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Rapid, Simple and Cost-Effective Molecular Method to Differentiate the Temperature Sensitive (ts+) MS-H Vaccine Strain and Wild-Type Mycoplasma synoviae Isolates.

Kreizinger Z, Sulyok KM, Pásztor A, Erdélyi K, Felde O, Povazsán J, Kőrösi L, Gyuranecz M - PLoS ONE (2015)

Bottom Line: The assays can be performed directly on clinical samples and they can be run simultaneously at the same annealing temperature.The assays can be performed in laboratories with limited facilities, using basic real-time PCR machine or conventional thermocycler coupled with agarose gel electrophoresis.The advantages of the described assays compared with previously used methods are simplicity, sufficient sensitivity, time and cost effectiveness and specificity.

View Article: PubMed Central - PubMed

Affiliation: Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Pest, Hungary.

ABSTRACT
Mycoplasma synoviae infection in chickens and turkeys can cause respiratory disease, infectious synovitis and eggshell apex abnormality; thus it is an economically important pathogen. Control of M. synoviae infection comprises eradication, medication or vaccination. The differentiation of the temperature sensitive (ts+) MS-H vaccine strain from field isolates is crucial during vaccination programs. Melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) are provided in the present study to distinguish between the ts+ MS-H vaccine strain, its non-temperature sensitive re-isolates and wild-type M. synoviae isolates based on the single nucleotide polymorphisms at nt367 and nt629 of the obg gene. The two melt-MAMAs and the two agarose-MAMAs clearly distinguish the ts+ MS-H vaccine strain genotype from its non-temperature sensitive re-isolate genotype and wild-type M. synoviae isolate genotype, and no cross-reactions with other Mycoplasma species infecting birds occur. The sensitivity of the melt-MAMAs and agarose-MAMAs was 103 and 104 copy numbers, respectively. The assays can be performed directly on clinical samples and they can be run simultaneously at the same annealing temperature. The assays can be performed in laboratories with limited facilities, using basic real-time PCR machine or conventional thermocycler coupled with agarose gel electrophoresis. The advantages of the described assays compared with previously used methods are simplicity, sufficient sensitivity, time and cost effectiveness and specificity.

No MeSH data available.


Related in: MedlinePlus

Amplification plot and melting-curves of MS-H2 melt-MAMA.Amplification plot (A) of dilution series of gBlock validation controls showing the sensitivity of the MS-H2 assay. Green line represents negative control. Melting curves (B) show melting temperatures for the temperature sensitive MS-H vaccine strain and a wild-type strain (Tm: 76.8°C; blue line) or non-temperature sensitive MS-H re-isolate (Tm: 70.9°C; orange line).
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pone.0133554.g003: Amplification plot and melting-curves of MS-H2 melt-MAMA.Amplification plot (A) of dilution series of gBlock validation controls showing the sensitivity of the MS-H2 assay. Green line represents negative control. Melting curves (B) show melting temperatures for the temperature sensitive MS-H vaccine strain and a wild-type strain (Tm: 76.8°C; blue line) or non-temperature sensitive MS-H re-isolate (Tm: 70.9°C; orange line).

Mentions: Both melt-MAMAs clearly differentiated the ts+ MS-H vaccine strain genotype, ts- MS-H re-isolate genotype and wild-type M. synoviae strain genotype (Table 4). The MS-H1 assay (nt367 SNP position) distinguished ts+ MS-H vaccine strain and ts- MS-H re-isolates from wild-type M. synoviae isolates with 80.1°C and 75.0°C melting temperatures, respectively (Fig 2). The MS-H2 assay (nt629 SNP position) distinguished ts+ MS-H vaccine strain and wild-type M. synoviae strains from ts- MS-H re-isolates with 76.8°C and 70.9°C melting temperatures, respectively (Fig 3). The agarose-MAMA versions of the MS-H1 and MS-H2 assays also differentiated the ts+ MS-H vaccine strain genotype, ts- MS-H re-isolate genotype and wild-type M. synoviae strain genotype based on the 19-20bp fragment size differences of the PCR products obtained from certain genotypes (Table 4 and Fig 4).


Rapid, Simple and Cost-Effective Molecular Method to Differentiate the Temperature Sensitive (ts+) MS-H Vaccine Strain and Wild-Type Mycoplasma synoviae Isolates.

Kreizinger Z, Sulyok KM, Pásztor A, Erdélyi K, Felde O, Povazsán J, Kőrösi L, Gyuranecz M - PLoS ONE (2015)

Amplification plot and melting-curves of MS-H2 melt-MAMA.Amplification plot (A) of dilution series of gBlock validation controls showing the sensitivity of the MS-H2 assay. Green line represents negative control. Melting curves (B) show melting temperatures for the temperature sensitive MS-H vaccine strain and a wild-type strain (Tm: 76.8°C; blue line) or non-temperature sensitive MS-H re-isolate (Tm: 70.9°C; orange line).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4514773&req=5

pone.0133554.g003: Amplification plot and melting-curves of MS-H2 melt-MAMA.Amplification plot (A) of dilution series of gBlock validation controls showing the sensitivity of the MS-H2 assay. Green line represents negative control. Melting curves (B) show melting temperatures for the temperature sensitive MS-H vaccine strain and a wild-type strain (Tm: 76.8°C; blue line) or non-temperature sensitive MS-H re-isolate (Tm: 70.9°C; orange line).
Mentions: Both melt-MAMAs clearly differentiated the ts+ MS-H vaccine strain genotype, ts- MS-H re-isolate genotype and wild-type M. synoviae strain genotype (Table 4). The MS-H1 assay (nt367 SNP position) distinguished ts+ MS-H vaccine strain and ts- MS-H re-isolates from wild-type M. synoviae isolates with 80.1°C and 75.0°C melting temperatures, respectively (Fig 2). The MS-H2 assay (nt629 SNP position) distinguished ts+ MS-H vaccine strain and wild-type M. synoviae strains from ts- MS-H re-isolates with 76.8°C and 70.9°C melting temperatures, respectively (Fig 3). The agarose-MAMA versions of the MS-H1 and MS-H2 assays also differentiated the ts+ MS-H vaccine strain genotype, ts- MS-H re-isolate genotype and wild-type M. synoviae strain genotype based on the 19-20bp fragment size differences of the PCR products obtained from certain genotypes (Table 4 and Fig 4).

Bottom Line: The assays can be performed directly on clinical samples and they can be run simultaneously at the same annealing temperature.The assays can be performed in laboratories with limited facilities, using basic real-time PCR machine or conventional thermocycler coupled with agarose gel electrophoresis.The advantages of the described assays compared with previously used methods are simplicity, sufficient sensitivity, time and cost effectiveness and specificity.

View Article: PubMed Central - PubMed

Affiliation: Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Pest, Hungary.

ABSTRACT
Mycoplasma synoviae infection in chickens and turkeys can cause respiratory disease, infectious synovitis and eggshell apex abnormality; thus it is an economically important pathogen. Control of M. synoviae infection comprises eradication, medication or vaccination. The differentiation of the temperature sensitive (ts+) MS-H vaccine strain from field isolates is crucial during vaccination programs. Melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) are provided in the present study to distinguish between the ts+ MS-H vaccine strain, its non-temperature sensitive re-isolates and wild-type M. synoviae isolates based on the single nucleotide polymorphisms at nt367 and nt629 of the obg gene. The two melt-MAMAs and the two agarose-MAMAs clearly distinguish the ts+ MS-H vaccine strain genotype from its non-temperature sensitive re-isolate genotype and wild-type M. synoviae isolate genotype, and no cross-reactions with other Mycoplasma species infecting birds occur. The sensitivity of the melt-MAMAs and agarose-MAMAs was 103 and 104 copy numbers, respectively. The assays can be performed directly on clinical samples and they can be run simultaneously at the same annealing temperature. The assays can be performed in laboratories with limited facilities, using basic real-time PCR machine or conventional thermocycler coupled with agarose gel electrophoresis. The advantages of the described assays compared with previously used methods are simplicity, sufficient sensitivity, time and cost effectiveness and specificity.

No MeSH data available.


Related in: MedlinePlus