Folia Microbiologica

, Volume 63, Issue 3, pp 373–380 | Cite as

Development and validation of a loop-mediated isothermal amplification assay for the detection of Mycoplasma bovis in mastitic milk

  • Aqeela Ashraf
  • Muhammad Imran
  • Tahir Yaqub
  • Muhammad Tayyab
  • Wasim Shehzad
  • Claro N. Mingala
  • Yung-Fu Chang
Original Article


Mycoplasma mastitis is often difficult to control due to a lack of rapid and accurate diagnostic tools. The aim of the current study was to develop a loop-mediated isothermal amplification (LAMP) assay for the detection of Mycoplasma bovis (M. bovis) in mastitic milk. The assay was developed using primers designed for three different target genes: uvrC, 16S rRNA, and gyrB, and validated using mastitic milk samples previously found positive for the target pathogen. Specificity of the developed assay was determined by testing cross-reactivity of LAMP primers against closely related bovine mastitis bacterial pathogens. The sensitivity was found to be higher compared to conventional polymerase chain reaction (PCR). The LAMP assay was also capable of detecting M. bovis in PCR-negative milk samples of cows with clinical mastitis. The uvrC primers were found to be more sensitive, while gyrB primers were more specific; however, 16S rRNA primers were less specific and sensitive compared to either uvrC or gyrB primers. Cohen’s kappa values for uvrC, gyrB, and 16S rRNA primers used in the LAMP assays were 0.940, 0.970, and 0.807, respectively. There was a high level of agreement between the test results and the true-disease status as indicated by the receiver operating characteristic (ROC) curve. Our findings suggest that the newly developed LAMP assays targeting the uvrC and gyrB genes could be a useful tool for rapid and accurate diagnosis of mastitis caused by M. bovis.



We are thankful to Dr. Peter Thomson, School of Life and Environmental Sciences, The University of Sydney, Australia, for providing assistance in statistical analysis. Special thanks to Quality Milk Production Services (QMP), Cornell University, Ithaca, NY, USA, for providing standard bacterial strains and mastitic milk samples.

Author contributions

AA carried out the study and wrote the manuscript; MI, MT, WS, TY, and CNM conceived and designed the experiments; MI and YFC supervised the work; MI and WS did proofreading of the paper; all the authors read and approved the final manuscript.


This work was supported by the Higher Education Commission of Pakistan under the International Research Support Initiative Program (IRSIP) grant #266.22.

Compliance with ethical standards

Competing interests

The authors declare that they have no competing interests.


  1. Ashraf A, Imran M, Yaqub T, Tayyab M, Shehzad W, Thomson PC (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. CrossRefPubMedGoogle Scholar
  2. Bai Z, Shi L, Hu C, Chen X, Qi J, Ba X, Peng Q, Chen Y, Chen H, Guo A (2011) Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of Mycoplasma bovis. Afr J Biotechnol 10:12333–12338. Google Scholar
  3. Boonyayatra S, Fox LK, Besser TE, Sawant A, Gay JM, Raviv Z (2012a) A PCR assay and PCR-restriction fragment length polymorphism combination identifying the 3 primary Mycoplasma species causing mastitis. J Dairy Sci 95(1):196–205. CrossRefPubMedGoogle Scholar
  4. Boonyayatra S, Fox LK, Gay JM, Sawant A, Besser TE (2012b) Discrimination between Mycoplasma and Acholeplasma species of bovine origin using digitonin disc diffusion assay, nisin disc diffusion assay, and conventional polymerase chain reaction. J Vet Diagn Investig 24(1):7–13. CrossRefGoogle Scholar
  5. Bosward KL, House JK, Deveridge A, Mathews K, Sheehy PA (2016) Development of a loop-mediated isothermal amplification assay for the detection of Streptococcus agalactiae in bovine milk. J Dairy Sci 99(3):2142–2150. CrossRefPubMedGoogle Scholar
  6. Cai HY, Bell-Rogers P, Parker L, Prescott JF (2005) Development of a real-time PCR for detection of Mycoplasma bovis in bovine milk and lung samples. J Vet Diagn Investig 17(6):537–545. CrossRefGoogle Scholar
  7. Centeno-Cuadros A, Abbasi I, Nathan R (2016) Sex determination in the wild: a field application of loop-mediated isothermal amplification successfully determines sex across three raptor species. Mol Ecol Resour 17(2):153–160. CrossRefPubMedGoogle Scholar
  8. Foddai A, Idini G, Fusco M, Rosa N, De La Fe C, Zinellu S, Corona L, Tola S (2005) Rapid differential diagnosis of Mycoplasma agalactiae and Mycoplasma bovis based on a multiplex-PCR and a PCR-RFLP. Mol Cell Probes 19(3):207–212. CrossRefPubMedGoogle Scholar
  9. Fowler VL, Howson ELA, Madi M, Mioulet V, Caiusi C, Pauszek SJ, Rodriguez LL, King DP (2016) Development of a reverse transcription loop-mediated isothermal amplification assay for the detection of vesicular stomatitis New Jersey virus: use of rapid molecular assays to differentiate between vesicular disease viruses. J Virol Methods 234:123–131. CrossRefPubMedGoogle Scholar
  10. Foysal MJ, Rahman MM, Prodhan MSH (2013) PCR based molecular detection of the gyr-B-2 gene from the Klebsiella Sp. isolates from patients who were suffering with pneumonia and urinary tract infections (UTIs). J Clin Diagn Res 7:23–25. PubMedPubMedCentralGoogle Scholar
  11. Fu S, Qu G, Guo S, Ma L, Zhang N, Zhang S, Gao S, Shen Z (2011) Applications of loop-mediated isothermal DNA amplification. Appl Biochem Biotechnol 163(7):845–850. CrossRefPubMedGoogle Scholar
  12. Fukuta S, Iida T, Mizukami Y, Ishida A, Ueda J, Kanbe M, Ishimoto Y (2003) Detection of Japanese yam mosaic virus by RT-LAMP. Arch Virol 148(9):1713–1720. CrossRefPubMedGoogle Scholar
  13. Gioia G, Werner B, Nydam DVD, Moroni P (2016) Validation of a mycoplasma molecular diagnostic test and distribution of mycoplasma species in bovine milk among New York State dairy farms. J Dairy Sci 99(6):4668–4677. CrossRefPubMedGoogle Scholar
  14. Greiner M, Pfeiffer D, Smith RD (2000) Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests. Prev Vet Med 45(1-2):23–41. CrossRefPubMedGoogle Scholar
  15. Higa Y, Uemura R, Yamazaki W, Goto S, Goto Y, Sueyoshi M (2016) An improved loop-mediated isothermal amplification assay for the detection of Mycoplasma bovis. J Vet Med Sci 78(8):1343–1346. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Kimura Y, de Hoon MJL, Aoki S, Ishizu Y, Daub CO, Lezhava A, Arner E, Hayashizaki Y, Kawai Y, Kogo Y (2011) Optimization of turn-back primers in isothermal amplification. Nucleic Acids Res 39(9):e59. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Kurosaki Y, Sakuma T, Fukuma A, Fujinami Y, Kawamoto K, Kamo N, Makino SI, Yasuda J (2009) A simple and sensitive method for detection of Bacillus anthracis by loop-mediated isothermal amplification . J Appl Microbiol 107(6):1947−1956.
  18. Le TH, Nguyen NT, Truong NH, Van De N (2012) Development of mitochondrial loop-mediated isothermal amplification for detection of the small liver fluke Opisthorchis viverrini (Opisthorchiidae; Trematoda; Platyhelminthes). J Clin Microbiol 1(50):1178–1184CrossRefGoogle Scholar
  19. Liu A, Wang C, Liang Z, Zhou ZW, Wang L, Ma Q, Wang G, Zhou SF, Wang Z (2015) High-throughput sequencing of 16S rDNA amplicons characterizes bacterial composition in cerebrospinal fluid samples from patients with purulent meningitis. Drug Des Devel Ther 9:4417–4429. PubMedPubMedCentralGoogle Scholar
  20. Maunsell FP, Woolums AR, Francoz D, Rosenbusch RF, Step DL, Wilson DJ, Janzen ED (2011) Mycoplasma bovis infections in cattle. J Vet Intern Med 25(4):772–783. CrossRefPubMedGoogle Scholar
  21. Modi M, Sharma K, Sharma M, Sharma A, Sharma N, Sharma S, Ray P, Varma S (2016) Multi targeted loop-mediated isothermal amplification for rapid diagnosis of tuberculous meningitis. Int J Tuberc Lung Dis 20(5):625–630. CrossRefPubMedGoogle Scholar
  22. Nagamine K, Hase T, Notomi T (2002) Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 16(3):223–229. CrossRefPubMedGoogle Scholar
  23. Nicholas RAJ, Ayling RD (2003) Mycoplasma bovis: disease, diagnosis, and control. Res Vet Sci 74(2):105–112. CrossRefPubMedGoogle Scholar
  24. Nitiss JL (2009) DNA topoisomerase II and its growing repertoire of biological functions. Mol Pharmacol 9(5):327–337. Google Scholar
  25. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:e63. 12, 63e, 663CrossRefPubMedPubMedCentralGoogle Scholar
  26. Parida MM, Sannarangaiah S, Dash PK, Rao PVL, Morita K (2008) Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. Rev Med Virol 18(6):407–421. CrossRefPubMedGoogle Scholar
  27. Subramaniam S, Bergonier D, Poumarat F, Capaul S, Schlatter Y, Nicolet J, Frey J (1998) Species identification of Mycoplasma bovis and Mycoplasma agalactiae based on the uvrC genes by PCR. Mol Cell Probes 12(3):161–169. CrossRefPubMedGoogle Scholar
  28. R Core Team (2013). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna.
  29. Thomas A, Dizier I, Linden A, Mainil J, Frey J, Vilei EM (2004) Conservation of the uvrC gene sequence in Mycoplasma bovis and its use in routine PCR diagnosis. Vet J 168(1):100–102. CrossRefPubMedGoogle Scholar
  30. Tie Z, Chunguang W, Xiaoyuan W, Xinghua Z, Xiuhui Z (2012) Loop-mediated isothermal amplification for detection of Staphylococcus aureus in dairy cow suffering from mastitis. J Biomed Biotechnol 2012:435982–435985. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Vidic J, Manzano M, Chang CM, Jaffrezic-Renault N (2017) Advanced biosensors for detection of pathogens related to livestock and poultry. Vet Res 48(1):11.
  32. Wang DG, Brewster JD, Paul M, Tomasula PM (2015) Two methods for increased specificity and sensitivity in loop-mediated isothermal amplification. Molecules 20(4):6048–6059.
  33. Wang J, Zhang Y, Cui Y, Yan Y, Wang X, Wang R, Jian F, Zhang L, Ning C (2017) A rapid, simple and sensitive loop-mediated isothermal amplification method to detect Anaplasma bovis in sheep and goats samples. Parasitol Int 16(1):30568–305672. Google Scholar

Copyright information

© Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2017

Authors and Affiliations

  • Aqeela Ashraf
    • 1
    • 2
  • Muhammad Imran
    • 2
  • Tahir Yaqub
    • 3
  • Muhammad Tayyab
    • 2
  • Wasim Shehzad
    • 2
  • Claro N. Mingala
    • 1
    • 4
  • Yung-Fu Chang
    • 1
  1. 1.Department of Population Medicine and Diagnostic Sciences, College of Veterinary MedicineCornell UniversityIthacaUSA
  2. 2.Institute of Biochemistry and BiotechnologyUniversity of Veterinary and Animal SciencesLahorePakistan
  3. 3.Department of MicrobiologyUniversity of Veterinary and Animal SciencesLahorePakistan
  4. 4.Philippine Carabao Center National Headquarters and Gene PoolScience City of MunozPhilippines

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