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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*Comments to Author 
Author affiliations: *University of Liverpool, Liverpool, United Kingdom; †National Public Health Laboratory, Kuala Lumpur, Malaysia; ‡University of Malaya, Kuala Lumpur, Malaysia

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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.

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