A whole-genome scan was performed on complete genomic sequences from B. henselae and B. quintana and shotgun sequences from B. bacilliformis available in GenBank. Each subsequence of 16, 17, 18, and 19 nucleotides present in published Bartonella genomes was compared with subsequences from other genomes present in GenBank, including genomes for bacteria that could infect human blood and tissues and potential mammalian hosts and arthropod vectors for bartonellae. The number of base changes necessary to convert each Bartonella subsequence to the closest subsequence in the background collection was calculated to identify potential primers with a reduced probability of hybridizing to and amplifying nontarget DNA.
In total, one ultraspecific, host-blind primer pair (the nuoG primer pair) was identified that met the following conditions: the pair (i) maintained at least a 2-base specificity among the complete GenBank sequence database, (ii) amplified fragments of identical sizes in the B. henselae and B. quintana genomes, (iii) had predicted amplicon sizes of less than 400 bp, and (iv) had primer melting temperatures (Tms) within 2°C. Although they did not conform to all of these conditions, the ftsZ and gltA primer sets were included in further comparisons due to the large amount of sequence data available for these genes.
Primer pairs were tested in reaction with three Bartonella species (B. henselae, B. quintana, and B. bacilliformis) and then with the use of ~30-fold excess competitor DNA from J774 (murine) and THP1 (human) tissue culture cells over the template DNA from B. henselae. Interestingly, despite their common use, the gltA primer set demonstrated high cross-reactivity both to potential Bartonella hosts (Rattus spp., Mus spp., and Homo sapiens) and to bacterial species, such as Ehrlichia spp., that could inhabit similar ecological niches (Table ).
Details of primers used in this studya