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|
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|
|Primer F||1 μM|
|Primer R||1 μM|
|Taq DNA polymerase||0.5 U|
- 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.
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.
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'.
Use the standard PCR protocol above.
~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.
Alternative ftsZ primers as described in Lo et al. 2001:
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.