2010/05/06

PCR primers for endosymbiotic bacteria



Traditionally, identification of bacteria was based on their morphology. So it was only be accessible for specialists on bacteriology. Now, development on molecular biology (i.e., PCR and DNA sequencing) has made it much easier even for non-bacteriologists. Below is a list of PCR primers for endosymbiotic bacteria of insects. Upon use, please reconfirm the sequence by checking the original papers
  1. Eubacteria (universal)

    2. 16S rDNA

    16SA1: AGAGTTTGATCMTGGCTCAG (for.)
    16SA2: GTGCCAGCAGCCGCGGTAATAC (for.)
    16SA3: TGCATGGYTGTCGTCAGCTCG (for.)
    16SB1: TACGGYTACCTTGTTACGACTT (rev.)
    16SB2: CGAGCTGACGACARCCATGCA (rev.)
    16SB3: GTATTACCGCGGCTGCTGGCAC (rev.)
    Eub925r: CCGYCAATTCCTTTGAGTTT (rev.)
    Eub1405r: GACGGGCGGTGTGTRCA (rev.)
    note: Fukatsu & Nikoh, 1998 AEM; Kikuchi & Fukatsu, 2005 Microb. Ecol.
    A1------> A2------> A3------>
    ----------16S(1.5kb)--------------
    <-----------B3 <--------B2 <---------B1

  2. Wolbachia

    3. wsp
    wsp81F: TGGTCCAATAAGTGATGAAGAAAC (for.)
    wsp691R: AAAAATTAAACGCTACTCCA (rev.)
    note: Zhou et al. (1998) Proc. R. Soc. B

    ftsZ
    ftsZf: GTATGCCGATTGCAGAGCTTG (for.)
    ftsZr: GCCATGAGTATTCACTTGGCT (rev.)
    note: 769bp, Holden et al. (1993) Mol. Gen. Genet.

    ftsZf1: GTTGTCGCAAATACCGATGC
    ftsZr1: CTTAAGTAAGCTGGTATATC
    note: Werren et al., 1995

    ftsZAdf: CTCAAGCACTAGAAAAGTCG (for.)
    ftsZAdr: TTAGCTCCTTCGCTTACGTG (rev.)
    note: A-group specific (Werren et al. 1995)

    ftsZBf: CCGATGCTCAAGGGTTAGAG (for.)
    ftsZBr: CCACTTAACTGTTTGGTTTG (rev.)
    note: B-group specific (Werren et al., 1995)

    16S rDNA
    WP1F: TTGTAGCCTGCTATGGTATAACT (for.)
    WP1R: GAATAGGTATGATTTTCATGT (rev.)
    note: 890 bp (O’Neill et al., 1992 PNAS)

    groE
    groEfl: TGTATTAGATGATAACGTGC (20mer)
    groErl: CCATTTGCAGAAATTATTGCA (21mer)
    note: ~820 bp (Masui et al.)

    groE (A-group-specific)
    groEAf: TGATCAAGCCTATTAGC (17mer)
    groEAr: GAGATTATTGCAACTTGTGCC (21mer)
    note: < 800 bp

    For MLST (Baldo et al., 2006 AEM)
    gatB_F1: GAKTTAAAYCGYGCAGGBGTT
    gatB_R1: TGGYAAYTCRGGYAAAGATGA
    coxA_F1: TTGGRGCRATYAACTTTATAG
    coxA_R1: CTAAAGACTTTKACRCCAGT
    hcpA_F1: GAAATARCAGTTGCTGCAAA
    hcpA_R1: GAAAGTYRAGCAAGYTCTG
    ftsZ_F1: ATYATGGARCATATAAARGATAG
    ftsZ_R1: TCRAGYAATGGATTRGATAT
    fbpA_F1: GCTGCTCCRCTTGGYWTGAT
    fbpA_R1: CCRCCAGARAAAAYYACTATTC
    wsp_F1: GTCCAATARSTGATGARGAAAC
    wsp_R1: CYGCACCAAYAGYRCTRTAAA
    94C 00:30
    (*) 00:45
    72C 01:30 (36 cycles)
    (*annealing temp, hcpA: 53C/ gatB:54C/ ftsZ: 54C/ coxA: 55C/ fbpA: 59C/ wsp: 59C)

  3. Spiroplasma

    4. 16S rDNA
    TKSSsp: TAGCCGTGGCTTTCTGGTAA (rev.)
    note: 16SA1(universal)+TKSSsp (Fukatsu et al., 2001), might not be specific

    SpoulF: GCTTAACTCCAGTTCGCC (for.)
    SpoulR: CCTGTCTCAATGTTAACCTC (rev.)
    note: specific to Spiroplasma poulsonii and its relatives. Montenegro et al. (2005) Insect Mol. Biol.

    dnaA
    SRdnaAF1: GGAGAYTCTGGAYTAGGAAA (for.)
    SRdnaAR1: CCYTCTAWYTTTCTRACATCA (rev.)
    note: designed by Hisashi Anbutsu

    dnaA109F: TTAAgAgCAgTTTCAAAATCggg
    dnaA246R: TgAAAAAAACAAACAAATTgTTATTACTTC
    note: for QPCR

    spoT
    spoT-f: CAAACAAAAGGACAAATTGAAG
    spoT-r: CACTGAAGCGTTTAAATGAC
    note: 94C, 2 min followed by 30cycles of [94C,20s/54C,60s/72C,55s]
    note: 513 bp, (Montenegro et al., 2005 Insect Mol. Biol.)

    P58
    P58-f: GTTGGTTGAATAATATCTGTTG
    P58-r: GATGGTGCTAAATTATATTGAC
    note: 793 bp (Montenegro et al., 2005 Insect Mol. Biol.)

  4. Rickettsia

    5. 16S rDNA
    Rick16SR: CATCCATCAGCGATAAATCTTTC (rev.)
    note: 16SA1 + Rick16SR, 200 bp

    Rb-F: GCTCAGAACGAACGCTATC (for.)
    Rb-R: GAAGGAAAGCATCTCTGC (rev.)
    note: 900bp, Gottlieb et al., 2006 AEM

    gltA
    CS1: GGGGGCCTGCTCACGGCGG (for.)
    CS2: ATTGCAAAAAGTACAGTGAACA (rev.)
    note: 360 bp

  5. Arsenophonus

    6. 16S rDNA
    ColArsfwd1: gctaataccgcmtaatctctaa (for.)  
    Ars16SR: ttagctccggaggccacagt (rev.)
    note: 16SA1+Ars16SR (###bp): not very specific
    note: ColArsfwd1+Ars16SR(16SrDNA, ###bp) might be too specific to aphids, from Takema Fukatsu

    23S rDNA
    Ars23S-1: CGTTTGATGAATTCATAGTCAAA (for.)
    Ars23S-2: GGTCCTCCAGTTAGTGTTACCCAAC (rev.)
    note: initial denaturation at 95C, 5 min; subsequent denaturation at 95C, 30 s/ annealing at 60.5C, 30 s/ elongation at 72C, 45 s (30 cycles), Thao & Baumann (2004) Curr. Micobiol.

  6. Cardinium
    16S rDNA
    Ch-F: TACTGTAAGAATAAGCACCGGC
    Ch-R: GTGGATCACTTAACGCTTTCG
    note: Zchori-Fein & Pearlman, 2004 Mol. Ecol., Annealing: 58C 
    Car-sp-F: CGGCTTATTAAGTCAGTTGTGAAATCCTAG
    Car-sp-R: TCCTTCCTCCCGCTTACACG
    note: 544 bp, Nakamura et al., 2009
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