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Table 1 Primer-sets, amplicon length, annealing temperature, amplification time and PCR-protocols

From: Appendiceal spirochaetosis in children

Primer-set

Name

5′–––3′

Amplicon

Bspp

B.spp FW

AACATggACTAATACCgCATATAC

147 bp

B.spp probe

LC610-TgAgCCTgCggCCTATTAgCC-BBQ

B.spp REV

CTCAggTCggCTACCTATC

β-globulin

β glob FW (GH20)

gAAgAgCCAAggACAggTAC

268 bp

β glob probe

Cy5-TCTgCCgTTACTgCCCTgT-BBQ

β glob REV (PC04)

CAACTTCATCCACgTTCACC

PhHV

PhHV FW

gggCgAATCACAgATTgAATC

89 bp

PhHV probe

LC640-TTTTTATgTgTCCgCCACCATCTggATC-BBQ

PhHV REV

gCggTTCCAAACgTACCAA

Step A

Step A FW

TggATAAgTTAgCggCgAACTg

212 bp

Step A REV

TCAggTCggCTACCTATCg

Step B

Step B FW

gAgCCTgCggCCTATTAgC

253 bp

Step B REV

gCCgAggCTTACATTATCTACTgTC

Step C

Step C FW

AgCgACATCgCgTgAgg

258 bp

Step C REV

TCCATCATCCCCTACAATATCCAAg

  1. PCR conditions.
  2. Primer concentration: 2,5 μM of both forward and reverse primers.
  3. Buffer composition.
  4. Probe PCR: LightCycler® 480 Probes Master (Version 09).
  5. Step A-C PCR: LightCycler® 480 SYBR Green I Master (Version 12).
  6. PCR volume.
  7. Probe: 13 μl buffer and 2 μl DNA-eluate.
  8. Step A-C: 15 μl buffer and 5 μl DNA-eluate.
  9. PCR consumables (probe and Step A-C).
  10. White 96-well plate (Roche Diagnostics, Almere, The Netherlands) sealed with transparent self-adhesive foil (Roche Diagnostics, Almere, The Netherlands).
  11. PCR protocol.
  12. Probe PCR: pre-incubation at 95°C for 10 minutes, denaturing at 95°C for 10 seconds, amplification at 60°C one minute, 45 cycles.
  13. Step A-C PCR: pre-incubation at 95°C for 10 minutes, denaturing at 95°C for 10 seconds, 10 seconds annealing at 62°C, amplification at 72°C one minute, 45 cycles. Melting-curve analysis: 5 seconds at 95°C, one minute at 65°C, 2.2°C per second increase of temperature until 97°C with five acquisitions per degree Celsius.