Development of a semi-nested PCR for the improved detection of Mycoplasma bovis from bovine milk and mucosal samples
Hayman, Bernadette, and Hirst, Robert (2003) Development of a semi-nested PCR for the improved detection of Mycoplasma bovis from bovine milk and mucosal samples. Veterinary Microbiology, 91 (2-3). pp. 91-100.
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A new forward primer, Mb-F, was designed to improve the sensitivity and reproducibility of the Mycoplasma bovis-specific PCR developed by Ghadersohi et al. [Vet. Microbiol. 56 (1997) 87] for testing clinical samples. A semi-nested (SN) PCR configuration was developed and this provided enhanced sensitivity and reproducibility. The detection limit of the SN PCR was in the range of 10–100 cfu/ml and the correct amplicon was amplified from 9.15 pg/μl of total extracted DNA (mixture of M. bovis and bovine cellular DNA). A dot blot assay was also developed and compared with the SN PCR on a number of randomly selected milk and mucosal samples. The dot blot had the same level of detection as the SN PCR. The specificity of the SN configuration was confirmed by Southern blot analysis and automated sequencing of the PCR product. The results from the tests on the samples from cattle, together with those from sheep, provided evidence that M. bovis is host-specific and that most cattle are colonised. The assay was shown to be specific, sensitive and reproducible and could be used successfully to detect M. bovis directly from clinical material without pre-enrichment.
|Item Type:||Article (Refereed Research - C1)|
|Keywords:||detection milk; diagnosis bacteria; mucus; Mycoplasma bovis; PCR|
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|Date Deposited:||30 Nov 2010 03:08|
|FoR Codes:||07 AGRICULTURAL AND VETERINARY SCIENCES > 0702 Animal Production > 070205 Animal Protection (Pests and Pathogens) @ 51%
07 AGRICULTURAL AND VETERINARY SCIENCES > 0799 Other Agricultural and Veterinary Sciences > 079999 Agricultural and Veterinary Sciences not elsewhere classified @ 49%
|SEO Codes:||83 ANIMAL PRODUCTION AND ANIMAL PRIMARY PRODUCTS > 8303 Livestock Raising > 830302 Dairy Cattle @ 100%|
|Citation Count from Web of Science||