fisheries Sciences University, Hebbal, Bengaluru, 560024, India.
authors.forename: Raveendra
authors.initials: R
authors.lastname: Hegde
authors.affiliation: Department of Microbiology, Karnataka Veterinary Animal and Fisheries Sciences University, Hebbal, Bengaluru, 560024, India.
authors.forename: D
authors.initials: D
authors.lastname: Rathnamma
authors.affiliation: Institute of Animal Health and Veterinary Biologicals, Karnataka Veterinary Animal and Fisheries Sciences University, Hebbal, Bengaluru, 560024, India.
authors.forename: Shivaraj
authors.initials: S
authors.lastname: Murag
authors.affiliation: Department of Microbiology, Karnataka Veterinary Animal and Fisheries Sciences University, Hebbal, Bengaluru, 560024, India.
authors.forename: B M
authors.initials: BM
authors.lastname: Veeregowda
authors.affiliation: Institute of Wildlife Veterinary Research, Kudige, Kodagu, 571232, India.
authors.forename: H A
authors.initials: HA
authors.lastname: Upendra
authors.affiliation: Ella Foundation, Genome Valley, Turkapally, Shameerpet Mandal, Hyderabad, 500078, India. hegden@ellafoundation.org.
authors.forename: Nagendra R
authors.initials: NR
authors.lastname: Hegde
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D002417:Cattle; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification*; D004927:Escherichia coli Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D005260:Female; D057230:Limit of Detection; D008414:Mastitis, Bovine / Q000175:diagnosis / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013203:Staphylococcal Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D013211:Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial