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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

Sequences of the gBlocks used as positive and validation controls in the study.The gBlocks (Integrated DNA Technologies Inc., Coralville, IA) contain 330 bp long fragment of the obg gene between nt342 and nt672.
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pone.0133554.g001: Sequences of the gBlocks used as positive and validation controls in the study.The gBlocks (Integrated DNA Technologies Inc., Coralville, IA) contain 330 bp long fragment of the obg gene between nt342 and nt672.

Mentions: In order to test the sensitivity of the assays, 10 fold dilutions of gBlocks (Integrated DNA Technologies Inc., Coralville, IA) containing 200 ng of a 330 bp long fragment of the obg gene (from nt342 to nt672) were used (Fig 1). The specificity of the assays was tested by including the following avian Mycoplasma species in the analysis: M. anatis, M. anseris, M. columbinasale, M. columborale, M. cloacale, M. gallinaceum, M. gallinarum, M. gallisepticum, M. gallopavonis, M. iners, M. iowae, M. meleagridis and M. sp. 1220. The temperature sensitive (ts+) MS-H vaccine strain (Vaxsafe MS-H), M. synoviae type strain (WVU 1835, NCTC 10124), seven clinical M. synoviae isolates (3 chicken and 4 turkey isolates), nine ts+ MS-H re-isolates and trachea swabs taken from 40 vaccinated and 40 clinically infected (seropositive) chickens were included in the analysis as well (Table 3). DNA was extracted from the isolated strains and trachea swabs with the Qiamp DNA Mini kit (Qiagen GmbH, Hilden, Germany). Ethical approval was not required for the study as all samples and strains were collected by the authors during routine diagnostic examinations.


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)

Sequences of the gBlocks used as positive and validation controls in the study.The gBlocks (Integrated DNA Technologies Inc., Coralville, IA) contain 330 bp long fragment of the obg gene between nt342 and nt672.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133554.g001: Sequences of the gBlocks used as positive and validation controls in the study.The gBlocks (Integrated DNA Technologies Inc., Coralville, IA) contain 330 bp long fragment of the obg gene between nt342 and nt672.
Mentions: In order to test the sensitivity of the assays, 10 fold dilutions of gBlocks (Integrated DNA Technologies Inc., Coralville, IA) containing 200 ng of a 330 bp long fragment of the obg gene (from nt342 to nt672) were used (Fig 1). The specificity of the assays was tested by including the following avian Mycoplasma species in the analysis: M. anatis, M. anseris, M. columbinasale, M. columborale, M. cloacale, M. gallinaceum, M. gallinarum, M. gallisepticum, M. gallopavonis, M. iners, M. iowae, M. meleagridis and M. sp. 1220. The temperature sensitive (ts+) MS-H vaccine strain (Vaxsafe MS-H), M. synoviae type strain (WVU 1835, NCTC 10124), seven clinical M. synoviae isolates (3 chicken and 4 turkey isolates), nine ts+ MS-H re-isolates and trachea swabs taken from 40 vaccinated and 40 clinically infected (seropositive) chickens were included in the analysis as well (Table 3). DNA was extracted from the isolated strains and trachea swabs with the Qiamp DNA Mini kit (Qiagen GmbH, Hilden, Germany). Ethical approval was not required for the study as all samples and strains were collected by the authors during routine diagnostic examinations.

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