Detection and enumeration of Staphylococcus aureus from bovine milk samples by real-time polymerase chain reaction.
The aim of this study was to develop and evaluate a real-time quantitative PCR (qPCR)-based method to detect and quantify Staphylococcus aureus in bronopol-preserved milk samples from subclinical intramammary infections (IMI). Serial dilutions of milk artificially inoculated with Staph. aureus ATCC 29213 were used to establish a standard curve (cfu/mL) of the qPCR assay targeting the Staph. aureus thermonuclease-encoding gene nuc according to the strain plate count. The analytical sensitivity, specificity, and repeatability of the qPCR assay were determined. A total of 60 milk samples, collected from mammary quarters without abnormal appearance and with positive isolation of Staph. aureus, were submitted to both the qPCR protocol and Staph. aureus plate counting and results from both methods were compared. Staphylococcus aureus from bronopol-preserved, subclinical IMI milk samples were not accurately enumerated by qPCR compared with plate counting of the nonpreserved, raw milk sample. The detection limit of the qPCR protocol of inoculated Staph. aureus ATCC 29213 in bronopol-preserved milk samples was 1.04 × 10(1) cfu/mL. The qPCR protocol can be a high-throughput and rapid diagnostic assay to accurately detect Staph. aureus IMI from bronopol-preserved milk samples compared with a traditional culturing method. However, the proposed qPCR protocol is not accurate for counting of Staph. aureus in bronopol-preserved milk samples from naturally infected mammary glands.
Department of Preventive Veterinary Medicine and Animal Health, 13635-900 Brazil.
B G
BG
Botaro
Department of Animal Sciences, School of Veterinary Medicine and Animal Sciences, University of São Paulo, 13635-900 Brazil.
C S
CS
Cortinhas
Department of Animal Sciences, School of Veterinary Medicine and Animal Sciences, University of São Paulo, 13635-900 Brazil.
L V
LV
Março
Department of Animal Sciences, School of Veterinary Medicine and Animal Sciences, University of São Paulo, 13635-900 Brazil.
J F G
JFG
Moreno
Department of Animal Sciences, School of Veterinary Medicine and Animal Sciences, University of São Paulo, 13635-900 Brazil.
L F P
LFP
Silva
Department of Preventive Veterinary Medicine and Animal Health, 13635-900 Brazil.
N R
NR
Benites
Department of Animal Sciences, School of Veterinary Medicine and Animal Sciences, University of São Paulo, 13635-900 Brazil. Electronic address: mveiga@usp.br.
M V
MV
Santos
D000818:Animals; D000890:Anti-Infective Agents; D002417:Cattle / Q000382:microbiology*; D005260:Female; D008414:Mastitis, Bovine / Q000382:microbiology*; D008836:Micrococcal Nuclease / Q000235:genetics; D008892:Milk / Q000382:microbiology*; D011409:Propylene Glycols; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013203:Staphylococcal Infections / Q000382:microbiology / Q000662:veterinary*; D013211:Staphylococcus aureus / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*
D000890:Anti-Infective Agents; D011409:Propylene Glycols; C006827:bronopol; D008836:Micrococcal Nuclease
bacteria; dairy cow; intramammary infection