Amplification and sequencing of single infected individuals

Use the following protocol for amplification and sequencing of Wolbachia in host species carrying a single strain infection.

Since individuals of the same species but coming from different populations can harbor different Wolbachia strains, please always use DNA extracted from a single individual, or from multiple individuals derived from a single female. For each strain perform PCRs using the same DNA for amplification of all five genes. This will ensure that all five alleles correspond to a single strain.

Standard primers (Baldo et al. 2006)

Gene Primers sequences (5'-3') Product size, bp
gatB gatB_F1: GAK TTA AAY CGY GCA GGB GTT 471
gatB_R1: TGG YAA YTC RGG YAA AGA TGA
coxA coxA_F1: TTG GRG CRA TYA ACT TTA TAG 487
coxA_R1: CT AAA GAC TTT KAC RCC AGT
hcpA hcpA_F1: GAA ATA RCA GTT GCT GCA AA 515
hcpA_R1: GAA AGT YRA GCA AGY TCT G
ftsZ ftsZ_F1: ATY ATG GAR CAT ATA AAR GAT AG 524
ftsZ_R1: TCR AGY AAT GGA TTR GAT AT
fbpA fbpA_F1: GCT GCT CCR CTT GGY WTG AT 509
fbpA_R1: CCR CCA GAR AAA AYY ACT ATT C

 

PCR protocol

This protocol is for guidance only and you may need to optimize it for your particular laboratory.

Perform PCR reactions in a 40 μL final volume using 2 μL DNA, H2O to volume and reagents at the following concentrations:

Reagents PCR final concentration
Buffer 1x
dNTPs 0.2 mM
MgCl2 1.5 mM
Primer F 1 μM
Primer R 1 μM
Taq DNA polymerase 0.5 U

 

Cycling conditions

  • 94°C for 2 min
  • 37 cycles at:
    • 94°C for 30 s
    • optimal annealing T for 45 s (54°C for hcpA, gatB and ftsZ and coxA, and 59°C for fbpA and wsp)
    • 72°C for 1 min 30 s
  • 1 cycle at:
    • 72°C for 10 min
    • 4°C hold

After cycling, check that amplification was successful by running 5 μl of each reaction on an agarose gel, with size standards.

Purify the remaining 35 μl and sequence using both forward and reverse primers. A double coverage of each sequence is required.

Use the allele templates provided as reference to verify the exact range of nucleotides for each gene sequence.

Alternative protocols

Given that Wolbachia are a diverse group of bacteria, circumstances may necessitate using alternative primers. In the case the standard primers fail to amplify we suggest the use of the following alternative protocols and primers.

  1. Standard primers (above) combined with M13 sequencing tags. These tags serve as anchors for the degenerate primers during amplification and are subsequently used for sequencing.

    M13 forward: TGTAAAACGACGGCCAGT
    M13 reverse: CAGGAAACAGCTATGACC

    M13f and M13r are used for all primers. Combine them with standard forward and standard reverse primers respectively.

    Below is an example: in bold are the tags, preceding the standard primer at the 5'.

    gatB_F1adp: TGTAAAACGACGGCCAGTGAKTTAAAYCGYGCAGGBGTT
    gatB_R1adp: CAGGAAACAGCTATGACCTGGYAAYTCRGGYAAAGATGA

    Use the standard PCR protocol above.

  2. ~64-fold degenerate primers. These primers are external to the MLST gene sequences, and have been found to work fairly broadly in Wolbachia. They can either be used solely, or in nested PCR with the standard MLST primers.

    Gene name Primer sequences (5'-3')
    gatB gatB_F2: CAGATAACNCARTTYTTYGARCC
    gatB_R2: ATTGTTCCATCNACDATRAARTC
    gatB_F3: ATTCAYYTAGARCAAGATGCAGG
    gatB_R3: AAGAGCTCKGAYAAAGCATYBGC
    coxA coxA_F2: GGAGGATTYGGNAAYTGGTTYGT
    coxA_R2: CCACCCCACATNGTNGCDATCCA
    coxA_F3: ATGATTGGCKCACCHGAYATGGC
    coxA_R3: ACTTTTACACCAGTWATMACRCC
    hcpA hcpA_F2: AAAGGCGCTCARGAYGCNAARCG
    hcpA_R2: ACATACTGNACRTCRTCRTTRTC
    hcpA_F3: ATTAGAGAAATARCAGTTGCTGC
    hcpA_R3: CATGAAAGACGAGCAARYTCTGG
    fbpA fbpA_F2: GTAGATCARGGNTTYGARCAYGG
    fbpA_R2: TTACCGCCACCYTGYTTDATYTC
    fbpA_F3: GTTAACCCTGATGCYYAYGAYCC
    fbpA_R3: TCTACTTCCTTYGAYTCDCCRCC

     

    F2/R2 and F3/R3 primers pairs are not specific to the Wolbachia lineage. F2/R2 primers have only been tested with sequencing adaptors (see above), whereas, F3/R3 have only been tested without sequencing adaptors.

    Amplicons are generated using standard PCR conditions with HotStarTaq (Qiagen), according to the manufacturer's recommendations, with 0.5 μM of each ~64-fold degenerate primer.

    PCR cycling conditions:

    • 95°C for 15 min
    • 50 cycles at:
      • 95°C for 15 s
      • 55°C for 30 s
      • 72°C for 1 min

    For sequencing we suggest the use of the sequencing adaptors when used, or alternatively, of the primers used for amplification.

  3. Alternative ftsZ primers as described in Lo et al. 2001:

    ftsZunif: 5'-GG(CT)AA(AG)GGTGC(AG)GCAGAAGA
    ftsZunif: 5'-ATC(AG)AT(AG)CCAGTTGCAAG

  4. A and B specific primers are available for each MLST locus. In some cases these could be used for single A and single B infections where the standard primers do not work.