- Email: [email protected]
Discrimination between Mycoplasma mycoides subsp. capri and Mycoplasma capricolum subsp. capricolum using PCR-RFLP and PCR
The Veterinary Journal 205 (2015) 421–423
Contents lists available at ScienceDirect
The Veterinary Journal j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t v j l
Short Communication
Discrimination between Mycoplasma mycoides subsp. capri and Mycoplasma capricolum subsp. capricolum using PCR-RFLP and PCR Grazia Cillara, Maria Giovanna Manca, Carla Longheu, Sebastiana Tola * Istituto Zooprofilattico Sperimentale della Sardegna G. Pegreffi, 07100 Sassari, Italy
A R T I C L E
I N F O
Article history: Accepted 17 May 2015 Keywords: Contagious agalactia Dihydrolipoyl dehydrogenase Mycoplasma mycoides subsp. capri Mycoplasma capricolum subsp. capricolum PCR-RFLP PCR
A B S T R A C T
In this study, the dihydrolipoyl dehydrogenase (lpdA) gene was used to distinguish Mycoplasma mycoides subsp. capri (Mmc) from Mycoplasma capricolum subsp. capricolum (Mcc), two of four Mycoplasma species that cause contagious agalactia in sheep and goats. After alignment of nucleotide sequences of both species, specific primer sets were designed from unchanging and variable gene segments. The first primer set LPD-C1-F/LPD-C1-R was used to amplify a 911 bp fragment that was subsequently co-digested with FastDigest PstI, SspI, EcoRI and ClaI enzymes. The PCR-RFLP profiles differentiated the two mycoplasma species. The second primer set was used to distinguish Mmc from Mcc by single tube PCR. Both methods were further applied to identify 54 isolates collected from dairy herds from different provinces in Sardinia. The results of this study showed that PCR-RFLP and PCR could be used in routine diagnosis for rapid and specific simultaneous discrimination of Mmc and Mcc. © 2015 Elsevier Ltd. All rights reserved.
Contagious agalactia (CA) is the most serious disease affecting small dairy ruminants (Corrales et al., 2007). CA is caused by four species of Mycoplasma, namely, M. agalactiae (the classic agent of the disease in sheep and goats), M. mycoides subsp. capri (Mmc), M. capricolum subsp. capricolum (Mcc) and M. putrefaciens. Among the four Mycoplasma species, Mmc and Mcc phylogenetically belong to the ‘M. mycoides’ cluster. Because of their importance in veterinary medicine (Gomez-Martin et al., 2013), it is important that specific and rapid diagnostic procedures are developed for their detection and discrimination. Unfortunately, PCR assays based on the amplification of housekeeping genes detect both Mmc and Mcc, but also detect all three other species within the ‘M. mycoides’ cluster (Le Grand et al., 2004; Woubit et al., 2007; Becker et al., 2012). Recently, we characterized Mmc isolates collected in Sardinia from contagious agalactia outbreaks using pulse-field gel electrophoresis (PFGE) and identified their immunogenic proteins using a proteomic approach (Corona et al., 2013a and b). In the present study, the dihydrolipoyl dehydrogenase (lpdA) gene was chosen as the target gene for differentiation of Mcc from Mmc because of the presence of polymorphic sites in the DNA sequence. Polymorphic sites can be highlighted by the analysis of PCR fragments with enzymes that detect interspecific restriction fragment length polymorphism (RFLP). Furthermore, a single and rapid PCR method, based on use of a primer set selected in variation sites of lpdA gene, was proposed. During the period 2004–2014, 54 mycoplasma isolates belonging to the ‘M. mycoides’ cluster were collected from dairy herds in
* Corresponding author. Tel.: +39 079 2892339. E-mail address: [email protected] (S. Tola). http://dx.doi.org/10.1016/j.tvjl.2015.05.013 1090-0233/© 2015 Elsevier Ltd. All rights reserved.
different provinces of Sardinia (see Appendix: Supplementary Table S1). All mycoplasma isolates and reference strains were grown at 37 °C in modified Hayflick medium supplemented with 8% equine serum. Genomic DNA was extracted and purified by standard protocols (Ausubel et al., 2013). In order to determine whether isolates belonged to the ‘M. mycoides’ cluster, isolates were first amplified by PCR using primers FusA-F and FusA-R for fusA gene (Table 1). lpdA gene sequences from Mmc strain GM12 (NCBI, nucleotide ID 256385136) and Mcc strain ATCC 27343 (NCBI, nucleotide ID 83319253:275328–277217) were aligned using the EMBOSS pairwise alignment algorithm.1 The primers LPD-C1-F and LPD-C1-R (Table 1) were selected based on the conserved region of the lpdA gene between the two Mycoplasma spp. PCR reactions were conducted in a DNA thermal cycler (GeneAmp 9700, Applied Biosystems). PCR products were purified and submitted to BMR Genomics2 for sequencing. The sequences of the lpdA gene from reference strains and isolates were compared with those of the GenBank database by using the BLASTN local alignment search tool.3 Enzymes and lengths of resulting restriction fragments were predicted with an on-line program.4 Twenty microlitres of PCR amplification were digested in a 40 μL volume containing 1× FastDigest Green buffer supplemented with tracking dyes, 1 μL of 20 mg/mL acetylated BSA
1 See: http://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html (accessed 17 May 2015). 2 See: http://www.bmr-genomics.it/bmr_it/BMR_home.html (accessed 17 May 2015). 3 See: http://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed 17 May 2015). 4 See: http://www.restrictionmapper.org (accessed 17 May 2015).
422
G. Cillara et al./The Veterinary Journal 205 (2015) 421–423
M
Table 1 Oligonucleotide primers used in this study. Target fusA
lpdA
lpdA
Sequence
Fragment size (bp)
FusA-F: 5′-TGAAATTTTTAGATGGTGGAGAA-3′ FusA-R: 5′-GGTAATTTAATAGTTTCACGATATGAA-3′ LPD-C1-F: 5′-AGGTGAAGCTGTTGCTTTAG-3′ LPD-C1-R: 5′-TTCCAATAATATGTGCACCTAA-3′ LPD-F: 5′-CGATGGAAAAGATCAAATGG-3′ LPD-R: 5′-TTGTTGTTCAGTTTTTCCT-3′
1
2
3
4
5
781
Manso-Silvan et al., 2007
911
This study
362/281
This study
6
2
3
4
5
M
Reference
(Ambion) and 1 μL of FastDigest PstI, SspI, EcoRI and ClaI enzymes (Thermo Scientific). Reaction mixtures were incubated at 37 °C for 15 min then directly loaded onto the gels. At the same time, 10 μL of digested fragments from lpdA gene was examined by electrophoresis in 2% agarose gel, containing 1% agarose for routine use (Sigma) and 1% agarose low melting point (Sigma). We tried to find a rapid and simple one-step PCR to differentiate Mmc strain GM12 from Mcc ATCC 27343. The primers LPD-F and LPD-R (Table 1) were selected from the variation sites of the lpdA gene. The lpdA gene was used as the target gene to distinguish Mmc from Mcc. The primer set for the amplification of both Mmc and Mcc (LPD-C1-F and LPD-C1-R) was designed for the conserved regions of the lpdA gene after preliminary sequence analysis. Additionally, type and reference strains were included in this study: M. agalactiae PG2T, M. putrefaciens KS1T, M. capricolum subsp. capripneumoniae F38, M. bovine group 7 PG50 and M. mycoides PGT. As shown in Fig. 1, amplicons of 911 bp were obtained from all type and reference strains except M. agalactiae PG2T and M. putrefaciens KS1T. PCR products of 911 bp were digested with PstI, SspI, EcoRI and ClaI restriction endonucleases and analyzed by SDS-PAGE electrophoresis. RFLP profiles allowed differentiation of all reference strains analyzed except M. mycoides PGT and M. bovine group 7 PG50 (Fig. 2). A detailed comparison of restriction maps identified that only two restriction enzymes, ClaI and EcoRI, produced distinct speciesspecific restriction profiles to reliably distinguish Mcc CKT from M. mycoides subsp. mycoides LC reference strain whereas PstI and SspI produced the same fragments in both species (see Appendix: Supplementary Table S2). PCR-RFLP assay was extended to the 54 field isolates included in this study and profiles were analyzed by both SDS-PAGE (Fig. 3A) and agarose gels (Fig. 3B). Thirty-eight of
M
1
7
M
Fig. 2. Restriction fragment length polymorphism (RFLP) patterns of PCR products from the lpdA gene of five Mycoplasma reference strains after digestion with PstI, SspI, EcoRI and ClaI enzymes for 15 min at 37 °C. Twenty microlitres of digested lpdA amplifications were loaded in 12% (wt/vol) polyacrylamide gels containing 0.1% sodium dodecyl sulfate (SDS) then electrophoresed in a Mini Protean Tetra Cell (Bio-Rad) containing 500 mL of running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS; pH 8.3) at 200 V for 40 min. After electrophoresis gels were stained with SYBR Gold (Invitrogen). Type and reference strains: Lane 1, M. mycoides PGT; lane 2, M. bovine group 7 PG50; lane 3, M. capricolum subsp. capripneumoniae F38; lane 4, M. capricolum subsp. capricolum CKT; lane 5, M. mycoides subsp. mycoides LC type strain. Lane M, marker VIII (Roche).
the 54 isolates had a fingerprint identical to M. mycoides subsp. mycoides LC reference strain while 16 had a profile identical to Mcc CKT (see Appendix: Supplementary Table S3). Two Mmc isolates (3866 and 9500) showed a PCR-RFLP profile slightly different from
M 1 2
3
4
5 6
7 8
9 10 11 12 13 M
A
B Fig. 1. PCR profile of the dihydrolipoyl dehydrogenase (lpdA) gene obtained from seven Mycoplasma reference strains. Amplification conditions were initial denaturation for 5 min at 95 °C followed by 30 cycles of 1 min at 95 °C, 1 min at 56 °C, and 1.5 min at 72 °C with a final extension step of 10 min at 72 °C. Amplicons were resolved by electrophoresis in 1% agarose gels, stained with SybrSafe DNA gel stain (Invitrogen). Type and reference strains: Lane 1, M. mycoides subsp. mycoides LC type; lane 2, M. capricolum subsp. capricolum CKT; lane 3, M. mycoides PGT; lane 4, M. bovine group 7 PG50; lane 5, M. capricolum subsp. capripneumoniae F38; lane 6, M. agalactiae PG2T and lane 7, M. putrefaciens KS1T. Lane M, marker VIII (Roche).
Fig. 3. Restriction fragment length polymorphism (RFLP) patterns of PCR products from the lpdA gene of 11 field isolates and reference strains after digestion with PstI, SspI, EcoRI and ClaI enzymes. Panel A: fragments were separated by 12% SDS-PAGE gel. Panel B: fragments were separated by 2% agarose gel. Isolates 55094 (lane 1), 74751 (lane 2), 11256 (lane 3), 26909 (lane 4), 41071 (lane 5), 61269 (lane 6), 3866 (lane 7), 9862 (lane 8), 13127 (lane 9), 18622 (lane 10) and 19002 (lane 11). M. mycoides subsp. mycoides LC type strain (lane 12) and M. capricolum subsp. capricolum CKT (lane 13). Lane M, marker VIII (Roche).
G. Cillara et al./The Veterinary Journal 205 (2015) 421–423
M 1
2
3 4
5 6
7
8
9 10 11 12 13 M
423
Acknowledgments The study was supported by the Istituto Zooprofilattico Sperimentale of Sardinia. Appendix: Supplementary material
Fig. 4. PCR amplification of 11 field isolates and reference strains using the primers set LPD-F/LPD-R. The amplification conditions used in this study were as follows: initial denaturation at 95 °C for 5 min followed by 30 cycles of 1 min at 95 °C, 1 min at 50 °C, and 1 min at 72 °C with a final 10 min extension step at 72 °C. The amplified samples were subjected to electrophoresis on 2% agarose gel. Isolates 55094 (lane 1), 74751 (lane 2), 11256 (lane 3), 26909 (lane 4), 41071 (lane 5), 61269 (lane 6), 3866 (lane 7), 9862 (lane 8), 13127 (lane 9), 18622 (lane 10) and 19002 (lane 11). M. mycoides subsp. mycoides LC type strain (lane 12) and M. capricolum subsp. capricolum CKT (lane 13). Lane M, marker VIII (Roche).
the type strain (Fig. 3, lane 7). On DNA sequencing of 911 bp products another SspI restriction site was identified in these isolates. All field isolates and type strains were additionally tested for amplification with a new primer set LPD-F and LPD-R, selected from polymorphic sites of the lpdA gene. The PCR was developed to allow the simultaneous detection of Mmc and Mcc in a single reaction, making it more applicable for routine diagnostic use. As shown in Fig. 4, a 362 bp fragment from Mmc isolates and a doublet (362 and 281 bp) from Mcc isolates were obtained. The results were reproducible and stable over time. This study has shown that the lpdA gene can be used for the correct identification of both Mmc and Mcc. An RFLP-based approach and single-tube PCR assay provide a relatively simple and cost-effective method for differential diagnosis of Mmc and Mcc. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of this paper.
Supplementary data to this article can be found online at doi:10.1016/j.tvjl.2015.05.013. References Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K. (Eds.), 2013. Current Protocols in Molecular Biology. Vol. I, Unit 2.1A: Purification and Concentration of DNA from Aqueous Solution. Wiley Interscience, New York, NY, USA. Becker, C.A.M., Ramos, F., Sellal, E., Moine, S., Poumarat, F., Tardy, F., 2012. Development of a multiplex real-time PCR for contagious agalactia diagnosis in small ruminants. Journal of Microbiological Methods 90, 73–79. Corona, L., Amores, J., Onni, T., De la Fe, C., Tola, S., 2013a. Characterization of Mycoplasma mycoides subsp. capri isolates by SDS-PAGE, immunoblotting and PFGE. Small Ruminant Research 115, 140–144. Corona, L., Cillara, G., Tola, S., 2013b. Proteomic approach for identification of immunogenic proteins of Mycoplasma mycoides subsp. capri. Veterinary Microbiology 167, 434–439. Corrales, J.C., Esnal, A., De la Fe, C., Sanchez, A., Assuncao, P., Poveda, J.B., Contreras, A., 2007. Contagious agalactia in small ruminants. Small Ruminant Research 68, 154–166. Gomez-Martin, A., Amores, J., Paterna, A., de la Fe, C., 2013. Contagious agalactia due to Mycoplasma spp. in small dairy ruminants: Epidemiology and prospects for diagnosis and control. The Veterinary Journal 198, 48–56. Le Grand, D., Saras, E., Blond, D., Solsona, M., Poumarat, F., 2004. Assessment of PCR for routine identification of species of the Mycoplasma mycoides cluster in ruminants. Veterinary Research 35, 635–649. Manso-Silvan, L., Perrier, X., Thiaucourt, F., 2007. Phylogeny of the Mycoplasma mycoides cluster based on analysis of five conserved protein-coding sequences and possible implications for the taxonomy of the group. International Journal of Systematic and Evolutionary Microbiology 57, 2247–2258. Woubit, S., Manso-Silvan, L., Lorenzon, S., Gaurivaud, P., Poumarat, F., Pellet, M.P., Singh, V.P., Thiaucourt, F., 2007. A PCR for the detection of mycoplasmas belonging to the Mycoplasma mycoides cluster: Application to the diagnosis of contagious agalactia. Molecular and Cellular Probes 21, 391–399.
Contents lists available at ScienceDirect
The Veterinary Journal j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t v j l
Short Communication
Discrimination between Mycoplasma mycoides subsp. capri and Mycoplasma capricolum subsp. capricolum using PCR-RFLP and PCR Grazia Cillara, Maria Giovanna Manca, Carla Longheu, Sebastiana Tola * Istituto Zooprofilattico Sperimentale della Sardegna G. Pegreffi, 07100 Sassari, Italy
A R T I C L E
I N F O
Article history: Accepted 17 May 2015 Keywords: Contagious agalactia Dihydrolipoyl dehydrogenase Mycoplasma mycoides subsp. capri Mycoplasma capricolum subsp. capricolum PCR-RFLP PCR
A B S T R A C T
In this study, the dihydrolipoyl dehydrogenase (lpdA) gene was used to distinguish Mycoplasma mycoides subsp. capri (Mmc) from Mycoplasma capricolum subsp. capricolum (Mcc), two of four Mycoplasma species that cause contagious agalactia in sheep and goats. After alignment of nucleotide sequences of both species, specific primer sets were designed from unchanging and variable gene segments. The first primer set LPD-C1-F/LPD-C1-R was used to amplify a 911 bp fragment that was subsequently co-digested with FastDigest PstI, SspI, EcoRI and ClaI enzymes. The PCR-RFLP profiles differentiated the two mycoplasma species. The second primer set was used to distinguish Mmc from Mcc by single tube PCR. Both methods were further applied to identify 54 isolates collected from dairy herds from different provinces in Sardinia. The results of this study showed that PCR-RFLP and PCR could be used in routine diagnosis for rapid and specific simultaneous discrimination of Mmc and Mcc. © 2015 Elsevier Ltd. All rights reserved.
Contagious agalactia (CA) is the most serious disease affecting small dairy ruminants (Corrales et al., 2007). CA is caused by four species of Mycoplasma, namely, M. agalactiae (the classic agent of the disease in sheep and goats), M. mycoides subsp. capri (Mmc), M. capricolum subsp. capricolum (Mcc) and M. putrefaciens. Among the four Mycoplasma species, Mmc and Mcc phylogenetically belong to the ‘M. mycoides’ cluster. Because of their importance in veterinary medicine (Gomez-Martin et al., 2013), it is important that specific and rapid diagnostic procedures are developed for their detection and discrimination. Unfortunately, PCR assays based on the amplification of housekeeping genes detect both Mmc and Mcc, but also detect all three other species within the ‘M. mycoides’ cluster (Le Grand et al., 2004; Woubit et al., 2007; Becker et al., 2012). Recently, we characterized Mmc isolates collected in Sardinia from contagious agalactia outbreaks using pulse-field gel electrophoresis (PFGE) and identified their immunogenic proteins using a proteomic approach (Corona et al., 2013a and b). In the present study, the dihydrolipoyl dehydrogenase (lpdA) gene was chosen as the target gene for differentiation of Mcc from Mmc because of the presence of polymorphic sites in the DNA sequence. Polymorphic sites can be highlighted by the analysis of PCR fragments with enzymes that detect interspecific restriction fragment length polymorphism (RFLP). Furthermore, a single and rapid PCR method, based on use of a primer set selected in variation sites of lpdA gene, was proposed. During the period 2004–2014, 54 mycoplasma isolates belonging to the ‘M. mycoides’ cluster were collected from dairy herds in
* Corresponding author. Tel.: +39 079 2892339. E-mail address: [email protected] (S. Tola). http://dx.doi.org/10.1016/j.tvjl.2015.05.013 1090-0233/© 2015 Elsevier Ltd. All rights reserved.
different provinces of Sardinia (see Appendix: Supplementary Table S1). All mycoplasma isolates and reference strains were grown at 37 °C in modified Hayflick medium supplemented with 8% equine serum. Genomic DNA was extracted and purified by standard protocols (Ausubel et al., 2013). In order to determine whether isolates belonged to the ‘M. mycoides’ cluster, isolates were first amplified by PCR using primers FusA-F and FusA-R for fusA gene (Table 1). lpdA gene sequences from Mmc strain GM12 (NCBI, nucleotide ID 256385136) and Mcc strain ATCC 27343 (NCBI, nucleotide ID 83319253:275328–277217) were aligned using the EMBOSS pairwise alignment algorithm.1 The primers LPD-C1-F and LPD-C1-R (Table 1) were selected based on the conserved region of the lpdA gene between the two Mycoplasma spp. PCR reactions were conducted in a DNA thermal cycler (GeneAmp 9700, Applied Biosystems). PCR products were purified and submitted to BMR Genomics2 for sequencing. The sequences of the lpdA gene from reference strains and isolates were compared with those of the GenBank database by using the BLASTN local alignment search tool.3 Enzymes and lengths of resulting restriction fragments were predicted with an on-line program.4 Twenty microlitres of PCR amplification were digested in a 40 μL volume containing 1× FastDigest Green buffer supplemented with tracking dyes, 1 μL of 20 mg/mL acetylated BSA
1 See: http://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html (accessed 17 May 2015). 2 See: http://www.bmr-genomics.it/bmr_it/BMR_home.html (accessed 17 May 2015). 3 See: http://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed 17 May 2015). 4 See: http://www.restrictionmapper.org (accessed 17 May 2015).
422
G. Cillara et al./The Veterinary Journal 205 (2015) 421–423
M
Table 1 Oligonucleotide primers used in this study. Target fusA
lpdA
lpdA
Sequence
Fragment size (bp)
FusA-F: 5′-TGAAATTTTTAGATGGTGGAGAA-3′ FusA-R: 5′-GGTAATTTAATAGTTTCACGATATGAA-3′ LPD-C1-F: 5′-AGGTGAAGCTGTTGCTTTAG-3′ LPD-C1-R: 5′-TTCCAATAATATGTGCACCTAA-3′ LPD-F: 5′-CGATGGAAAAGATCAAATGG-3′ LPD-R: 5′-TTGTTGTTCAGTTTTTCCT-3′
1
2
3
4
5
781
Manso-Silvan et al., 2007
911
This study
362/281
This study
6
2
3
4
5
M
Reference
(Ambion) and 1 μL of FastDigest PstI, SspI, EcoRI and ClaI enzymes (Thermo Scientific). Reaction mixtures were incubated at 37 °C for 15 min then directly loaded onto the gels. At the same time, 10 μL of digested fragments from lpdA gene was examined by electrophoresis in 2% agarose gel, containing 1% agarose for routine use (Sigma) and 1% agarose low melting point (Sigma). We tried to find a rapid and simple one-step PCR to differentiate Mmc strain GM12 from Mcc ATCC 27343. The primers LPD-F and LPD-R (Table 1) were selected from the variation sites of the lpdA gene. The lpdA gene was used as the target gene to distinguish Mmc from Mcc. The primer set for the amplification of both Mmc and Mcc (LPD-C1-F and LPD-C1-R) was designed for the conserved regions of the lpdA gene after preliminary sequence analysis. Additionally, type and reference strains were included in this study: M. agalactiae PG2T, M. putrefaciens KS1T, M. capricolum subsp. capripneumoniae F38, M. bovine group 7 PG50 and M. mycoides PGT. As shown in Fig. 1, amplicons of 911 bp were obtained from all type and reference strains except M. agalactiae PG2T and M. putrefaciens KS1T. PCR products of 911 bp were digested with PstI, SspI, EcoRI and ClaI restriction endonucleases and analyzed by SDS-PAGE electrophoresis. RFLP profiles allowed differentiation of all reference strains analyzed except M. mycoides PGT and M. bovine group 7 PG50 (Fig. 2). A detailed comparison of restriction maps identified that only two restriction enzymes, ClaI and EcoRI, produced distinct speciesspecific restriction profiles to reliably distinguish Mcc CKT from M. mycoides subsp. mycoides LC reference strain whereas PstI and SspI produced the same fragments in both species (see Appendix: Supplementary Table S2). PCR-RFLP assay was extended to the 54 field isolates included in this study and profiles were analyzed by both SDS-PAGE (Fig. 3A) and agarose gels (Fig. 3B). Thirty-eight of
M
1
7
M
Fig. 2. Restriction fragment length polymorphism (RFLP) patterns of PCR products from the lpdA gene of five Mycoplasma reference strains after digestion with PstI, SspI, EcoRI and ClaI enzymes for 15 min at 37 °C. Twenty microlitres of digested lpdA amplifications were loaded in 12% (wt/vol) polyacrylamide gels containing 0.1% sodium dodecyl sulfate (SDS) then electrophoresed in a Mini Protean Tetra Cell (Bio-Rad) containing 500 mL of running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS; pH 8.3) at 200 V for 40 min. After electrophoresis gels were stained with SYBR Gold (Invitrogen). Type and reference strains: Lane 1, M. mycoides PGT; lane 2, M. bovine group 7 PG50; lane 3, M. capricolum subsp. capripneumoniae F38; lane 4, M. capricolum subsp. capricolum CKT; lane 5, M. mycoides subsp. mycoides LC type strain. Lane M, marker VIII (Roche).
the 54 isolates had a fingerprint identical to M. mycoides subsp. mycoides LC reference strain while 16 had a profile identical to Mcc CKT (see Appendix: Supplementary Table S3). Two Mmc isolates (3866 and 9500) showed a PCR-RFLP profile slightly different from
M 1 2
3
4
5 6
7 8
9 10 11 12 13 M
A
B Fig. 1. PCR profile of the dihydrolipoyl dehydrogenase (lpdA) gene obtained from seven Mycoplasma reference strains. Amplification conditions were initial denaturation for 5 min at 95 °C followed by 30 cycles of 1 min at 95 °C, 1 min at 56 °C, and 1.5 min at 72 °C with a final extension step of 10 min at 72 °C. Amplicons were resolved by electrophoresis in 1% agarose gels, stained with SybrSafe DNA gel stain (Invitrogen). Type and reference strains: Lane 1, M. mycoides subsp. mycoides LC type; lane 2, M. capricolum subsp. capricolum CKT; lane 3, M. mycoides PGT; lane 4, M. bovine group 7 PG50; lane 5, M. capricolum subsp. capripneumoniae F38; lane 6, M. agalactiae PG2T and lane 7, M. putrefaciens KS1T. Lane M, marker VIII (Roche).
Fig. 3. Restriction fragment length polymorphism (RFLP) patterns of PCR products from the lpdA gene of 11 field isolates and reference strains after digestion with PstI, SspI, EcoRI and ClaI enzymes. Panel A: fragments were separated by 12% SDS-PAGE gel. Panel B: fragments were separated by 2% agarose gel. Isolates 55094 (lane 1), 74751 (lane 2), 11256 (lane 3), 26909 (lane 4), 41071 (lane 5), 61269 (lane 6), 3866 (lane 7), 9862 (lane 8), 13127 (lane 9), 18622 (lane 10) and 19002 (lane 11). M. mycoides subsp. mycoides LC type strain (lane 12) and M. capricolum subsp. capricolum CKT (lane 13). Lane M, marker VIII (Roche).
G. Cillara et al./The Veterinary Journal 205 (2015) 421–423
M 1
2
3 4
5 6
7
8
9 10 11 12 13 M
423
Acknowledgments The study was supported by the Istituto Zooprofilattico Sperimentale of Sardinia. Appendix: Supplementary material
Fig. 4. PCR amplification of 11 field isolates and reference strains using the primers set LPD-F/LPD-R. The amplification conditions used in this study were as follows: initial denaturation at 95 °C for 5 min followed by 30 cycles of 1 min at 95 °C, 1 min at 50 °C, and 1 min at 72 °C with a final 10 min extension step at 72 °C. The amplified samples were subjected to electrophoresis on 2% agarose gel. Isolates 55094 (lane 1), 74751 (lane 2), 11256 (lane 3), 26909 (lane 4), 41071 (lane 5), 61269 (lane 6), 3866 (lane 7), 9862 (lane 8), 13127 (lane 9), 18622 (lane 10) and 19002 (lane 11). M. mycoides subsp. mycoides LC type strain (lane 12) and M. capricolum subsp. capricolum CKT (lane 13). Lane M, marker VIII (Roche).
the type strain (Fig. 3, lane 7). On DNA sequencing of 911 bp products another SspI restriction site was identified in these isolates. All field isolates and type strains were additionally tested for amplification with a new primer set LPD-F and LPD-R, selected from polymorphic sites of the lpdA gene. The PCR was developed to allow the simultaneous detection of Mmc and Mcc in a single reaction, making it more applicable for routine diagnostic use. As shown in Fig. 4, a 362 bp fragment from Mmc isolates and a doublet (362 and 281 bp) from Mcc isolates were obtained. The results were reproducible and stable over time. This study has shown that the lpdA gene can be used for the correct identification of both Mmc and Mcc. An RFLP-based approach and single-tube PCR assay provide a relatively simple and cost-effective method for differential diagnosis of Mmc and Mcc. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of this paper.
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