Multiplex PCR assay for species identification of bovine mastitis pathogens

被引：96
作者：
Shome, B. R. [2 ]
Das Mitra, S. [2 ]
Bhuvana, M. [2 ]
Krithiga, N. [2 ]
Velu, D. [2 ]
Shome, R. [2 ]
Isloor, S. [3 ]
Barbuddhe, S. B. [1 ]
Rahman, H. [2 ]
机构：
[1] ICAR Res Complex Goa, Ela 403402, Old Goa, India
[2] Project Directorate Anim Dis Monitoring & Surveil, Bangalore, Karnataka, India
[3] Karnataka Vet & Fishery Sci Univ, Vet Coll, Bangalore, Karnataka, India
来源：
JOURNAL OF APPLIED MICROBIOLOGY | 2011年 / 111卷 / 06期
关键词：
bacteria; bovine mastitis; detection; multiplex PCR; POLYMERASE-CHAIN-REACTION; MILK SAMPLES; STAPHYLOCOCCUS-AUREUS; STREPTOCOCCAL CAUSES; ESCHERICHIA-COLI; RIBOSOMAL-RNA; BACTERIA; WATER; RAW;
D O I：
10.1111/j.1365-2672.2011.05169.x
中图分类号：
Q81 [生物工程学（生物技术）]; Q93 [微生物学];
学科分类号：
071005 ; 0836 ; 090102 ; 100705 ;
摘要：
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.
引用
收藏
页码：1349 / 1356
页数：8
相关论文
共 24 条
[1]   DETECTION OF ESCHERICHIA-COLI AND SHIGELLA SPP IN WATER BY USING THE POLYMERASE CHAIN-REACTION AND GENE PROBES FOR UID [J].
BEJ, AK ;
DICESARE, JL ;
HAFF, L ;
ATLAS, RM .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1991, 57 (04) :1013-1017
[2]   Survey of the incidence and aetiology of mastitis on dairy farms in England and Wales [J].
Bradley, A. J. ;
Leach, K. A. ;
Breen, J. E. ;
Green, L. E. ;
Green, M. J. .
VETERINARY RECORD, 2007, 160 (08) :253-258
[3]   Comparison of a commercialized phenotyping system, antimicrobial susceptibility testing, and tuf gene sequence-based genotyping for species-level identification of coagulase-negative staphylococci isolated from cases of bovine mastitis [J].
Capurro, A. ;
Artursson, K. ;
Waller, K. Persson ;
Bengtsson, B. ;
Ericsson-Unnerstad, H. ;
Aspan, A. .
VETERINARY MICROBIOLOGY, 2009, 134 (3-4) :327-333
[4]   Improved method for rapid DNA extraction of mastitis pathogens directly from milk [J].
Cremonesi, P ;
Castiglioni, B ;
Malferrari, G ;
Biunno, I ;
Vimercati, C ;
Moroni, P ;
Morandi, S ;
Luzzana, M .
JOURNAL OF DAIRY SCIENCE, 2006, 89 (01) :163-169
[5]   Pathogen detection in milk samples by ligation detection reaction-mediated universal array method [J].
Cremonesi, P. ;
Pisoni, G. ;
Severgnini, M. ;
Consolandi, C. ;
Moroni, P. ;
Raschetti, M. ;
Castiglioni, B. .
JOURNAL OF DAIRY SCIENCE, 2009, 92 (07) :3027-3039
[6]  
DINSMORE RP, 1992, J DAIRY SCI, V75, P2706, DOI 10.3168/jds.S0022-0302(92)78032-1
[7]   cpn60 gene based multiplex-PCR assay for simultaneous identification of streptococcal species [J].
Dmitriev, A. ;
Bhide, M. ;
Mikula, I. .
ACTA VETERINARIA BRNO, 2006, 75 (02) :235-240
[8]   Identification of staphylococcal and streptococcal causes of bovine mastitis using 16S-23S rRNA spacer regions [J].
Forsman, P ;
TilsalaTimisjarvi, A ;
Alatossava, T .
MICROBIOLOGY-UK, 1997, 143 :3491-3500
[9]   Genetic classification and distinguishing of Staphylococcus species based on different partial gap, 16S rRNA, hsp60, rpoB, sodA, and tuf gene sequences [J].
Ghebremedhin, B. ;
Layer, F. ;
Koenig, W. ;
Koenig, B. .
JOURNAL OF CLINICAL MICROBIOLOGY, 2008, 46 (03) :1019-1025
[10]   Simultaneous detection of mastitis pathogens, Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae by multiplex real-time polymerase chain reaction [J].
Gillespie, BE ;
Oliver, SP .
JOURNAL OF DAIRY SCIENCE, 2005, 88 (10) :3510-3518
123