title: Multiplex PCR assay for species identification of bovine mastitis pathogens.
abstract: OBJECTIVE
  To develop and evaluate a multiplex PCR (mPCR) assay for simultaneous detection of 10 bacterial species causing bovine mastitis namely, Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus sciuri, Staphylococcus haemolyticus, Staphylococcus simulans, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Escherichia coli in milk.
  
  
  RESULTS
  A two-tube mPCR assay was developed. The accuracy of the mPCR was evaluated using 56 standard reference strains and 705 strains comprising of E. coli (n = 99), staphylococci (n = 522) and streptococci (n = 84). The threshold of detection of the mPCR assay was 10 fg of genomic DNA and <10(3) CFU ml(-1). A comparative evaluation of mPCR with culture method using 115 milk samples from subclinical mastitis showed mPCR to be more efficacious. Subsequently, the mPCR showed successful detection of target bacteria, when applied directly for the assessment of 36 bulk milk samples.
  
  
  CONCLUSIONS
  The developed mPCR assay was found to be simple, rapid, reliable and specific in species identification of 10 bacteria at a time.
  
  
  CONCLUSIONS
  The assay will be useful for the detection of mastitis, testing bacteriological safety of milk and for species level differentiation. The assay will be of value in the dairy sector for diagnosis and research. The early and accurate identification of pathogens will enable timely interventions for the treatment and control of bovine mastitis.
authors.affiliation: Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bangalore, Karnataka, India.
authors.forename: B R
authors.initials: BR
authors.lastname: Shome
authors.forename: S
authors.initials: S
authors.lastname: Das Mitra
authors.forename: M
authors.initials: M
authors.lastname: Bhuvana
authors.forename: N
authors.initials: N
authors.lastname: Krithiga
authors.forename: D
authors.initials: D
authors.lastname: Velu
authors.forename: R
authors.initials: R
authors.lastname: Shome
authors.forename: S
authors.initials: S
authors.lastname: Isloor
authors.forename: S B
authors.initials: SB
authors.lastname: Barbuddhe
authors.forename: H
authors.initials: H
authors.lastname: Rahman
mesh_terms: D000818:Animals; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D002417:Cattle; D004269:DNA, Bacterial / Q000032:analysis; D005260:Female; D005506:Food Contamination / Q000032:analysis; D057230:Limit of Detection; D008414:Mastitis, Bovine / Q000175:diagnosis* / Q000382:microbiology; D008892:Milk / Q000382:microbiology; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D004269:DNA, Bacterial