University of Veterinary and Animal Sciences, Pakistan
Posters & Accepted Abstracts: J Veterinar Sci Techno
Mycoplasma mastitis is always difficult to control due to lack of rapid and accurate diagnostic tool. The diagnostic methods available are mostly time consuming due to laborious culturing requirement, expensive, non-specific and less sensitive like biochemical tests and conventional PCR assay. A loop mediated isothermal amplification (LAMP) assay was developed for detection of Mycoplasma bovis directly from clinical mastitic milk samples. The LAMP assay was developed and validated on clinical samples obtained from M. bovis and other mastitis-causing pathogens detected by MALDI-TOF. Three different set of primers were used targeting different gene regions of M. bovis. The genes selected were UvrC, 16S rRNA and GyrB region. LAMP conditions were optimized for each of these and the efficiency, sensitivity and specificity of these LAMP primers were evaluated and compared. The result of 16S rRNA primers was more sensitive while GyrB primers were more specific. To confirm the specificity of the developed assay, other bacterial strains used were Mycoplasma agalactiae, Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis. No cross reactivity was observed in all of the primer sets. Results were also compared to conventional PCR assay with primers chosen from the same genes and confirmed by sequencing. For the evaluation of LAMP assay sensitivity, culture-positive milk samples were subjected to the assay. LAMP assay detected M. bovis in some of those milk samples which were PCR negative. In the present study we have developed, validated and evaluated LAMP assay for detection of M. bovis from mastitis milk samples. The assay is authentic, rapid and sensitive.
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