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

Abstract

Mycoplasmas comprise a conglomerate of pathogens and commensals occurring in humans and animals. The genus Mycoplasma 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 tuf gene target regions. Following a PCR amplification and biotinylation step, hybridization on the array was shown to specifically identify 31 Mycoplasma spp., as well as 3 Acholeplasma spp. and 3 Ureaplasma spp. Members of the Mycoplasma mycoides cluster can be recognized at subgroup level. This procedure enables parallel detection of Mollicutes 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 Mycoplasma spp. This phenomenon deserves more attention, particularly its implications for epidemiology and treatment.

Validation of a mycoplasma molecular diagnostic test and distribution of mycoplasma species in bovine milk among New York State dairy farms

“…Rebelo et al (2011) reported a relatively rapid method that employed PCR amplification of ITS followed by high resolution melt analysis; however, this method only identified Mycoplasma and analysis is difficult. Recently, a matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry method for identification of ruminant Mycoplasma (Pereyre et al, 2013) and a rapid DNA microarray assay for identification of both Mycoplasma and Acholeplasma (Schnee et al, 2012) have been reported. However, both methods require specialized and expensive equipment, and the matrix-assisted laser desorption/ionization time-offlight mass spectrometry method is intended for a high density mycoplasma specimen, requiring additional culture.…”

Point prevalence of infection with Mycoplasma bovoculi and Moraxella spp. in cattle at different stages of infectious bovine keratoconjunctivitis

“…bovoculi was further verified in 14 and 18 randomly selected samples from herds 2 and 3, respectively, using a Mycoplasma spp. microarray assay (Schnee et al, 2012).…”

“…(Schnee et al, 2012), 12/14 were positive for Myc. bovoculi and 1/14 was positive for Ureaplasma diversum.…”

Comparison of PCR tests for the detection of Mycoplasma agalactiae in sheep and goats

“…One disadvantage of the PCR/ DGGE method is the cost of the specialised equipment and the specialist technical skills required to perform the test and analyse the results. As of yet, no other test has delivered similar sensitivity and flexibility in identifying and detecting a range of Mycoplasma species, although a microarray described by Schnee et al (19) may be the method of choice in the future.…”