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Updated: Aug 13, 2026
Optimized PCR-based Detection of Mycoplasma
Published on: June 20, 2011
A multiplex PCR to identify porcine mycoplasmas present in broth cultures
T Stakenborg1, J Vicca, P Butaye
1Veterinary and Agrochemical Research Centre, Groeselenberg 99, 1180, Brussels. tista@var.fgov.be
Abstract:
Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Mycoplasma flocculare can be present in the lungs of pigs at the same time. These three mycoplasma species all require similar growth conditions and can be recovered from clinical samples using the same media. We have developed a multiplex PCR as a helpful tool for rapid differentiation of these three species in the course of isolation. Based on the 16S ribosomal DNA sequences, three different forward primers and a single reverse primer were selected. Each forward primer was compared to available mycoplasma sequences, showing the primers to be specific. The three amplification products observed of 1129 bp (M. hyorhinis), 1000 bp (M. hyopneumoniae) and 754 bp (M. flocculare) were clearly distinguishable on a 1% agarose gel. In addition, no cross-reaction with Mycoplasma hyosynoviae, another porcine mycoplasma, was noted. This multiplex PCR using the proposed set of primers is the first reported assay that allows the simultaneous identification of the different Mycoplasma species isolated from the lungs of pigs.
Insights
A new multiplex PCR accurately identifies three common Mycoplasma species in pigs. This rapid diagnostic tool differentiates Mycoplasma hyopneumoniae, M. hyorhinis, and M. flocculare, aiding in swine respiratory disease management.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Swine Pathology
Background:
- Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, and Mycoplasma flocculare frequently co-infect swine lungs.
- These pathogens share similar growth requirements, complicating their individual identification during isolation.
- Accurate differentiation is crucial for effective diagnosis and treatment of porcine respiratory diseases.
Purpose of the Study:
- To develop a rapid and reliable method for simultaneously differentiating three common Mycoplasma species in pigs.
- To create a diagnostic tool for use in the course of bacterial isolation from clinical samples.
Main Methods:
- Development of a multiplex Polymerase Chain Reaction (PCR) assay.
- Selection of specific forward and a single reverse primers based on 16S ribosomal DNA sequences.
- Validation of primer specificity against known Mycoplasma sequences and clinical isolates.
Main Results:
- The multiplex PCR successfully amplified distinct DNA fragments for M. hyorhinis (1129 bp), M. hyopneumoniae (1000 bp), and M. flocculare (754 bp).
- Amplification products were clearly distinguishable on a 1% agarose gel.
- No cross-reactivity was observed with Mycoplasma hyosynoviae, another significant swine pathogen.
Conclusions:
- The developed multiplex PCR is the first assay enabling simultaneous identification of M. hyopneumoniae, M. hyorhinis, and M. flocculare from swine lung samples.
- This assay provides a valuable tool for rapid differentiation, improving diagnostic efficiency in swine respiratory disease investigations.
