Development of real-time PCR assay for the detection of Mycoplasma bovis
Mycoplasma bovis is one of the important bovine mycoplasma involved in economically important clinical conditions like respiratory diseases, otitis media, and mastitis. The present study was undertaken with the objective of developing a SYBR Green dye-based real-time PCR assay targeting uvrC gene for the diagnosis of M. bovis. The analytical sensitivity and specificity of the assay were evaluated. The test showed 103-fold more sensitivity than conventional PCR and detected down to 100 fg level of DNA. It was found to be specific, as no cross reactivity was shown with other related bacteria and Mycoplasma species. The developed assay was able to detect down to 40 copies of uvrC gene from spiked bovine milk samples. At present, this developed assay may be used as a valuable diagnostic tool for the detection of Mycoplasma bovis.
KeywordsMycoplasma bovis uvrC gene Real-time PCR
The authors are thankful to the Director, IVRI, for providing necessary facilities to carry out the study.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
- Cremonesi, P., Vimercati, C., Pisoni, G., Perez, G., Miranda Ribera, A., Castiglioni, B., Luzzana, M., Ruffo, G. and Moroni, P., 2007. Development of DNA extraction and PCR amplification protocols for detection of Mycoplasma bovis directly from milk samples, Veterinary Research Communications, 31, 225–227.CrossRefPubMedGoogle Scholar
- Dua K., 2003. Comparative disease susceptibility of cattle and buffalo in Punjab (India). International Symposia on Veterinary Epidemiology and Economics proceedings, ISVEE 10: Proceedings of the 10th Symposium of the International Society for Veterinary Epidemiology and Economics, Clinical epidemiolgy session, Vina del Mar, p 92, Nov 2003.Google Scholar
- Eissa, S.I., Hassan, A.M., Hashem, Y.M. and Shaker, M.M., 2012. Comparative molecular study of Mycoplasma bovis isolates from Egyptian buffaloes and cows suffered from mastitis. Europian Journal of Biological Sciences, 4 (4): 114–120Google Scholar
- Fox, L. K., 2012. Mycoplasma mastitis causes, transmission, and control, Veterinary Clinics: Food and Animal Practices, 28, 225–237.Google Scholar
- Fu, J., Liu, Q., Xu, M., Shi, D., He, X., Pan, Y., Gou, R., Gao, Q., Yi, S. and Si, H., 2011. Seroprevalence of Mycoplasma bovis infection in dairy cows in subtropical southern China, African Journal of Biotechnology, 10, 1313–1316.Google Scholar
- Mustafa, R., Qi, J., Ba, X., Chen, Y., Hu, C., Liu, X., Tu, L., Peng, Q., Chen, H. and Guo, A., 2013. In vitro quinolones susceptibility analysis of Chinese Mycoplasma bovis isolates and their phylogenetic scenarios based upon QRDRs of DNA topoisomerases revealing a unique transition in ParC, Pakistan Veterinary Journal, 33, 364–369.Google Scholar
- Naikare, H., Bruno, D., Mahapatra, D., Reinisch, A., Raleigh, R. and Sprowls, R., 2015. Development and evaluation of a novel Taqman real-time PCR assay for rapid detection of Mycoplasma bovis: comparison of assay performance with a conventional PCR assay and another Taqman real-time PCR assay, Veterinary Science, 2, 32–42.CrossRefGoogle Scholar
- Szacawa, E., Szymańska-Czerwińska, M., Niemczuk, K., Dudek, K., Bednarek, D. and Ayling, R. D., 2016. Comparison of serological, molecular and cultural diagnostic methods for the detection of Mycoplasma bovis infections in cattle, Animal Science Papers and Reports, 34, 351–359.Google Scholar
- Yoshida, T., Deguchi, T., Ito, M., Maeda, S.I., Tamaki, M. and Ishiko, H., 2002. Quantitative detection of Mycoplasma genitalium from first-pass urine of men with urethritis and asymptomatic men by real-time PCR, Journal of Clinical Microbiology, 40, 1451–1455.CrossRefPubMedPubMedCentralGoogle Scholar
- Ziv, R., Rajala, P., 2012. The development of a dual target Mycoplasma bovis Taqman real-time PCR system for the rapid analysis of bovine semen. https://scholar.google.co.in/ assessed 10.09.2016.