A Multiplex PCR to Identify Porcine Mycoplasmas Present in Broth Cultures

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.

Multilocus Sequence Typing of Mycoplasma hyorhinis Strains Identified by a Real-Time TaqMan PCR Assay

“…or from samples collected from live animals (nasal cavities, tonsils, tracheal and bronchiolar mucus, oral fluids), but isolation and identification are tedious and time-consuming. The difficulty in culturing M. hyorhinis has led to the development of other diagnostic assays: three PCR tests were developed to detect this Mycoplasma species in pure cultures (15)(16)(17). In our study, a new PCR test-a real-time TaqMan PCR (qPCR)-was developed to rapidly detect small quantities of M. hyorhinis.…”

“…The difficulty in culturing M. hyorhinis has led to the development of other diagnostic assays. Several specific endpoint PCR tests were developed to detect M. hyorhinis in pure cultures or in cell cultures (15)(16)(17). The detection thresholds of these gel-based PCR tests varied from 1,000 to 10,000 microorganisms per reaction.…”

Mycoplasma hyorhinis infection in early cases of mycoplasmal pneumonia in swine and evaluation of diagnostic assays

“…For the detection of the Mycoplasmas, the initiator oligonucleotides that amplify the 16s portion of the rRNA were used: 5'-CGGGATGTAGCAATACTTCAG-3' for M. hyorhinis and 5'-TTCAAAGGAGCCTTCAAGCTTC-3' for M. hyopneumoniae and a reverse oligonucleotide 5'-AGAGGCATGATTTGACGTC-3', common to all the initiators present in the reaction. These initiators am-plified a fragment of 1129 base pairs (bp) for M. hyorhinis and 1000 bp for M. hyopneumoniae within the coding region of the 16S rRNA gene (Stakenborg et al 2006). Total DNA was subjected to PCR using a final volume of 25 µL.…”

“…The mixture was dispensed into 200 µL microtubes and mixed slowly. The PCR reactions were performed in a PXE 0.2 THERMO (Thermo Electron Corporation) thermocycler with an initial temperature of 94 °C for 1 minute, followed by 30 cycles of 30 seconds at 94 °C, 15 seconds at 54.6°C, and 1 minute at 68°C with an extension of 10 minutes at 72°C (Stakenborg et al 2006).…”

“…M. hyorhinis). Therefore, the search for diagnostic methods with high sensitivity such as PCR is an interesting option (Calsamiglia et al 1999, Stakenborg et al 2006. Alternatively, other techniques can be used such as immunohistochemistry and, more recently, fluorescence in situ hybridization (FISH).…”