High-resolution melting curve analysis: a novel method for identification of Mycoplasma species isolated from clinical cases of bovine and porcine respiratory disease

  • Ania Ahani AzariEmail author
  • Reza Amanollahi
  • Razi Jafari Jozani
  • Darren J. Trott
  • Farhid Hemmatzadeh
Regular Articles


Mycoplasma species cause wide ranges of infectious diseases in human and animals. The aim of the present study was to evaluate a real-time polymerase chain reaction (RT-PCR) followed by a high-resolution melting curve assay (HRM) for rapid differentiation of Mycoplasma species isolated from clinical cases of bovine and porcine respiratory disease. Lung samples from suspected cases to respiratory infections from cows and pigs were cultured on specific media, and the extracted DNA were tested by conventional polymerase chain reaction (PCR) assays for Mycoplasma. A set of universal primers specific for the 16S ribosomal RNA gene was designed and used for RT-PCR and HRM. The HRM analysis was able to differentiate between five different species of Mycoplasmas, namely, M. hyopneumoniae, M. bovis, M. hyorhinis, M. hyosynoviae and other uncultured Mycoplasma. All results were confirmed based on 16S rRNA gene sequencing. This rapid and reliable assay was as a simple alternative to PCR and sequencing, differentiating bovine and porcine mycoplasmas in species level.


Mycoplasma Identification Bovine Porcine Respiratory disease 



The authors would like to express their special thanks to Andrea McWhorter and Abd Al-Bar Ahmed Noori Al-Farha for their contribution in Lab work.

Funding information

This study was financially supported by the Research council of Faculty of Biological sciences of Gorgan branch, Islamic Azad University and research branch of University of Adelaide.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Al-Farha, A., Petrovski, K., Jozani, Razi, Hoare A. and Hemmatzadeh, Farhid. (2018) Discrimination between some Mycoplasma spp. and Acholeplasma laidlawii in bovine milk using high resolution melting curve analysis. BMC Res Notes. 11:107.CrossRefGoogle Scholar
  2. Caswell, J. L. and Archambault M. (2008) Mycoplasma bovis pneumonia in cattle. Animal Health Research Reviews. 8(2); 161–186.CrossRefGoogle Scholar
  3. Ghorashi, S. A., Noormohammadi A. H. and Markham P. F. (2010) Differentiation of Mycoplasma gallisepticum strains using PCR and high-resolution melting curve analysis. Microbiology. 156, 1019–1029.CrossRefGoogle Scholar
  4. Hashemi, S., Mahzounieh, M., Sheikhi, N., Ebrahimi, A. (2018) Application of high-resolution melting-curve analysis on pvpA gene for detection and classification of Mycoplasma gallisepticum strains. Microbial Pathogenesis. 124,365–371.CrossRefGoogle Scholar
  5. Jeffery, N., Gasser, R. B., Steer P. A. and Noormohammadi A. H. (2007) Classification of Mycoplasma synoviae strains using single-strand conformation polymorphism and high-resolution melting-curve analysis of the vlhA gene single-copy region. Microbiology. 153, 2679–2688.CrossRefGoogle Scholar
  6. Landolt, P., Stephan, R. and Scherrer, S. (2019) Development of a new high resolution melting (HRM) assay for identification and differentiation of Mycobacterium tuberculosis complex samples. Scientific Reports 9, Article number: 1850.Google Scholar
  7. McAuliffe, L., Ellis R. J., Ayling R. D. and Nicholas, R. A. J. (2003) Differentiation of Mycoplasma species by 16S ribosomal DNA PCR and denaturing gradient gel electrophoresis fingerprinting. Journal of clinical Microbiology. p. 4844–4847.CrossRefGoogle Scholar
  8. McAuliffe, L., Kokotovic, B., Ayling, R.D. and Nicholas, R. A. J. (2004) Molecular epidemiological analysis of Mycoplasma bovis isolates from the United Kingdom shows two genetically distinct clusters. J. Clin. Microbial. 42: 4556–4565.CrossRefGoogle Scholar
  9. Miller, M., Zorn, J., Brielmeier, M. (2015) High-resolution melting curve analysis for identification of Pasteurellaceae species in experimental animal facilities. PLoS ONE 10(11): e0142560. Scholar
  10. Nicholas, R. A. J. (2004). Recent developments in the diagnosis and control of mycoplasma infections in cattle. Proceedings of the WBC Congress, Québec, Canada.Google Scholar
  11. Rebelo, A., Parker, L., Cai, H.Y. (2011) Use of high-resolution melting curve analysis to identify Mycoplasma species commonly isolated from ruminant, avian, and canine samples. Journal of Veterinary Diagnostic Investigation. 23(5) 932–936.CrossRefGoogle Scholar
  12. Reed, GH. , Kent, JO. , Wittwer, CT. (2007) High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics. 8(6):597.CrossRefGoogle Scholar
  13. Ren, X., Fu, Y., Xu, C., Feng, Z., Li, M., Zhang, L., Zhang, J., Liao, M. (2017) High resolution melting (HRM) analysis as a new tool for rapid identification of Salmonella enterica serovar Gallinarum biovars Pullorum and Gallinarum. Poult Sci. 96 (5):1088–93.PubMedGoogle Scholar
  14. Jakub Surýnek, Irena Vrtková, Aleš Knoll. (2016) Mycoplasma bovis was not detected in milk from dairy cattle in the Czech Republic. ACTA Universittatis Agriculturae et Silviculturae Mendelianae Brunensis. Volume 64 19 Number 1, p.165-168.Google Scholar
  15. Tong, SY. And Gifford, PM. (2012) Microbiological applications of high-resolution melting analysis. J Clin Microbial. 50 (11):3418–21.CrossRefGoogle Scholar
  16. Zahidi J. M., Yong T. B., Hashmi R., Noor A. M. Hamza, S. h., Ahmad N. (2015) Identification of Brucella spp. isolated from human brucellosis in Malaysia using high-resolution melt (HRM) analysis. Diagnostic Microbiology and Infectious Disease .81: 227–233.CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Microbiology, Gorgan branchIslamic Azad UniversityGorganIran
  2. 2.Department of Clinical Sciences, School of Veterinary MedicineShiraz UniversityShirazIran
  3. 3.Department of Clinical SciencesTabriz UniversityTabrizIran
  4. 4.Australian Centre for Antimicrobial Resistance EcologyThe University of AdelaideAdelaideAustralia
  5. 5.School of Animal and Veterinary Sciences, Roseworthy CampusThe University of AdelaideRoseworthyAustralia

Personalised recommendations