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. 2021 Apr 17;17(1):170.
doi: 10.1186/s12917-021-02870-5.

MALDI-TOF mass spectrometry and high-resolution melting PCR for the identification of Mycoplasma bovis isolates

Aric J McDaniel  1 , Rachel J Derscheid  2
Affiliations

MALDI-TOF mass spectrometry and high-resolution melting PCR for the identification of Mycoplasma bovis isolates

Aric J McDaniel et al. BMC Vet Res. .

Abstract

Background: Mycoplasma bovis is an important pathogen of cattle worldwide. Many different clinical manifestations of infection can occur, including respiratory disease, arthritis, and mastitis, causing heavy losses to beef and dairy industries. Because Mycoplasma species are slow-growing and fastidious, traditional identification methods are not cost- or time-effective, and improved methods are sought to streamline laboratory processes. High-resolution melting PCR (HRM-PCR) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) are 2 relatively recent tools that are rapid and inexpensive to use; we tested 9 isolates of M. bovis using both assays. The HRM-PCR assay used universal mycoplasma primers for the 16S-23S intergenic spacer region (IGSR).

Results: The resulting melting profiles of the field isolates were indistinguishable from the reference strain, indicating accurate identification. For the MALDI-TOF MS, each M. bovis isolate was accurately identified. Mycoplasma arginini and Mycoplasma alkalescens isolates did not identify as M. bovis when tested by either assay.

Conclusions: Our work shows that either assay could be used to identify unknown M. bovis isolates. For future work, the MALDI-TOF MS library should be expanded to include more mycoplasmas, and the HRM-PCR assay should be tested on additional mycoplasmas to ensure that the melting profiles are sufficiently distinctive.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Identification of Mycoplasma bovis isolates by high-resolution melting PCR. HRM-PCR was done in triplicate for each isolate. The PCR threshold was determined automatically by the Qiagen Rotor-Gene Q Series Software
Fig. 2
Fig. 2
Mean MALDI-TOF MS score of the four spots for each Mycoplasma bovis isolate. The Dunnett test was performed comparing the field isolates to B9 (gray), the reference strain. An asterisk indicates p ≤ 0.05
See this image and copyright information in PMC

References

    1. Maunsell FP, Woolums AR, Francoz D, Rosenbusch RF, Step DL, Wilson DJ, et al. Mycoplasma bovis infections in cattle. J Vet Intern Med. 2011;25(4):772–83. 10.1111/j.1939-1676.2011.0750.x. - PubMed
    1. Tully J. Methods in Mycoplasmology V2: diagnostic Mycoplasmology. New York; Academic Press, Inc; 2012. Elsevier Science.
    1. Register KB, Boatwright WD, Gesy KM, Thacker TC, Jelinski MD. Mistaken identity of an open reading frame proposed for PCR-based identification of mycoplasma bovis and the effect of polymorphisms and insertions on assay performance. J Vet Diagnostic Invest. 2018;30(4):637–641. doi: 10.1177/1040638718764799. - DOI - PMC - PubMed
    1. Holland RD, Wilkes JG, Rafii F, Sutherland JB, Persons CC, Voorhees KJ, et al. Rapid identification of intact whole bacteria based on spectral patterns using matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry. Rapid Commun Mass Spectrom. 1996;10(10):1227–32. 10.1002/(SICI)1097-0231(19960731)10:10<1227::AID-RCM659>3.0.CO;2-6. - PubMed
    1. Bernardo K, Pakulat N, Macht M, Krut O, Seifert H, Fleer S, et al. Identification and discrimination of Staphylococcus aureus strains using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Proteomics. 2002;2(6):747–53. 10.1002/1615-9861(200206)2:6<747::AID-PROT747>3.0.CO;2-V. - PubMed

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