TABLE 3.

Primer sequences and specificities

Gene and primer set	Primer sequence (5′→3′)a	Amplicon (bp)b	Specificityd	Reference
16S rRNA gene
16SIGF     16SIGR	CGG CGT GGA TGA GGC AT TCA GTC CCA GTG TTG GC	298	Chlamydiales 5′ 16S rRNA gene	29
ccF	CCT CGG GTT GTA AAG CAC TTT CGC	512	Chlamydialesf	61
ccR	CCC CGT CAA TTC TTT TGA GTT T
CF1	CGT GGA TGA RGC ATG CRA GTC G	1,445	Chlamydiales	17
CR6	GTC ATC RGC CYY ACC TTD SRC RYY TCT
FOR2	CGT GGA TGA GGC ATG CAA GTC GA	264	Chlamydiales	80
REV2	CAA TCT CTC AAT CCG CCT AGA CGT CTT AG
ZpF	AAA GGT AAC GAA TAA TTG CCT	405	S. negevensis	60
ZpR	GCA CAG TCG GGG TTG AGA CCG ACT
23S rRNA gene
23SAPF2	GAA CCT GAA ACC ART AGC	92	Chlamydiales	90
23SAPR	CCT TTT GCA TGA TGA GCC AG
AFc	CAC AGG TAG GCA TGA TGA	1,099	S. negevensis	30
BR	CTA GCT GCG GGT AAA CG
IntF     IntR	TTA GAT GCA CAA TGG ATA GTT GGA CCA TCA GCG CTC ATG TGC TCA	338	S. negevensis     intron I	30
Other genes
TS1F     TS1R	ATG CTT TCG TTC TGG TCT AC CCT GCA CGG AGA CGG TTG AC	180	S. negevensis Hsp60 gene	81
Adp81Fe     Adp84R	TAG TGA TCT GCT ACG GGA TTT TTG GAT TAG GAT ATT GCT TAA A	81	P. acanthamoebae ATP/ADP translocase gene	51
Adp_probe	6-FAM-5′-AACCTTGTAGAAGTAACCTGGAAGAACCAGC-3′-TAMRA

^aFor degenerate primers, D is A, G, or C; R is A or G; S is C or G; and Y is T or C.

^bLengths of amplicons may vary slightly depending on the chlamydia taxon.

^cThe IntF/IntR set used in nested PCR on AF/AR amplicons. There are four mismatches with the F. bemisiae sequence for primers AF and IntF but no mismatch for primer AR. There are three to seven mismatches with sequences of other Chlamydiales for primers AF and AR.

^dSpecificity was determined with the sequences available at the time of primer design. For pan-chlamydia primers, such specificity may not cover all the newly determined sequences.

^eThis primer set was applied in a real-time PCR with an internal probe.

^fPrimer ccF matches exactly the last seven nucleotides at the 3′ end but has two to five mismatches at the 5′ end for sequences other than S. negevensis.