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Table 4 Primers and probes for detection and sequencing in this study

From: Decontamination of mycoplasma-contaminated Orientia tsutsugamushistrains by repeating passages through cell cultures with antibiotics

Targets

Assay

Name

Primers and probes

Mycoplasmas

   

tuf genea)

real-time PCR

Mollicutes 414F

5'-TCCAGGWCAYGCTGACTA-3'

  

Mollicutes 541R

5'-ATTTTWGGAACKCCWACTTG-3'

  

Probe 451Fa)

5'-GGTGCTGCACAAATGGATGG-3'

tuf gene

Sequencing 1st

Myco-tuf-F1

5'-HATHGGCCAYRTTGAYCAYGGKAAAA-3'

  

Myco-tuf-F2

5'-ATGATYACHGGDGCWGCHCAAATGGA-3'

 

Sequencing 2nd

Myco-tuf-R1

5'-CCRCCTTCRCGRATDGAGAAYTT-3'

  

Myco-tuf-R2

5'-TKTRTGACGDCCACCTTCYTC-3'

16s-23s rRNA intergenic spacer region

nested PCR 1st

MCGpF11

5'-ACACCATGGGAGYTGGTAAT-3'

  

R23-1R

5'-CTCCTAGTGCCAAGSCATYC-3'

 

nested PCR 2nd

R16-2

5'-GTGSGGMTGGATCACCTCCT-3'

  

MCGpR21

5'-GCATCCACCAWAWACYCTT-3'

Orientia tsutsugamushi

   

47kDa common antigen coding gene

real-time PCR

Ots-47k-F

5'-AATTCGTCGTGGTATGTTAAATG-3'

  

Ots-47k-R

5'-AGCAATTCCACATTGTGCTG-3'

  

Ots-47k-P b)

5'-TGCTTAATGAATTAACTCCAGAATT-3'

  1. a) Locked nucleic acid (LNA) bases (underlined) and was synthesized with the fluorescent reporter 6-carboxyfluorescein (FAM) covalently coupled to the 5’ end and a dark quencher to the 3’ end.
  2. b) TaqMan probe was synthesized with the fluorescent reporter 6-carboxyfluorescein (FAM) covalently coupled to the 5’ end and a dark quencher to the 3’ end.