We used a whole-genome scanning technique to identify the NADH dehydrogenase

We used a whole-genome scanning technique to identify the NADH dehydrogenase gamma subunit (species in a wide range of environmental samples yet maintains minimal cross-reactivity to mammalian host and arthropod vector organisms. been shown to cause disease in humans including Carrion’s disease (13) cat scratch disease (7 14 25 endocarditis (6 11 and recently a febrile illness in humans from Thailand (caused by species are considered potential emerging pathogens (1 26 28 identification requires the ability to detect bacteria in both mammalian hosts and arthropod vectors. Although bacterial culture is considered ideal the difficulty and time involved make it impractical for large-scale use. Additionally nucleic acid-based detection techniques may be hindered by inhibitors in environmental and clinical samples low sensitivity and the absence of genus-specific primers (10 27 To address these issues we used whole-genome scanning based on the complete genomes of to identify host- and vector-blind primer sets for real-time PCR detection of in various field-collected samples. We identified a primer set based on the NADH dehydrogenase gamma subunit (species and sensitive enough to detect in both mammalian hosts and arthropod vectors. Identification of host-blind primer sets. A whole-genome scan was performed on complete genomic sequences from and and shotgun sequences from available in GenBank. Each subsequence of 16 CHR2797 17 18 and 19 nucleotides present in published genomes was compared with subsequences from other genomes present in GenBank including CHR2797 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 subsequence to the closest subsequence in the background collection was calculated to identify potential primers with a reduced probability of CHR2797 hybridizing to and amplifying nontarget DNA. In total one ultraspecific host-blind primer pair (the 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 CHR2797 sizes in the and genomes (iii) had predicted amplicon sizes of less than 400 bp and (iv) had primer melting temperatures (and 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 species (primer set exhibited high cross-reactivity both to potential hosts (spp. spp. and spp. that could inhabit comparable ecological niches (Table ?(Table11). TABLE 1. Details of primers used in this studyprimer sets against reference DNA and environmental samples. The primer sets were used Mouse monoclonal to CD53.COC53 monoclonal reacts CD53, a 32-42 kDa molecule, which is expressed on thymocytes, T cells, B cells, NK cells, monocytes and granulocytes, but is not present on red blood cells, platelets and non-hematopoietic cells. CD53 cross-linking promotes activation of human B cells and rat macrophages, as well as signal transduction. to amplify reference DNAs from 11 species chosen for their distant phylogenetic relationships under conditions optimized for each primer set. The amplification results differed considerably between primer sets and species of being amplified (Table ?(Table2)2) and are as follows: CHR2797 the primer set performed best (amplifying first with the lowest threshold cycle [set performed best on 7 of the 11 species and the set performed best on 1 of the 11 species. Although the primer set performed best for the highest number of reference species only the set successfully amplified all 11 species. TABLE 2. values for the three primer sets resulting from amplification of 11 reference DNAs derived from culture samplesDNA in field-collected hosts and vectors. Consistent with the predicted specificities from the whole-genome scans the primer set demonstrated significantly higher sensitivity and specificity for than the other primer sets by consistently yielding more sequence-confirmed PCR-positive results (Table ?(Table3).3). For the 61 total ticks sampled the primer set yielded 7 and sets respectively. Of 24 total rodent liver samples tested 18 were found to be positive by the primer set compared to 10 and 2 for the and sets respectively. TABLE 3. and and related hosts including subspecies (subsp. subsp. subsp. primer set provides better phylogenetic estimation with closely related species. was placed extremely distant to the other species with strong statistical support; conversely was placed more centrally within the phylogeny than is seen with other CHR2797 genes though this placement did not have strong statistical support. These placements which are different from those generated with multiple concatenated sequences (Fig. ?(Fig.1)1) (17) are likely due to the genetic rearrangements and horizontal gene transfer events that commonly.

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