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Multiplex PCR assay for species identification of bovine mastitis pathogens
Author(s) -
Shome B.R.,
Das Mitra S.,
Bhuvana M.,
Krithiga N.,
Velu D.,
Shome R.,
Isloor S.,
Barbuddhe S.B.,
Rahman H.
Publication year - 2011
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.2011.05169.x
Subject(s) - mastitis , streptococcus dysgalactiae , streptococcus uberis , streptococcus agalactiae , microbiology and biotechnology , biology , multiplex polymerase chain reaction , staphylococcus aureus , staphylococcus , multiplex , streptococcus , bacteria , polymerase chain reaction , biochemistry , genetics , gene , bioinformatics
Abstract Aim: 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. Methods and 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. Conclusion: The developed mPCR assay was found to be simple, rapid, reliable and specific in species identification of 10 bacteria at a time. Significance and Impact of the Study: 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.