DNA extraction from standard isolates
Standard isolates were provided by Dr. Higuchi, Rakuno Gakuen University, Hokkaido, Japan. Isolates were cultured in mycoplasma culture broth (Kanto Kagaku, Japan) at 37°C for 72 h. DNA was extracted by boiling the isolates and then adjusting the concentrations of the respective isolates as follows: 10−2 ng/μL of M. bovis (ATCC 25523), 0.6 ng/μL of M. arginini (ATCC 23838), 10−1 ng/μL of M. bovigenitalium, 10−3 ng/μL of M. bovirhinis (ATCC, 27748), 10−3 ng/μL of M. alkalescens (ATCC 29103), 10−3 ng/μL of M. canadense (ATCC 29418), 10−2 ng/μL of M. californicum, and 10−3 ng/μL of M. dispar (ATCC 27140). In this study, 10−1 ng/μL was equal to 0.76 pmol/tube. The DNA from all the positive control isolates were extracted according to the procedure for the AxyPrep™ Bacterial Genomic DNA Miniprep Kit (Corning Inc., USA).
Sensitivity test using bulk and mastitis milk samples
Bulk milk samples, either infected with (+) or not infected with (−) Mycoplasma bovis, and milk samples from cows with mastitis, which had been previously identified using primers designed by Bashiruddin, Frey et al. 2005, were collected from Taiwan dairy farms to evaluate the sensitivity of the new set of primers. Milk samples (1 mL) were centrifuged at 3,000 rpm for 10 min, and 100 μL of the liquid supernatant was collected. DNA from the milk samples were prepared according to the protocol described for the AxyPrep™ Bacterial Genomic DNA Miniprep Kit (Corning Inc.).
Highly specific primers were designed using NCBI’s Primer-BLAST based on the 16S rRNA gene sequences of M. bovis PG 45, M. arginini, M. bovigenitalium, M. californicum, M. alkalescens, M. canadense, M. dispar, and other Mycoplasma. species. The main sequence was chosen from the partial 16S rRNA sequence (bases 618 to 850) of M. bovisrhinis PG43 (Accession: LC158834.1), and the sequence was 90% to 99% homologous to the 16S rRNA sequence of M. bovis, between 316,750 bp and 316,976 bp (Fig. 1). The most similar part was then compared with the region with the highest dissimilarity in other bacteria and animal (including human) genomes, using the U.S. National Library of Medicine, BLAST® (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Finally, the size of the specific amplicon, based on Mycoplasma bovis 2093 (taxid: 28903, rr13 and rr14 genes, GenBank: KX462439.1), was 233 bp, and the chosen oligonucleotide sequences (5’–3’) for the newly designed universal primers were as follows:
Forward primer, 5’-TGT AGA GGT TAG CGG AAT TCC-3’; reverse primer, 5’-GAG CAT ACT ACT CAG GC-3’.
Optimization of PCR for Mycoplasma spp.
PCR was performed in a total reaction volume of 20 μL containing 2 μL of each forward and reverse primer (10 pmol), 1 μL of DDW, 10 μL of 2X Ampdirect solution (Shimadzu, Japan), and 5 μL of the DNA sample. The PCR conditions used were as follows: initial denaturation at 94°C for 7 min followed by 40 cycles of denaturation at 94°C for 1 min, annealing at 46°C for 40 seconds, extension at 72°C for 1 min, and then a final extension step of 72°C for 7 min. The PCR products were separated through electrophoresis on 2% (w/v) agarose gels, stained with ethidium bromide solution or with HealthyView™ Nucleic Acid Stain (JSB bio Inc., Taiwan), and then visualized with a UV transilluminator.
Limit of detection
To understand the limit of detection for each standard isolate, 10-fold series dilution was conducted for the DNA of each standard isolate. Then, 5 μL of each serial DNA dilution was used in PCR. The minimum concentration that could be detected for each amplicon was recorded as the limit of detection.
To test the primers’ specificity, DNA was extracted from Escherichia coli, Streptococcus uberis, Klebiella spp., Korucia rosea, Staphylococcus aureus, and coagulase-negative staphylococci, including Staphylococcus epidermitis, S, chromogenes, S. lugdunensis, S. xylous, and S. simulans. The DNA samples were then used as templates to test the specificity of the primer pair. Based on the sequence of the positive amplicons, this set of primers could not detect any species of Acholeplasma.