A Novel Rapid DNA Microarray Assay Enables Identification of 37 Mycoplasma Species and Highlights Multiple Mycoplasma Infections

Abstract

<div><p>Mycoplasmas comprise a conglomerate of pathogens and commensals occurring in humans and animals. The genus <i>Mycoplasma</i> alone contains more than 120 species at present, and new members are continuously being discovered. Therefore, it seems promising to use a single highly parallel detection assay rather than develop separate tests for each individual species. In this study, we have designed a DNA microarray carrying 70 oligonucleotide probes derived from the 23S rRNA gene and 86 probes from the <i>tuf</i> gene target regions. Following a PCR amplification and biotinylation step, hybridization on the array was shown to specifically identify 31 <i>Mycoplasma</i> spp., as well as 3 <i>Acholeplasma</i> spp. and 3 <i>Ureaplasma</i> spp. Members of the Mycoplasma mycoides cluster can be recognized at subgroup level. This procedure enables parallel detection of <i>Mollicutes</i> spp. occurring in humans, animals or cell culture, from mono- and multiple infections, in a single run. The main advantages of the microarray assay include ease of operation, rapidity, high information content, and affordability. The new test's analytical sensitivity is equivalent to that of real-time PCR and allows examination of field samples without the need for culture. When 60 field samples from ruminants and birds previously analyzed by denaturing-gradient gel electrophoresis (DGGE) were tested by the microarray assay both tests identified the same agent in 98.3% of the cases. Notably, microarray testing revealed an unexpectedly high proportion (35%) of multiple mycoplasma infections, i.e., substantially more than DGGE (15%). Two of the samples were found to contain four different <i>Mycoplasma</i> spp. This phenomenon deserves more attention, particularly its implications for epidemiology and treatment.</p></div