Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bangalore, Karnataka, India.
J Appl Microbiol. 2011 Dec;111(6):1349-56. doi: 10.1111/j.1365-2672.2011.05169.x. Epub 2011 Oct 31.
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.
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.
The developed mPCR assay was found to be simple, rapid, reliable and specific in species identification of 10 bacteria at a time.
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.
开发和评估一种多重 PCR（mPCR）检测方法，用于同时检测引起奶牛乳腺炎的 10 种细菌，即金黄色葡萄球菌、产色葡萄球菌、表皮葡萄球菌、松鼠葡萄球菌、溶血性葡萄球菌、模仿葡萄球菌、无乳链球菌、停乳链球菌、乳房链球菌和大肠杆菌。
建立了两管 mPCR 检测方法。使用 56 株标准参考菌株和 705 株包括大肠杆菌（n=99）、葡萄球菌（n=522）和链球菌（n=84）的菌株评估 mPCR 的准确性。mPCR 检测的阈值为 10 fg 基因组 DNA 和<103 CFU ml-1。用该 mPCR 与培养方法对 115 份亚临床乳腺炎奶样进行比较评估显示，mPCR 更有效。随后，该 mPCR 成功地直接用于评估 36 份牛奶样品中的目标细菌。
该方法快速、可靠且具有特异性，可同时鉴定 10 种细菌。
该检测方法将有助于乳腺炎的检测、牛奶的细菌安全性测试以及种水平的鉴别。该检测方法将在乳制品行业的诊断和研究中具有价值。早期和准确地识别病原体将使及时干预治疗和控制奶牛乳腺炎成为可能。
