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Table 1 Primers used for PCR amplification (and prior to 454 pyrosequencing)

From: 16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice

Primer name

Primer sequence

Target group/specificity

Reference

Forward primers

 27fa

AGAGTTTGATCCTGGCTCAG

Universal

Methé et al. [21, 53]

 27f-YMb

AGAGTTTGATYMTGGCTCAG

Universal

Satokari et al. [35]

 27f-Chlc

AGAATTTGATCTTGGTTCAG

Universal/Chlamydiales

Frank et al. [32]

 27f-Borc

AGAGTTTGATCCTGGCTTAG

Universal/Borrelia

Frank et al. [32]

 27f-Bifc

AGGGTTCGATTCTGGCTCAG

Universal/Bifidobacteriales

Frank et al. [32]

 27f-Atoc

AGAGTTCGATCCTGGCTCAG

Universal/Atopobium group

Frank et al. [32]

 Bif164-f

GGGTGGTAATGCCGGATG

Bifidobacteria

Satokari et al. [35]

Reverse primersd

 Bif662-r

CCACCGTTACACCGGGAA

Bifidobacteria

Satokari et al. [35]

 534r

5′ ATTACCGCGGCTGCTGG

Universal

Muyzer et al. [55]

Q-PCR primerse

 Bif spp for

TCGCGTCYGGTGTGAAAG

Bifidobacteria

Rinttilä et al. [56]

 Bif spp rev

CCACATCCAGCRTCCAC

Bifidobacteria

Rinttilä et al. [56]

 UniF

GTGSTGCAYGGYYGTCGTCA

Universal

Fuller et al. [57]

 UniR

ACGTCRTCCMCNCCTTCCTC

Universal

Fuller et al. [57]

 Bac303F

GAAGGTCCCCCACATTG

Bacteroides spp.

Bartosch et al. [58]

 Bfr-Fmrev

CGCKACTTGGCTGGTTCAG

Bacteroides spp.

Ramirez-Farias et al. [51]

 Erec482F

CGGTACCTGACTAAGAAGC

Cluster XIVa

Rinttilä et al. [56]

 Erec870R

AGTTTYATTCTTGCGAACG

Cluster XIVa

Rinttilä et al. [56]

  1. aPrimer 27f was not used in this study but is shown for comparison and to indicate the positions of the three mismatches with the Bifidobacteriales 16S rRNA gene (in bold)
  2. bSame as primer 7-f in Satokari et al. [35] and 27f-YM in Frank et al. [32]. Contains two degenerate positions but still has two mismatches with the Bifidobacteriales 16S rRNA gene (in bold). The fusion primer used also contained the 454 adaptor “A” sequence—see “Methods” section for full details
  3. c27f-Chl—optimised for Chlamydiales; 27f-Bor—optimised for Borrelia group; 27f-Bif—optimised for Bifidobacteriales; 27f-Ato—optimised for Atopobium group. The fusion primers used also contained the 454 adaptor “A” sequence—see “Methods” section for full details
  4. dThe reverse primers for sequencing also contained the 454 adaptor “B” sequence and 12-base Golay barcodes. See “Methods” section and Additional file 1: Table S1 for full details
  5. eThe Q-PCR annealing temperatures used were 60 °C