A species-specific PCR for Lactobacillus iners demonstrates a relative specificity of this species for vaginal colonization

Figures & data

Table I.  Bacterial strains used in this study.

Strain specification	Strain name	L. iners-specific PCR
Lactobacillus iners	CCUG 28746^T	+
Lactobacillus iners	BVS011	+
Lactobacillus iners	FB077-03	+
Lactobacillus iners	FB088-01	+
Lactobacillus iners	FB094-05b	+
Lactobacillus iners	FB123-CAN-4	+
Lactobacillus iners	PB2003/087-T2-1	+
Lactobacillus iners	PB2003/195-T1-1	+
Lactobacillus brevis	LB4	−
Lactobacillus delbrueckii	LB31	−
Lactobacillus crispatus	ATCC 33199 ^T	−
Lactobacillus crispatus	33820	−
Lactobacillus casei	ATCC 393^T	−
Lactobacillus fermentum	SAV1	−
Lactobacillus fermentum	SAV 3	−
Lactobacillus fermentum	SAV 5	−
Lactobacillus fermentum	ATCC 11739^T	−
Lactobacillus fermentum	ATCC 23271	−
Lactobacillus gasseri	ATCC 33323^T	−
Lactobacillus gasseri	33323	−
Lactobacillus jensenii	ATCC 25258^T	−
Lactobacillus johnsonii	DSM 20553	−
Lactobacillus johnsonii	100-100	−
Lactobacillus johnsonii	NCK 088	−
Lactobacillus plantarum	ATCC 8014	−
Lactobacillus plantarum	ATCC 10012	−
Lactobacillus plantarum	ATCC 14917^T	−
Lactobacillus paracasei	LTH 2570	−
Lactobacillus reuteri	ATCC 23272^T	−
Lactobacillus reuteri	RC-14	−
Lactobacillus reuteri	100-23	−
Lactobacillus reuteri	DSM 20016^T	−
Lactobacillus reuteri	TMW 1.106	−
Lactobacillus rhamnosus	ATCC 7469^T	−
Lactobacillus rhamnosus	GR-1	−
Lactobacillus rhamnosus	GG	−
Lactobacillus salivarius	SAV 6	−
Lactobacillus sakei	23K	−
Enterococcus faecalis	ATCC 19433^T	−
Escherichia coli	ATCC 25922	−
Lactococcus lactis	I6	−
Lactococcus lactis	1404	−
Lactococcus lactis	V1	−
Lactococcus lactis	V5	−
Lactococcus lactis	S1	−
Lactococcus lactis	S5	−
Lactococcus lactis	V63	−
Micrococcus luteus	I1	−
Proteus vulgatus	ATCC 13315	−
Staphylococcus aureus	ATCC 25923	−
Staphylococcus saprophyticus	ATCC 15305^T	−
Streptococcus agalactiae	P3	−
Streptococcus agalactiae	211B	−
Streptococcus anginosus	I3	−
Streptococcus anginosus	SAV4	−
Streptococcus equisimilis	I9	−
Streptococcus pyogenes	I2	−
Streptococcus pyogenes	I5	−
Streptococcus pyogenes	I7	−
Streptococcus pyogenes	I8	−
Streptococcus pyogenes	P2	−
Streptococcus salivarius	SAV 2	−
Streptococcus uberis	I4	−

Table II.  Alignment of 16S rRNA sequences.

Sequence	Source
5′-G TCT GCC TTG AAG ATC GG-3′	AY283264 (position 42-101); InersFw
GAG TCT GCC TTG AAG ATC GGA GTG	Consensus of 15 L. iners sequences*
--- CT- --- -A- -T- -AT TTG ---	AF243144; L. gasseri strain BLB1b
--- CT- --- -A- -T- -AT TTG ---	AF243151; L. gasseri strain KC36a
--- CTA --- -A- -T- -AT TTG ---	AF243165; L. gasseri strain KC5a
--- CT- --- -A- -T- –T TTG ---	AF371478; uncultured bacterium
--- CT- --- -A- -T- --T TTG ---	AF371479; uncultured bacterium
--- CT- --- -A- -T- -AT TTG ---	AF519171; L. gasseri strain ATCC 33323
--- CT- --T -A- -T- -AT TTG ---	AY094066; Lactobacillus sp. IDSAc
--- CT- --- -A- -T- -AT TTG ---	AY730721; L. gasseri strain 130704-6172
--- CT- --- -A- -T- –T TT- ---	AY862434; Lactobacillus sp. ID920
--- CT- --- -A- -T- -AT TTG ---	M58820; L. gasseri (library: DSM 20243)
--- CT- --- -A- -T- –T TT- ---	M99704; L. acidophilus strain BMF 6Lb6
--- CT- --- -A- -T- -AT TTG ---	AE017198; L. johnsonii strain NCC 533
Sequence	Source
3′-CTA CTA CGG ATA GTT GAC A-5′	InersRev (position 162-221 of AY283264)
ACA GAT GAT GCC TAT CAA CTG TT	Consensus of 15 L. iners sequences*
— CTA –C –A -G- -T- GA- –	Consensus of 12 non-L. iners sequences†

*Consensus of the L. iners sequences AY283264, AY283265, AY283266, AY283267, AY283268, AY23269, AY283270, AY283271, AY283272, AY283273, AY283274, AY283275, AY526083, AY995250 and Y16329.

†Consensus of non-L. iners sequences AF243144, AF243151, AF243165, AF371478, AF371479, AF519171, AY094066, AY730721, AY862434, M58820, M99704, AE017198.

Table III.  Primers used in this study.

Primer	Sequence	Reference
InersFw	5′-GTC TGC CTT GAA GAT CGG-3′	This study
InersRev	5′-ACA GTT GAT AGG CAT CAT C-3′	This study
CbsA2F	5′-GTA CCA AGC CAA AGC AAG AC-3′	[12]
CbsA2R	5′-GTT TGA AGC CTT TAC GTA AGT C-3′	[12]
Lac1	5′-AGC AGT AGG GAA TCT TCC A-3′	[23]
Lac2	5′-GC-Clamp-ATT YCA CCG CTA CAC ATG-3′	[23]
16S Fw	5′-GGA CTA HAG GGT ATC TAA T -3′	[11]
16S Re	5′-AGA GTT TGA TCM TGG-3′	[11]

Figure 1.  Lactobacillus iners-specific PCR assay of vaginal specimens. Lanes 1–9, PCR products of vaginal specimen DNA amplified from subjects 1–9; lane 10, negative control; lane 11, positive control using L. iners DNA; lane M, GeneRuler™ DNA ladder.

Horie M, Kajikawa HS, Toba T. Identification of Lactobacillus crispatus by polymerase chain reaction targeting S-layer protein gene. Lett Appl Microbiol 2002; 35: 57–61

 Web of Science ®Google Scholar

Walter J, Hertel C, Tannock GW, Lis CM, Munro K, Hammes WP. Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis. Appl Environ Microbiol 2001; 67: 2578–85

 PubMed Web of Science ®Google Scholar

Wilson KH, Blitchington RB, Greene RC. Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction. J Clin Microbiol 1990; 28: 1942–6

 PubMed Web of Science ®Google Scholar