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Table 1 Primers and optimized procedures for MLST, P146 gene and MLVA analyses of M. hyopneumoniae isolates

From: Genotyping and biofilm formation of Mycoplasma hyopneumoniae and their association with virulence

Methods

Target genes

Sequence (5′→3′)

10×PCR buffer (Mg2 + free, µL)

MgCl2

(25mM, µL)

dNTP

(2.5mM, µL)

Primer (10µM, µL)

Taq DNA polymerase

(5U/µL, µL)

Annealing temperature (℃)

References

MLST

adk

GGAGCTCCTGGCTCAGGTAAAG

2

2

2

0.8

0.5

60

[33]

  

GTTTCTTCAAGGGTTTGCTCG

 

rpoB

AAACGGATAGTTAGTGTTGGCG

2

2

2

0.8

0.5

60

  

TGTTCGGCATCAAGGACAAG

   
 

tpiA

GAAATTGAAAAATGAATAAAACCGTAAG

2

2

2

0.8

0.5

60

  

GATGCTTTTCTGGGATACTAACTCG

      

P146 sequencing

P146

TCCAAGACGAAGATCTTGACTATC

2

2

2

0.8

0.5

56

[34]

TTAGAACTTGCAAGATAAAGCTTG

       

MLVA

P97 R1

GAAGCTATCAAAAAAGGGGAAACTA

2

3

3

0.8

0.8

59.7

[11]

GGTTTATTTGTAAGTGAAAAGCCAG

      

P97 R2

AGCGAGTATGAAGAACAAGAA

2

3

3

0.8

0.8

50.5

GGTTTATTTGTAAGTGAAAAGCCAG

      

H2R2

TCAATAACAAAGCACCTTTC

2.5

2

2

1.1

0.8

50.3

[8]

GAGCTATTAATCCAGGCATC

      

H3

ATTACCGGAAATAAGAAGTG

2

2.5

2.5

0.8

0.8

55.9

AGTTAAAAAAGCGGCTTTTC

      

H5R1/R2

AAGTAAAAAAGACCTCGAAG

2

2.5

2

1.4

0.8

50.3

CCAAAGAATTAATCCAAGTC

      

MLVA

H6R3

CAGATCAAATGGCTGTAACA

3

1.5

2.5

1.1

0.8

59.7

Designed in this study

CCTTCACGGATTGCTTCA

      

CH

GGCAAAAAAATGTGAATGTC

2

2

2

1.1

1.1

52.4

[8]

GGCTTTTTGGTATATTCAGTTC

      

P95

ATTTATCCTTTACTTAGCGG

2.5

2

2

1.4

0.8

50.3

AAGACAAGTGGATATTTTGC

      

P146R1

AGTCACAAAAACCTCAAAGTG

2

2

2

1.1

0.8

52.3

AGGAGAGCTTTGAATTTGAG

      

P146R2

AAGACCAAAAAGTAGGACATAC

3

2

2

1.1

1.1

48.6

AACTCAAGCATCTAAAAGTG

      

P216

GCAAAGCCAAAATGTAAATG

2

2

2

0.8

0.8

52.4

CCAGTTCTTCTTCTTTTCTAAC

      

P146R3

AAAACCCAAAGTAGTGATTC

2

2

2

1.1

0.8

50.3

TGTATCGGTTTCAGAAGAAG

      
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Veterinary Research

ISSN: 1297-9716