J. Microbiol. Biotechnol. (2009), 19(3), 265–270 doi: 10.4014/jmb.0806.347 First published online 10 October 2008 Oligonucleotide Array-based Detection and Genotyping of Mollicutes (Acholeplasma, Mycoplasma, and Ureaplasma) Jang, Hyunjung1, Hyomyeung Kim1, Byeongchul Kang3, Cheolmin Kim1, 2, and Heekyung Park4* 1Institute for Genomic Medicine, GeneIn Co., Ltd, Busan 617-030, Korea 2Department of Biochemistry, Pusan National University Medical School, Busan 602-739, Korea 3Institute of Supportive Bioinformatics, Insilicogen Inc., Suwon 441-813, Korea 4PANAGENE Inc., 816 Tamnip-dong, Yuseong-gu, Daejeon 305-510, Korea Received: June 2, 2008 / Accepted: July 24, 2008 An oligonucleotide array was developed to detect and genotype Bacterial Strains and Preparation of Genomic DNAs mollicutes based on the internal transcribed spacer (ITS) Acholeplasma laidlawii ATCC 25937, Mycoplasma arginini sequence. The results of the assay were compared with ATCC 23838, Mycoplasma arthritidis ATCC 19611, those of a PCR-RFLP assay. The proposed oligonucleotide Mycoplasma bovis ATCC 27368, Mycoplasma cloacale array containing 5 genus- and 23 species-specific probes was ATCC 35276, Mycoplasma falconis ATCC 51372, Mycoplasma able to detect Mycoplasma species, including M. penetrans faucium ATCC 25293, Mycoplasma fermentans ATCC and M. spermatophilum, that were not detected by the PCR- 19989, Mycoplasma genitalium ATCC 33530, Mycoplasma RFLP assay. Therefore, the results demonstrated that hominis ATCC 23114, Mycoplasma hyorhinis ATCC 17981, the proposed oligonucleotide array was effective for the Mycoplasma hyosynoviae ATCC 25591, Mycoplasma detection and discrimination of 23 species, including an neurolyticum ATCC 19988, Mycoplasma opalescens ATCC acholeplasma, 21 mycoplasmas, and a ureaplasma, and 27921, Mycoplasma orale ATCC 23714, Mycoplasma showed promise as a countermeasure to ensure that biological penetrans ATCC 55252, Mycoplasma pirum ATCC 25960, products are safe and of good quality. Mycoplasma pneumoniae ATCC 15531, Mycoplasma Keywords: Oligonucleotide array, mollicute, internal transcribed primatum ATCC 15497, Mycoplasma salivarium ATCC 23064, spacer, genotyping Mycoplasma spermatophilum ATCC 49695, Mycoplasma synoviae ATCC 25204, and Ureaplasma urealyticum ATCC 27618 were all purchased from the American Type Culture Collection (ATCC), whereas the other bacteria, Bacillus The contamination of biological materials by mollicutes subtilis KCTC 1103, Lactobacillus casei KCTC 2180, (including Acholeplasma, Mycoplasma, and Ureaplasmas) Clostridium pasteurianum KCTC 1674, Escherichia coli can lead to unreliable experimental results and unsafe biological KCTC 1042, and Enterococcus faecalis KCTC 2011, were products. Mycoplasmas are phenotypically distinguished from obtained from the Korean Collection for Type Cultures other bacteria by their minute size and total lack of a cell (KCTC). The genomic DNAs were extracted using an wall. The methods for detecting pathogenic or contaminant InstaGene matrix kit (Bio-Rad Laboratories Inc., Hercules, mycoplasmas include microbiological cultures, immunological CA, U.S.A.) according to the manufacturer’s protocol. assays, DNA staining techniques, nucleic acid hybridization, and PCRs [4, 11]. Various laboratories have already developed PCR and Sequencing of ITS Regions species-specific PCR assays to detect mycoplasmas [12]. The primers used for the amplification of the ITS region of However, PCR assays can only detect a single or limited number the mycoplasmas (F2 and R2) and acholeplasmas (M78 and of mollicute species [5, 10]. Accordingly, this study analyzed R34) have already been described [10]. The PCR mixture a phylogeny tree of the ITS sequences of mollicutes and (50 µl) contained 1 U of Taq DNA polymerase, 1× Qiagen developed an oligonucleotide array that can genotype PCR buffer (Qiagen Inc., Valencia, CA, U.S.A.), and 20 pmol 23 mollicutes (an acholeplasma, 21 mycoplasmas, and a of each primer. The PCR conditions included 4 min at ureaplasma). 94oC; 30 cycles of 1 min at 94oC, 1 min at 55oC, and 1 min at 72oC; plus a further 10 min at 72oC. The PCR products *Corresponding author Phone: +82-42-936-9298; Fax: +82-42-861-9297; were sequenced after subcloning using a TA Cloning Kit E-mail: [email protected] (Promega, Madison, WI, U.S.A.), and compared with existing 266 Jang et al. sequences in the GenBank database. The ITSs of M. bovis, digestion with VspI (MBI Fermentas, Hanover, MD, U.S.A.), M. cloacale, M. falconis, M. faucium, M. spermatophilum, SspI and HindIII (New England BioLabs, Beverly, MA, and M. synoviae were newly sequenced in this study. U.S.A.), ClaI, DraI, and HinfI (DCC-BIONET, Korea), and the digested patterns determined by agarose gel electrophoresis. Phylogenetic Analysis The distances for each ITS were calculated using the PHYLIP Phylogenetic Relationships of Mycoplasmas Based on ITS package [2] and Kimura 2 parameter [4]. The phylogenetic Sequences trees were also constructed with the PHYLIP package using An acholeplasma, 21 mycoplasmas, and a ureaplasma were the neighbor-joining method [8]. The significance of the clustered in 5 groups based on the ITS sequences (Fig. 1). The resulting tree topologies was tested by performing a bootstrap overall ITS sequence similarity ranged from 32.0% to 99.0% analysis with resampling 1,000 times. (data not shown). The lowest similarity was 32.0% between M. hyorhinis and M. pirum, whereas the highest similarity was Oligonucleotide Array for Genotyping of Mollicutes 99% between M. arthritidis and M. faucium (data not shown). Genus- and species-specific probes were designed from the Therefore, these results indicated that the phylogenetic analysis conserved and polymorphic regions of the ITSs for using the ITS sequences was capable of distinguishing between Acholeplasma, Mycoplasma, and Ureaplasma, respectively. an acholeplasma, 21 mycoplasmas, and an ureaplasma, with The specificity of the probes was evaluated by testing using the exception of M. arthritidis and M. faucium. mollicutes and closely related bacteria (B. subtilis, Lb. casei, Cl. pasteurianum, E. coli, and En. faecalis). Genotyping of Mycoplasmas Using Oligonucletide Array: The fabrication, hybridization, and scanning processes of Specificity and Sensitivity the oligonucleotide array have been described previously [3, 6]. The ITS sequences of mollicutes contain both conserved and A schematic showing the layouts of the oligonucleotide polymorphic regions. Thus, the conserved regions in the ITS probes is shown in Figs. 2Y and 2Z. sequences of Acholeplasma and Mycoplasma were used to design several genus-specific probes. First, the optimal genus- PCR-Restriction Enzyme Analysis (PRA) specific probes were selected for 5 groups based on the ITS The unique restriction patterns predicted by the NEB cutter, sequences. The MP-CP probe was hybridized with the version 2.0 program (http://www.neb.com/rebase), were verified mycoplasmas in groups 1 and 3. The MP-CB probe was using the PCR products amplified with the F2 and R2 primers hybridized with the mycoplasmas in group 2. The MP-CE probe for the restriction enzyme analysis of the mollicutes. was hybridized with the mycoplasmas in groups 4 and 5. In Differentiation of the mollicute species was accomplished by addition, since there was no single genus-specific probe to Fig. 1. Phylogenic tree derived from ITS region sequences of mycoplasmas. The numbers on the tree are the percentages of occurrence in 1,000 bootstrapped trees; only values greater than 70% are shown. OLIGONUCLEOTIDE ARRAY TO DETECT MOLLICUTES 267 Fig. 2. Hybridization results for mycoplasma species when using an oligonucleotide array. A to W. Each species of mycoplasma exhibited a unique pattern of reactivity to each set of probes. X. Hybridization results for other pathogenic bacteria. Y. Image of QC probe before hybridization. Z. Layout of probes in the oligonucleotide array. The probes were patterned in duplicate. The probe names are as explained in Table 1. hybridize all the mycoplasmas, a combination of the four differentiation of an acholeplasma, 21 mycoplasmas, and a probes (MP-CP and MP-CB or MP-CP and MP-CE) was ureaplasma, yet was unable to differentiate M. arthritidis used to detect the mycoplasmas at the genus level. Finally, and M. faucium, owing to the 99% similarity of their the AP-CB probe was hybridized with the acholeplasma. respective ITS sequences. The oligonucleotide array also The presence of several polymorphic regions within the allowed for the identification of a number of mycoplasma ITS sequences enabled the design of individual species-specific species in one reaction. The genus-specific probes showed probes. The genus- and species-specific probes only detected a unique hybridization pattern with mycoplasmas (Figs. 2A their target, and there was no cross-hybridization with other to 2W) and a mismatch with other bacteria containing B. bacteria (Fig. 2). The oligonucleotide array allowed for the subtilis, Lb. casei, Cl. pasteurianum, E. coli, and En. faecalis, 268 Jang et al. Table 1. Probes designed in this study. Strain Probe Sequence Positionc a Acholeplasma AP-CB 5'-T15-AGTCTTTGAAAAGTAGATAAA-3' 134-154 d MP-CP 5'- T15-RWTCTTTVAAAACTRRATWN-3' 074-93 0 b d Mycoplasma MP-CB 5'- T15-GGKYAATTTGTTTWGAT-3' 028-44 0 e MP-CE 5'- T15-AATAAGTTACTAAGGGCTTAT-3' 201-221 M. arginini MP-arg 5'- T15-AGATTATATCATACAATAGA-3' 145-164 f M. arthritidis-faucium MP-arf 5'- T15-GAATACAAAATCAATACAATA-3' 146-166 M. cloacale MP-clo 5'-T15-AGTACAATTCTCACTGTTATG-3' 011-310