Comparison and evaluation of two rapid diagnostic assays for the identification of Mycoplasma species

Members of the class Mollicutes are unique among bacteria in that they are unable to synthesize a cell wall. These organisms are also difficult to culture and very slow-growing with some species requiring up to 40 days to culture. Many of these bacteria are also significant pathogens in humans and animals, so rapid identification of isolates is a necessary diagnostic step. Many different tests are used for identification of pathogenic Mollicutes (primarily Mycoplasma and Ureaplasma species), however most tests are not cost or time efficient. Two relatively recent diagnostic tools are matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and high-resolution melting polymerase chain reaction (HRM-PCR). Nine isolates of Mycoplasma bovis, a pathogen in cattle, were grown and used first in a MALDI-TOF assay and then in an HRM-PCR assay utilizing universal primers for the 16S-23S rRNA intergenic spacer region (IGSR) of Mycoplasma species. The HRM-PCR assay compared the melt curve profile of the control isolate to the other eight for species determination. Both assays were able to successfully identify all nine isolates. Next, a new HRM-PCR assay also targeting the 16S-23S rRNA IGSR was developed and compared to the original assay. Reference and field strains of six Mycoplasma species were cultured along with reference strains for two additional Mycoplasma species and one Ureaplasma species. All of these isolates were tested in both the first (HRM-PCR-1) and second (HRM-PCR-2) HRM assay, and melt curve profiles of the field isolates were compared to the controls for identification. HRM-PCR-1 failed to amplify Mycoplasma gallisepticum and Ureaplasma diversum and was unable to identify the majority of Mycoplasma hyorhinis and Mycoplasma hyosynoviae isolates. HRM-PCR-2 identified the majority of M. hyorhinis and all Mycoplasma canis isolates, but failed to identify M. gallisepticum. All isolates of Mycoplasma bovis and Mycoplasma hyopneumoniae were successfully identified in both methods. HRM-PCR is a promising tool for identification, but more validation is necessary to provide consistent and accurate results. Future work should focus on expanding the MALDI-TOF database and testing different species in the HRM-PCR assays. Additionally, the use of HRM-PCR directly on tissue samples should be explored