Development of multiplex polymerase chain reaction for the simultaneous detection of bovine mastitis-associated pathogens

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Pennapa Takam
Saengtong Pongjaroenkit
Thararat Chitov
Panwad Sillapawattana
Parkorn Limlenglert
Wasin Charerntantanakul


A multiplex polymerase chain reaction (mPCR) assay was developed for the simultaneous detection of five bovine mastitis-associated pathogens including Staphylococcus simulans, Staphylococcus hominis, Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus genus-specific group. Results showed that the optimal annealing temperature for mPCR was 58 °C. The sensitivity of mPCR to detect the least DNA template concentration was 0.625 ng. The nucleotide sequences of PCR products were identified using the NCBI database. Results showed 99.15-100% homology to the gap gene of Staphylococcus simulans, 100% homology to the peptidase gene of Staphylococcus hominis, 98.76-99.69% homology to the clumping factor A gene of Staphylococcus aureus, 99.09-99.64% homology to the CAMP factor gene of Streptococcus agalactiae, and 99.13% to the elongation factor tu gene of the Streptococcus genus-specific group. This developed mPCR technique showed potential as a rapid, specific, highly sensitive, and low-cost diagnostic tool for the simultaneous detection of bovine mastitis-associated pathogens.

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Takam, P., Pongjaroenkit, S., Chitov, T., Sillapawattana, P., Limlenglert, P., & Charerntantanakul, W. . (2024). Development of multiplex polymerase chain reaction for the simultaneous detection of bovine mastitis-associated pathogens: Veterinary Integrative Sciences, 23(1), 1–11. Retrieved from
Research Articles


Amin, A., Hamouda, R., Abdel-All, A.A., 2011. PCR assays for detecting major pathogens of mastitis in milk samples. World. J. Dairy. Food. Sci. 6(2), 199-206.

Ashraf, A., Imran, M., Yaqub, T., Tayyab, M., Shehzad, W., Thomson, P.C., 2017. A novel multiplex PCR assay for simultaneous detection of nine clinically significant bacterial pathogens associated with bovine mastitis. Mol. Cell. Probes. 33, 57-64.

Bleul, U., Sacher, K., Corti, S., Braun, U., 2006. Clinical findings in 56 cows with toxic mastitis. Vet. Rec. 159(20), 677-679.

Bramley, A., Cullor, L., Erskine, R., Fox, L., Harmon, R., Hogan, J., Nickerson, S., Oliver, S., Sordillo, L., 1996. Current concepts of bovine mastitis. National Mastitis Council, Madison, WI.

Chiang, Y.C., Pai, W.Y., Chen, C.Y., Tsen, H.Y., 2008. Use of primers based on the heat shock protein genes hsp70, hsp40, and hsp10, for the detection of bovine mastitis pathogens Streptococcus agalactiae, Streptococcus uberis, and Streptococcus bovis. Mol. Cell. Probes. 22(4), 262-266.

Chotar, M., Vidova, B., Godany, A., 2006. Development of specific and rapid detection of bacterial pathogens in dairy products by PCR. Folia. Microbiol. 51(6), 639-646.

El-Behiry, A., Elsayed, M., Marzouk, E., Bathich, Y., 2015. Detection of virulence genes in Staphylococcus aureus and Streptococcus agalactiae isolated from mastitis in the Middle East. Microbiol. Res. J. Int. 10(3), 1-9.

El-Sayed, A., Awad, W., Abdou, N.E., Vázquez, H.C., 2017. Molecular biological tools applied for identification of mastitis causing pathogens. Int. J. Vet. Sci. Med. 5(2), 89-97.

Gillespie, B., Oliver, S., 2005. Simultaneous detection of mastitis pathogens, Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae by multiplex real-time polymerase chain reaction. J. Dairy Sci. 88(10), 3510-3518.

González, R.N., Wilson, D.J., 2003. Mycoplasmal mastitis in dairy herds. Vet. Clin. North Am. Food Anim. Pract. 19(1), 199-221.

Gosiewski, T., Brzychczy Włoch, M., Heczko, P.B., 2012. The application of multiplex PCR to detect seven different DNA targets in group B streptococci. Folia. Microbiol. 57(3), 163-167.

Graber, H.U., Casey, M., Naskova, J., Steiner, A., Schaeren, W., 2007. Development of a highly sensitive and specific assay to detect Staphylococcus aureus in bovine mastitic milk. J. Dairy Sci. 90(10), 4661-4669.

Henegariu, O., Heerema, N., Dlouhy, S., Vance, G., Vogt, P., 1997. Multiplex PCR: critical parameters and step-by-step protocol. BioTechniques. 23(3), 504-511.

Hogeveen, H. 2005. Mastitis is an economic problem. In British Mastitis Conference 2005, Warwickshire, UK, 12 October 2005.

Kalin, R., Karahan, M., Acik, M.N., Tasdemir, B., Cetinkaya, B., 2017. Development of a multiplex PCR method for direct detection of common mastitis pathogens in bovine milk samples. Kafkas. Univ. Vet. Fak. Derg. 23(6), 925-931.

Kalorey, D.R., Shanmugam, Y., Kurkure, N.V., Chousalkar, K.K., Barbuddhe, S.B., 2007. PCR-based detection of genes encoding virulence determinants in Staphylococcus aureus from bovine subclinical mastitis cases. J. Vet. Sci. 8(2), 151-154.

Ke, D., Ménard, C., Picard, F.J., Boissinot, M., Ouellette, M., Roy, P.H., Bergeron, M.G., 2000. Development of conventional and real-time PCR assays for the rapid detection of group B streptococci. Clin. Chem. 46(3), 324-331.

Kim, C.H., Khan, M., Morin, D., Hurley, W., Tripathy, D., Kehrli Jr, M., Oluoch, A., Kakoma, I., 2001. Optimization of the PCR for detection of Staphylococcus aureus nuc gene in bovine milk. J. Dairy Sci. 84(1), 74-83.

Kuang, Y., Tani, K., Synnott, A.J., Ohshima, K., Higuchi, H., Nagahata, H., Tanji, Y., 2009. Characterization of bacterial population of raw milk from bovine mastitis by culture-independent PCR–DGGE method. Biochem. Eng. J. 45(1), 76-81.

Lane, D., 1991. 16S/23S rRNA sequencing. In: Stackebrandt, E., Goodfellow, M., (Eds.), Nucleic acid techniques in bacterial systematic, John Wiley and Sons, New York, pp. 115-175.

Lee, K.H., Lee, J.W., Wang, S.W., Liu, L.Y., Lee, M.F., Chuang, S.T., Shy, Y.M., Chang, C.L., Wu, M.C., Chi, C.H., 2008. Development of a novel biochip for rapid multiplex detection of seven mastitis-causing pathogens in bovine milk samples. J. Vet. Diagn. Invest. 20(4), 463-471.

Madico, G., Quinn, T.C., Boman, J., Gaydos, C.A., 2000. Touchdown enzyme time release-PCR for detection and identification of Chlamydia trachomatis, C. pneumoniae, and C. psittaci using the 16S and 16S-23S spacer rRNA genes. J. Clin. Microbiol. 38(3), 1085-1093.

Meiri Bendek, I., Lipkin, E., Friedmann, A., Leitner, G., Saran, A., Friedman, S., Kashi, Y. 2002. A PCR-based method for the detectionof Streptococcus agalactiae in milk. J. Dairy. Sci. 85(7), 1717-1723.

Menzies, F., McBride, S., McDowell, S., McCoy, M., McConnell, W., Bell, C., 2000. Clinical and laboratory findings in cases of toxic mastitis in cows in Northern Ireland. Vet. Rec. 147(5), 123-128.

Phuektes, P., Mansell, P., Browning, G., 2001. Multiplex polymerase chain reaction assay for simultaneous detection of Staphylococcus aureus and streptococcal causes of bovine mastitis. J. Dairy. Sci. 84(5), 1140-1148.

Picard, F.J., Ke, D., Boudreau, D.K., Boissinot, M., Huletsky, A., Richard, D., Ouellette, M., Roy, P.H., Bergeron, M.G., 2004. Use of tuf sequences for genus-specific PCR detection and phylogenetic analysis of 28 streptococcal species. J. Clin. Microbiol. 42(8), 3686-3695.

Podbielski, A., Blankenstein, O., Lütticken, R., 1994. Molecular characterization of the cfb gene encoding group B streptococcal CAMP-factor. Med. Microbiol. Immunol. 183(5), 239-256.

Radostits, O., Gay, C., Blood, D., Hinchcliff, K., 2000. Mastitis. In: Veterinary medicine, a textbook of the diseases of cattle, sheep, pigs, goats and horses, 9th edition. W.B. Saunders, London, pp. 611-613.

Riffon, R., Sayasith, K., Khalil, H., Dubreuil, P., Drolet, M., Lagacé, J., 2001. Development of a rapid and sensitive test for identification of major pathogens in bovine mastitis by PCR. J. Clin. Microbiol. 39(7), 2584-2589.

Shome, B., Das Mitra, S., Bhuvana, M., Krithiga, N., Velu, D., Shome, R., Isloor, S., Barbuddhe, S., Rahman, H., 2011. Multiplex PCR assay for species identification of bovine mastitis pathogens. J. Appl. Microbiol. 111(6), 1349-1356.

Smith, B., 1996. Large animal internal medicine. Mosby, USA, pp. 666-667.

Steele, N.M., 2015. Mastitis pathogen identification using polymerase chain reaction in New Zealand milk samples (Thesis). Massey University, Palmerston North, New Zealand.

Tran, H.D., Nguyen, T.N., 2023. Identification of prevalence and antibiotic resistance property as a basis for establishing an efficient treatment of bacteria causing mastitis in beef cows. Vet. Integr. Sci. 21(3), 783-797.

Vieira da Motta, O., Folly, M.M., Sakyiama, C.C.H., 2001. Detection of different Staphylococcus aureus strains in bovine milk from subclinical mastitis using PCR and routine techniques. Braz. J. Microbiol. 32, 27-31.

Zadoks, R.N., Middleton, J.R., McDougall, S., Katholm, J., Schukken, Y.H., 2011. Molecular epidemiology of mastitis pathogens of dairy cattle and comparative relevance to humans. J. Mammary. Gland. Biol. Neoplasia. 16(4), 357-372.