The B. henselae MLST scheme uses internal fragments of the following seven house-keeping genes and 16s loci:
- 16S rRNA (small subunit rRNA)
- batR (regulatory protein of batR/S two component regulatory system)
- ribC (riboflavin synthase)
- groEL (Hsp60 chaperone)
- gltA (citrate synthase)
- nlpD (glycoprotein)
- ftsZ (cell division protein)
- rpoB (RNA polymerase beta subunit)
PCR Amplification of genes from isolates
The primer pairs used for the PCR amplification of internal fragments of these genes are:
|gene||product size (bp)||forward primer||reverse primer|
PCR Conditions for MLST housekeeping genes
Amplification of each of the MLST housekeeping genes from isolates is carried out using a single round PCR assay that amplifies a fragment of each of the loci in the table above. Each reaction mixture comprised 12.5 µl of 2x PCR mastermix (Abgene), 0.5 µl of a 20 ρmol/μl solution of both forward and reverse primers, 10.5 µl sterile, distilled water and 1 µl of bacterial cell suspension. Reaction mixtures are exposed to a thermal cycle consisting of 96°C for 5 min followed by 40 cycles of 96 °C for 10 s, 55 °C for 10 s and 72 °C for 50 s, with a final extension step of 72 °C for 10 min.
Due to the difficulty in obtaining isolates of B. henselae from humans, alternative primers have been suggested in the table below for use in the first round of a nested PCR to allow amplification of the loci from human clinical DNA extracts. The second round primers are the primers from the original B. henselae MLST scheme. Reaction mixes and thermal cycling conditions are the same as those described above, with 1 µl of 1st round post-amplification PCR mix being incorporated as template into a second round reaction mix.
|gene||nested PCR round||forward primer||reverse primer|
The same primer pairs as used for amplification (or the second round of amplification when nested PCRs are employed are also used for sequencing.