Table 2.
DNA oligonucleotides toolbox for detection and characterization of different bacteria causing fever of intermediate duration
| Bacterial genus | Oligonucleotides | Amplicon length (bp) | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Useb | Binding Region | Positions (5’-to-3’)c | Sequenced, e | Length (nt) | Modification | Tm (ºC)f | |||
| Coxiellaa | A | Transposase gene (IS1111) | 200-221 | TATGTATCCACCGTAGCCAGTC | 22 | FAM | 64.0 | 686 | |
| A | 885-865 | CCCAACAACACCTCCTTATTC | 21 | 63.2 | |||||
| P | 240-223 | ACCACGCAGCCCACCTTA | 18 | Cy5 | 65.6 | ||||
| S | 240-256 | TGATGGAAGCGTGTGGA | 17 | 58.3 | |||||
| Rickettsia | A | 23S rRNA gene | 32-56 | TGTAGCTAACYGATACTAATAGCTC | 25 | FAM | 58.8-63.0 | 302-372 | |
| A | 5S rRNA gene | 379-359 | GATCGTGTGTTTCACTCATGC | 21 | 62.2 | ||||
| P | 23S rRNA gene/ intergenic spacer | 81-57 | AATCTCACAGCAAAGTAAATCAATC | 25 | Cy5 | 64.7 | |||
| S | 5S rRNA gene/ intergenic spacer | 365-346 | CTCATGCTATRACCACCAAG | 20 | 57.2-59.7 | ||||
| Bartonella | A | 23S rRNA gene | 1,434,678-1,434,696 | GSATCCACCAAATGCCCTT | 19 | FAM | 61.8-62.5 | 245-559 | |
| A | intergenic spacer | 1,435,059-1,435,035 | GTGAAGAGAAGATATATTCARACAT | 25 | 57.2-58.7 | ||||
| P | 23S rRNA gene/ intergenic spacer | 1,434,744-1,434,721 | GGCAATGAGAACGATCAAGTGTCT | 24 | Cy5 | 65.2 | |||
| S | 1,434,690-1,434,712 | TGCCCTTAAGACACTTGATCGTT | 23 | 62.8 | |||||
| Anaplasma/Ehrlichia | A | 16S rRNA gene | 89-118/ 131-160 | GRATAGCCAYTAGAAATGATGGGTAATACT | 30 | 64.4-66.6 | 572 | ||
| A | 660-637/ 702-679 | AGTGTCAGTATCGRACCAGAYAGC | 24 | 63.0-67.0 | |||||
| P | 220-243/ 262-285 | GGTCTGAGAGGACGATCAGCCACA | 24 | Cy5 | 69.8 | ||||
| P | 248-277/ 290-319 | AACTGAGATACGGTCCAGACTCCTACGGGAA | 30 | 5’-FAM, 3’-P | 72.8 | ||||
| S | 634-615/ 663-644 | CCTTCGCCACTGGTGTTCCT | 20 | 63.8 | |||||
aAmplification primers described in Willems et. al [23]
bA, amplification primer; S, sequencing primer; P, probe
cNucleotide positions relative to reference sequences from GenBank of C. burnetii (M80806), R. conorii (AY125012), B. henselae (CP020742), A. phagocytophilum (AF470701) and E. chaffeensis (U60476)
dNucleotides with an attached fluorescent dye (Cy5/FAM) or blocked with phosphate (P) are highlighted in bold
eDegenerate positions: R (A or G); S (G or C); Y (C or T)
fTheoretical melting temperature (Tm) assuming 100% complementarity between the oligonucleotide and its target. Minimum and maximum Tm values are indicated for degenerate primers