title: Detection and Enumeration of Streptococcus agalactiae from Bovine Milk Samples by Real-Time Polymerase Chain Reaction.
abstract: The aim of this study was to evaluate the use of real-time polymerase chain reaction (qPCR) combined with DNA extraction directly from composite milk and bulk tank samples for detection and enumeration of Streptococcus agalactiae (SAG) causing subclinical mastitis. Dilutions of sterile reconstituted skim milk inoculated with SAG ATCC 13813 were used to establish a standard curve (cfu/mL) for the qPCR assay targeting SAG. The analytical sensitivity and repeatability of the qPCR assay were determined. Bulk tank (BTM; n = 38) and composite milk samples (CM; n = 26) collected from lactating cows with positive isolation of SAG were submitted to the qPCR protocol and SAG plate counting, with results from both methods compared. Amplification of DNA was not possible in two out of 64 samples, indicating that qPCR was able to detect SAG in 96 and 97% of BTM and CM samples, respectively. The inter-assay coefficient of variation was <5%, showing that the technique had adequate repeatability. The qPCR protocol can be a high-throughput and rapid diagnostic assay to accurately detect SAG from BTM and CM samples compared with conventional microbiological culture method. However, the evaluated qPCR protocol is not accurate for enumerating SAG in milk samples, probably due to quantification of DNA of non-viable cells.
authors.affiliation: Department of Animal Sciences, School of Veterinary Medicine and Animal Sciences, University of São Paulo (USP), Avenue Duque de Caxias Norte, 225, Pirassununga, SP, 13635-900, Brazil, naralcarvalho@gmail.com.
authors.forename: Nara Ladeira
authors.initials: NL
authors.lastname: de Carvalho
authors.forename: Juliano Leonel
authors.initials: JL
authors.lastname: Gonçalves
authors.forename: Bruno Garcia
authors.initials: BG
authors.lastname: Botaro
authors.forename: Luis Felipe de Prada E
authors.initials: LF
authors.lastname: Silva
authors.forename: Marcos Veiga
authors.initials: MV
authors.lastname: dos Santos
mesh_terms: D000818:Animals; D058345:Asymptomatic Infections; D058491:Bacterial Load / Q000379:methods*; D002417:Cattle; D008414:Mastitis, Bovine / Q000382:microbiology; D008892:Milk / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013292:Streptococcus agalactiae / Q000235:genetics / Q000302:isolation & purification*