Investigation of infectious agents associated with arthritis by reverse transcription PCR of bacterial rRNA

Table 3 Primers and amplification conditions used in the study

Forward primer (5'-3')	Reverse primer (5'-3')	Specific for	Annealing temperature/time	MgCl₂ concentration (mmol/l)	Size of product (bp)
AGTAGTTTACTACTTTGCCG	ACTGCTGCCTCCCGTAGGAG	Universal (R1 and R2)	58°C/60 secs	1.5	350
CATAACGTCGCAAGACCAAA	GTGCAATATTCCCCACTGCT	E. coli	58°C/45 secs	1.5	187
TTGGGAATAACGGTTGGAAA	TGTCTCAGTCCCAGTGTTGG	Chlamydia spp.	59°C/30 secs	1.5	203
CGCACGGGTGAGTAAGGTA	GCGTCATAGCCTTGGTAAGC	Campylobacter spp.	66°C/1 min	2.5	170
AGTAGTTTACTACTTTGCCG	CCGATGGCGTGAGGCCCTAA	Yersinia spp.	65°C/30 secs	3.5	154
CGCACGGGTGAGTAAGGTA	GCTTAACACAAGTTGACTAG	Campylobacterjejuni	63°C/30 secs	2.5	70
CGCACGGGTGAGTAAGGTA	GTCTTACATAAGTTAGATA	Campylobacter concisus	55°C/30 secs	2.0	70
CGCACGGGTGAGTAAGGTA	ATACCTCATACTCCTATTTAAC	Bacteroides ureolyticus	55°C/30 secs	2.0	70

Specific oligonucelotides were designed as described here. Standard reaction mix was used with each set of oligonucleotides (see Materials and method), although annealing conditions varied, as did MgCl₂ concentration. Denaturation conditions were always 94°C for 1 min and extension was performed at 72°C for 1 min (10 min final extension). Typically 35–40 cycles were performed. If no PCR product was detected, then a second round of amplification was performed using 2 μl of PCR product as template.

ISSN: 1478-6362