Volume 11, Number 2—February 2005
Research
Isolation of Waddlia malaysiensis, A Novel Intracellular Bacterium, from Fruit Bat (Eonycteris spelaea)
Paul K.B. Chua*†, John E. Corkill*, Poh Sim. Hooi‡, Soo Choon Cheng‡, Craig Winstanley*, and C. Anthony Hart*
Table 1
Oligonucleotide primers for PCR and sequencing*
| Gene target | Primer sequence |
|---|---|
| PCR | |
| 16S rRNA (1,526 bp from ref. 20) | |
| 16S-FOR | 5′ AGA GTT TGA TCC TGG 3′ |
| 16S-REV | 5′ TAC CTT GTT ACG ACT T 3′ |
| Tm = 55ºC | |
| 16S rRNA signature sequence (298 bp from ref. 21) | |
| 16S1GF | 5′ CGG CGT GGA TGA GGC AT 3′ |
| 16S1GR | 5′ TCA GTC CCA GTG TTG GC 3′ |
| Tm = 51º C | |
| 16S – 23S rRNA signature sequence (1 kbp from ref. 21) | |
| 16SF2 | 5′ CCG CCC GTC ACA TCA TGG 3′ |
| 23S1GR | 5′ TGG CTC ATC ATG CAA AAG GCA 3′ |
| Tm = 61º C | |
| 23S rRNA signature sequence (627 bp: domain I from ref. 21) | |
| 23S1GR | 5′ TGG CTC ATC ATG CAA AAG GCA 3′ |
| Tm = 61º C | |
| MurA signature sequence (690 bp from ref. 22) | |
| murA-for | 5′ GTN GGN GCN ACN GAR AA 3' |
| murA-rev | 5′ GCC ATN ACR TAN GCR AAN CCN GC 3′ |
| Tm = 55º C | |
| sctN (331 bp) | |
| sctN FOR | 5' AGA RGG AAT GAA ACG TTC 3' |
| sctN REV | 5' GGC TCR TTC ATA TCA TC 3' |
| Tm = 58º C | |
| Sequencing | |
| 16S rRNA (1,526 bp) | |
| Forward: | |
| F1 (M13) | 5 GTT TTC CCA GTC ACG ACG TTG TA 3′ |
| F2 | 5′ GCT CAC CAA GGC TAA GAC GTC 3′ (277-298) |
| F3 | 5′ CTA GCT TTG ACC TGA CGC TGA T 3′ (752-774) |
| F4 | 5′ GAA TCT GCA ACT CGG CTC CAT G 3′ (1323-1345) |
| Reverse: | |
| R1 (M13) | 5′ TTG TGA GCG GAT AAC AAT TTC 3′ |
| R2 | 5′ CAT CCT AAA TGC TGG CAA C 3' (392-373) |
| R3 | 5′ CAC CGC TAC ATG TGG AAT TCC 3′ (843-822) |
| R4 | 5′ GAT CCT CTC TAG CAC CAT ATC C 3′ (1358-1336) |
*PCR, polymerase chain reaction; Tm, melting temperature.
