Previous Article | Next Article 
Journal of Clinical Microbiology, January 1998, p. 269-272, Vol. 36, No. 1
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Species-Specific Plasmid Sequences for PCR
Identification of the Three Species of Borrelia burgdorferi
Sensu Lato Involved in Lyme Disease
Marie-Christine
Misonne and
Philippe Pierre
Hoet*
Microbial Pathogenesis Unit, Catholic
University of Louvain Medical School, International Institute of
Cellular and Molecular Pathology, B-1200 Brussels, Belgium
Received 30 April 1997/Returned for modification 12 June
1997/Accepted 19 September 1997
 |
ABSTRACT |
Species-specific sequences were shown to be carried by plasmids of
the three main species of Borrelia burgdorferi sensu lato involved in Lyme disease. Libraries of the 16-, 33-, and 25-kb plasmids
of B. burgdorferi sensu stricto, Borrelia
garinii, and Borrelia afzelii, respectively, were
then built and used to isolate species-specific sequences. After
sequencing of the cloned inserts, three sets of primers were designed.
They were shown to determine species-specific PCR amplification
products. The sensitivities of the PCR assay with these primers were
100 spirochetes for B. burgdorferi sensu stricto and 1,000 spirochetes for B. garinii and B. afzelii. The
usefulness of these primers for the identification of species in
biological samples (tick, serum, and cerebrospinal fluid samples) was
ascertained.
| |
TEXT |
Borrelia burgdorferi
sensu lato spirochetes, which cause Lyme disease, are transmitted to a
wide range of vertebrates by the bite of infected ticks of the genus
Ixodes. Genetic and immunological studies (4, 7, 11,
19) have led to the delineation of three distinct species among
pathogenic B. burgdorferi sensu lato isolates: B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii. Recently, Borrelia japonica and
Borrelia andersonii were recognized as two novel species of
B. burgdorferi sensu lato (16, 20, 25). Up to
now, these two species were not found in Lyme disease patients. The
identification of the different species of B. burgdorferi
sensu lato might be clinically relevant, since they seem to cause
different complications (2, 3) by distinct pathogenic
mechanisms (13, 14).
B. burgdorferi sensu lato strains have a small linear
chromosome of approximately 950 kb. In addition, they harbor several circular and linear plasmids. The contribution of plasmids, considered minichromosomes, to the genetic makeup and variability of
Borrelia strains has long been recognized (5, 8, 31,
32). In view of the fact that different clinical outcomes depend
on the species of the infecting spirochete, the isolation of
species-specific sequences might help provide an understanding of the
distinct pathogenic mechanisms. We recently isolated a species-specific sequence of B. burgdorferi sensu stricto repeated on several
plasmids of this species (21).
Among the methods able to distinguish the B. burgdorferi
sensu lato species, PCRs have the advantage of not requiring previous cultivation of the organisms. In the present study, we extended the
isolation of phylogenetically significant plasmid target sequences to
the three pathogenic B. burgdorferi sensu lato genospecies. These sequences were exploited for the development of PCR tests. The
chosen primers were tested for their ability to amplify DNA from
cultured bacteria, biological fluids from patients, and infected ticks
in order to facilitate the study of risk and clinical outcome of Lyme
disease in areas inhabited by ticks.
Isolation of species-specific B. burgdorferi sensu lato
plasmid sequences.
The Borrelia strains used in this
study, listed in Table 1, were provided
by I. Saint Girons (Institut Pasteur, Paris, France). Their isolation
has been described previously (4, 6). We previously showed
in each species plasmids with distinct pulsed-field gel electrophoretic
profiles, allowing us to identify for each species a plasmid that was
present in all tested strains of a given species and that had no
prevalent counterpart with an equivalent molecular weight in strains
belonging to the other two species (21). These plasmids were
chosen as possible carriers of species-specific sequences: a 16-kb
plasmid of B. burgdorferi sensu stricto IP1, a 33-kb plasmid
of B. garinii N34, and a 25-kb plasmid of B. afzelii UO1. They were excised from the gel, radiolabeled by
random priming, and hybridized onto a dot blot with DNAs of different
strains of each species (28). As indicated in Fig.
1, the 16-kb plasmid of the B. burgdorferi sensu stricto strain hybridized strongly with DNA of
this species and poorly with the DNAs of B. garinii and
B. afzelii. The 33-kb plasmid of the B. garinii
strain hybridized strongly with the DNAs of the other strains of this
species and moderately with the DNAs of B. burgdorferi sensu
stricto B31 and B. afzelii VS461 and UMO1. It hybridized
poorly with the DNAs of other strains of B. burgdorferi
sensu stricto and B. afzelii species. The 25-kb plasmid of
the B. afzelii strain hybridized strongly with the DNAs of
the other strains of B. afzelii but also hybridized with the
DNA of B. garinii 20047. It hybridized poorly with DNAs of
other strains of B. burgdorferi sensu stricto and B. garinii. It seemed that these three plasmids contained largely
species-specific sequences.
View larger version (30K):
[in this window]
[in a new window]
|
FIG. 1.
Hybridization of the 16-kb plasmid of B. burgdorferi sensu stricto, the 33-kb plasmid of B. garinii, and the 25-kb plasmid of B. afzelii with DNAs
of the three species of B. burgdorferi sensu lato involved
in Lyme disease. DNAs (100 ng) of the B31 (dot 1), IP1 (dot 2), IP2
(dot 3), IP3 (dot 4), and IRS (dot 5) strains of B. burgdorferi sensu stricto, the 20047 (dot 6), N34 (dot 7), G25
(dot 8), and P/Bi (dot 9) strains of B. garinii, and the
VS461 (dot 10), UO1 (dot 11), UMO1 (dot 12), and Iper3 (dot 13) strains
of B. afzelii were denatured and spotted onto a nylon
membrane. The membrane was incubated in the presence of the 16-kb
radiolabeled plasmid of B. burgdorferi sensu stricto IP1
(A), the 33-kb radiolabeled plasmid of B. garinii N34 (B),
or the 25-kb radiolabeled plasmid of B. afzelii UO1 (C). The
membrane was autoradiographed.
|
|
For isolation of those specific sequences, the chosen plasmids, excised
from the agarose gel, cleaved with
Sau3A1, and separately
ligated into the
BamHI site of the pBluescript
SK
+ vector. These recombinant plasmids were introduced in
Escherichia coli DH5
F'. Recombinant colonies were
hybridized in parallel
with total cleaved radiolabeled DNA of
B. burgdorferi sensu stricto
IP1,
B. garinii N34, and
B. afzelii UO1 (
28). Three species-specific
recombinants were further studied. Plasmid pMC52, carrying cloned
sequences of the 16-kb plasmid of a
B. burgdorferi
sensu stricto
strain, hybridized in a Southern blot analysis to several
plasmids
of strains belonging to the same species (
21).
Plasmids pMC159
and pMC18, carrying sequences of the 33-kb plasmid of
B. garinii N34 and of the 25-kb plasmid of
B. afzelii UO1, respectively,
hybridized exclusively to the
equivalent plasmid of strains belonging
to their respective species
(data not shown).
Both strands of the spirochetal inserts were sequenced (
29).
The insert of plasmid pMC52, specific for
B. burgdorferi
sensu
stricto, contained 1,271 bp. The inserts of plasmid pMC159,
specific
for
B. garinii, and plasmid pMC18, specific for
B. afzelii, contained
254 and 347 bp, respectively. These
sequences, analyzed by the
DNA Strider (
17) and oligo4-s
(National Biosciences, Inc., Plymouth,
Minn.) programs, were analyzed
in a database search with the BLAST
program for homology with
nucleotide and amino acid sequences
(
1). They did not show
homology with any other sequences (BLAST
[release, June 1997]).
Selection of primers and specificity of the PCR amplification
products.
To select PCR primers suitable for the differentiation
of the three species of B. burgdorferi sensu lato involved
in Lyme disease, we have chosen a set of primers in each specific
sequence derived from each recombinant plasmid (pMC52, pMC159, and
pMC18). The nucleotide sequences of the primers and their target
species are presented in Table 2.
All primers were tested for their ability to amplify DNA from 10 B. burgdorferi sensu lato isolates and 3 relapsing fever
agents.
For PCR, the reaction mixture (50 µl) contained 1.5 mM
MgCl
2, 175 µM (each) deoxyribonucleotide triphosphates
(dATP, dCTP,
dTTP, and dGTP), 0.1% Triton X-100, 50 mM KCl, 10 mM
Tris-HCl
(pH 8.0), 2.5 U of
Thermus brockianus DNA
polymerase (Dynazyme;
Techgen), 50 pmol of each primer, and 10 ng of
spirochetal DNA
(or volumes of biological samples, as described below).
Samples
were overlaid with 50 µl of mineral oil (Sigma) and amplified
for 35 cycles (with the c/c' and MC16 primers), 30 cycles (with
MC33
primers), or 40 cycles (with MC25 primers) in a thermocycler
(New
Brunswick Scientific, Benelux b.v.) under the following conditions:
1 min at 96°C; 1 min at 54°C (with the c/c' and MC16 primers),
60°C (with MC33 primers), or 48°C (with MC25 primers); and 1 min
at
72°C. The last cycle was terminated by elongation for 10 min
at
72°C. The PCR amplification products were resolved by electrophoresis
in a 1.5% agarose gel containing 0.5 µg of ethidium bromide per
ml
and were visualized under UV light. Possible inhibition of
DNA
polymerase, yielding a false-negative result for any given
sample, was
checked by adding 10 ng of DNA of
B. burgdorferi sensu
lato
to the reaction mixture.
PCR with the MC16 primers amplified an expected 395-bp fragment from
the DNA of
B. burgdorferi sensu stricto B31, IP1, and
IRS
but not from the DNA of
B. garinii and
B. afzelii
isolates
(Fig.
2A). Fragments of 236 bp
from
B. garinii 20047, N34, and
G25 were amplified in the
PCR assay with the MC33 primers but
not from the strains of
B. burgdorferi sensu stricto or
B. afzelii (Fig.
2B).
Finally, the MC25 primers amplified an expected 125-bp
fragment from
the DNA of
B. afzelii VS461, UO1, and Iper3 but
not from the
DNA of
B. burgdorferi sensu stricto or
B. garinii (Fig.
2C). The MC16, MC33, and MC25 primers did not amplify the
DNA of
B. japonica Cow611C,
B. hermsii,
B. parkeri, or
B. turicatae.
No signal was observed when
template DNA was omitted from the
amplification reaction mixture (faint
signals are nucleotides
and oligonucleotides not used in the reaction).
View larger version (46K):
[in this window]
[in a new window]
|
FIG. 2.
Specificity of the chosen primers for amplification of
B. burgdorferi sensu stricto, B. garinii, or
B. afzelii DNA. DNAs from B. burgdorferi sensu
stricto B31 (lanes 3), IP1 (lanes 4), and IRS (lanes 5), B. garinii 20047 (lanes 6), N34 (lanes 7), and G25 (lanes 8),
B. afzelii VS461 (lanes 9), UO1 (lanes 10), and Iper3 (lanes
11), B. japonica Cow611C (lanes 12), B. hermsii
(lanes 13), B. parkeri (lanes 14), and B. turicatae (lanes 15) were amplified with the MC16 (A), MC33 (B),
or MC25 (C) primers. The amplified products were separated on a 1.5%
agarose gel and revealed under UV light after the addition of ethidium
bromide (10 µg/ml). Lanes 2, control without DNA; lanes 1, DNA
molecular mass markers (587, 540, 504, 458, 434, 267, 234, 213, 192, 184, and 124 bp).
|
|
The species-specific primers described here recognize different
plasmids. Since only strains with high passage numbers were
analyzed,
these plasmids must be stably maintained and the primers
should be able
to distinguish most if not all
B. burgdorferi sensu
lato
strains in nature belonging to the three pathogenic species.
Other
species-specific primers were described on chromosomal (
fla,
orfX,
rRNA) (
15,
18,
24,
27) or
plasmid (
ospA) genes (
10,
22). These primers were
able to distinguish the species by their
ability to recognize specific
sequences within a given gene shared
by all spirochetes. A current
comparison of two different sets
of primers for species identification
underlines the genetic heterogeneity
of spirochetes in nature
(
20a).
Sensitivity of the PCR amplification assay.
The detection
threshold of the PCR assay was determined by performing amplification
reactions with serially diluted samples of DNA from B. burgdorferi sensu stricto, B. garinii, or B. afzelii. Amplification reactions were performed with aliquots
containing amounts of DNA equivalent to given numbers of spirochetes
(from 107 to 1) and with the MC16 (Fig. 3A), MC33 (Fig.
3B), and MC25 (Fig. 3C) primers. It was
found that a template DNA input corresponding to 100 spirochetes was
sufficient for the detection of the amplified fragment of B. burgdorferi sensu stricto and that 1,000 spirochetes was
sufficient for the detection of the amplified fragments of B. garinii and B. afzelii.
View larger version (58K):
[in this window]
[in a new window]
|
FIG. 3.
Sensitivity of the B. burgdorferi sensu
stricto, B. garinii, and B. afzelii
species-specific primers. DNA corresponding to 107 (lanes
2), 106 (lanes 3), 105 (lane 4),
104 (lanes 5), 103 (lanes 6), 102
(lanes 7), 10 (lanes 8), 1 (lanes 9), or 0 (lanes 10) spirochetes of
B. burgdorferi sensu stricto IP1 (A), B. garinii
N34 (B), or B. afzelii UO1 (C) was amplified with the MC16
(A), MC33 (B), or MC25 (C) primers. The amplified products were
electrophoretically separated in a 1.5% agarose gel and revealed under
UV light after the addition of ethidium bromide (10 µg/ml). Lanes 1, DNA molecular mass markers (587, 540, 504, 458, 434, 267, 234, 213, 192, 184, and 124 bp).
|
|
The usefulness of these primers for the identification of species in
biological samples was ascertained. Biological samples
came from
patients with Lyme disease diagnosed during the summer
of 1996 at the
Belgian Reference Center for Borreliosis (G. Bigaignon,
Infectious
Serology Laboratory, Saint-Luc Hospital, Brussels,
Belgium) by positive
enzyme-linked immunosorbent assay (Diagast,
Lille, France) and PCR
(Biocode, Sclessin, Belgium) test results.
Sera and cerebrospinal
fluids (150 µl) were centrifuged (13,000
×
g, 20 min), and the pellets were washed three times with phosphate-buffered
saline and resuspended in 60 µl of H
2O. After heat
denaturation
(10 min at 100°C), 20 µl was added to the PCR mixture.
Ticks were collected in July 1996 from vegetation on the ground in the
forest near Matagne-la-Petite, Namur Province, Belgium.
Ticks were kept
at 4°C in alcohol until use. Ticks were dried
and incubated in 100 µl of TE (10 mM Tris-HCl [pH 7.8], 1 mM EDTA)
containing 200 µg
of proteinase K (Boehringer Mannheim, Mannheim,
Germany) per ml. After
overnight incubation at room temperature,
the ticks were crushed with a
pipette tip, boiled for 10 min,
and then placed on ice for 10 min. The samples were centrifuged
at 13,000 ×
g for 10 min, and supernatants were collected and
stored at
20°C. A total of
5 µl of the supernatants was added
to the PCR mixture.
Three serum samples, two cerebrospinal fluid samples, and four ticks
containing
B. burgdorferi sensu lato DNA, as shown by
amplification with the c/c' primers of Rosa et al. (
27),
were
analyzed further to identify the
Borrelia species in
these samples
(Table
3). Only one sample
from a human contained DNA from a
unique species, whereas the four
others contained DNA originating
from at least two different species of
B. burgdorferi sensu lato.
PCR with the MC16 or the MC33
primer amplified DNA from three
samples from patients, whereas PCR with
the MC25 primer, amplified
DNA from four samples. Of the four ticks,
one contained the DNA
of only one species and the three others
contained the DNAs of
two or three
B. burgdorferi sensu lato
species. The three primer
sets were thus able to amplify borrelial DNA
in biological samples.
The sensitivities of the PCR amplification assays with the primers
designed in this work seemed adequate and at least equivalent
to those
with the general c/c' primers (
27). Indeed, the infecting
species could be identified with the primers being studied in
all the
samples, which were selected by the ability of the c/c'
primers to
amplify DNA. The detection of
B. burgdorferi sensu
stricto
seemed more sensitive than the detection of the other
two species (Fig.
3). This is probably due to the presence of
the target sequence of the
corresponding primers (MC16) in several
plasmids of this species
(
21). Interestingly, homologous regions
of DNA have been
found in different plasmids of
Borrelia strains
(
9,
30,
31,
33). These shared sequences are not specific
for any one of
the described
B. burgdorferi sensu lato genospecies,
however.
The PCR data indicated the usefulness of these primers with tick and
clinical samples (Table
3). This analysis revealed several
cases of
infection with multiple
Borrelia species, in agreement
with
observations with ticks (
12,
23,
26) and biological
fluids
from patients with Lyme disease (
10).
Nucleotide sequence accession numbers.
The accession
numbers for the nucleotide sequences (EMBL Nucleotide Sequence
Database) reported here are U12332 for B. burgdorferi sensu
stricto, U83998 for B. garinii, and U84145 for B. afzelii.
| |
ACKNOWLEDGMENTS |
M. C. Misonne received a predoctoral grant from Institut
d'Encouragement de la Recherche Scientifique dans
l'Industrie et l'Agriculture. P. Hoet is research director of FNRS
(National Fund for Scientific Research, Brussels, Belgium).
We thank P. Gilot for critically reading the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Microbial
Pathogenesis Unit, Catholic University of Louvain Medical School,
International Institute of Cellular and Molecular Pathology, 74, Avenue
Hippocrate, B-1200 Brussels, Belgium. Phone and Fax: 32 2 764 74 51. E-mail: hoet{at}mipa.ucl.ac.be.
| |
REFERENCES |
| 1.
|
Altschul, S. F.,
W. Gish,
W. Miller,
E. W. Myers, and D. J. Lipman.
1990.
Basic local alignment search tool.
J. Mol. Biol.
215:403-410[Medline].
|
| 2.
|
Anthonissen, F. M.,
M. De Kesel,
P. P. Hoet, and G. H. Bigaignon.
1994.
Evidence for the involvement of different genospecies of Borrelia in the clinical outcome of Lyme disease in Belgium.
Res. Microbiol.
145:327-331[Medline].
|
| 3.
|
Assous, M. V.,
D. Postic,
G. Paul,
P. Névot, and G. Baranton.
1993.
Western blot analysis of sera from Lyme borreliosis patients according to the genomic species of the Borrelia strains used as antigens.
Eur. J. Clin. Microbiol. Infect. Dis.
12:261-268[Medline].
|
| 4.
|
Baranton, G.,
D. Postic,
I. Saint Girons,
P. Boerlin,
J. C. Piffaretti,
M. Assous, and P. A. D. Grimont.
1992.
Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis.
Int. J. Syst. Bacteriol.
42:378-383[Abstract/Free Full Text].
|
| 5.
|
Barbour, A. G.
1988.
Plasmid analysis of Borrelia burgdorferi, the Lyme disease agent.
J. Clin. Microbiol.
26:475-478[Abstract/Free Full Text].
|
| 6.
|
Boerlin, P.,
O. Peter,
A.-G. Bretz,
D. Postic,
G. Baranton, and J.-C. Piffaretti.
1992.
Population genetic analysis of Borrelia burgdorferi isolates by multilocus enzyme electrophoresis.
Infect. Immun.
60:1677-1683[Abstract/Free Full Text].
|
| 7.
|
Canica, M. M.,
F. Nato,
L. du Merle,
J. C. Mazie,
G. Baranton, and D. Postic.
1993.
Monoclonal antibodies for identification of Borrelia afzelii sp. nov. associated with late cutaneous manifestations of Lyme borreliosis.
Scand. J. Infect. Dis.
25:441-448[Medline].
|
| 8.
|
Casjens, S.,
M. Delange,
H. L. Ley III,
P. Rosa, and W. M. Huang.
1995.
Linear chromosomes of Lyme disease agent spirochetes: genetic diversity and conservation of gene order.
J. Bacteriol.
177:2769-2780[Abstract/Free Full Text].
|
| 9.
|
Casjens, S.,
R. van Vugt,
K. Tilly,
P. A. Rosa, and B. Stevenson.
1997.
Homology throughout the multiple 32-kilobase circular plasmids present in Lyme disease spirochetes.
J. Bacteriol.
179:217-227[Abstract/Free Full Text].
|
| 10.
|
Demaerschalck, I.,
A. Ben Messaoud,
M. De Kesel,
B. Hoyois,
Y. Lobet,
P. Hoet,
G. Bigaignon,
A. Bollen, and E. Godfroid.
1995.
Simultaneous presence of different Borrelia burgdorferi genospecies in biological fluids of Lyme disease patients.
J. Clin. Microbiol.
33:602-608[Abstract].
|
| 11.
|
Fukunaga, M.,
M. Sohnaka, and Y. Yanagihara.
1993.
Analysis of Borrelia species associated with Lyme disease by rRNA gene restriction fragment length polymorphism.
J. Gen. Microbiol.
139:1141-1146.
|
| 12.
|
Guttman, D. S.,
P. W. Wang,
I.-N. Wang,
E. M. Bosler,
B. J. Luft, and D. E. Dykhuizen.
1996.
Multiple infections of Ixodes scapularis ticks by Borrelia burgdorferi as revealed by single-strand conformation polymorphism analysis.
J. Clin. Microbiol.
34:652-656[Abstract].
|
| 13.
|
Isogai, E.,
H. Isogai,
K. Kimura,
S. Hayashi,
T. Kubota,
T. Nishikawa,
A. Nakane, and N. Fujii.
1996.
Cytokines in the serum and brain in mice infected with distinct species of Lyme disease Borrelia.
Microb. Pathog.
21:413-419[Medline].
|
| 14.
|
Isogai, E.,
K. Kimura,
N. Fujii,
T. Nishikawa,
N. Ishii,
D. Postic,
G. Baranton, and H. Isogai.
1996.
Platelet-activating-factor-mediated pathogenesis in Lyme disease.
Infect. Immun.
64:1026-1029[Abstract].
|
| 15.
|
Johnson, B. J. B.,
C. M. Happ,
L. W. Mayer, and J. Piesman.
1992.
Detection of Borrelia burgdorferi in ticks by species-specific amplification of the flagellin gene.
Am. J. Trop. Med. Hyg.
47:730-741.
|
| 16.
|
Kawabata, H.,
T. Masuzawa, and Y. Yanagihara.
1993.
Genomic analysis of Borrelia japonica sp. nov. isolated from Ixodes ovatus in Japan.
Microbiol. Immunol.
37:843-848[Medline].
|
| 17.
|
Marck, C.
1988.
"DNA strider": a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers.
Nucleic Acids Res.
16:1829-1836[Abstract/Free Full Text].
|
| 18.
|
Marconi, R. T., and C. F. Garon.
1992.
Development of polymerase chain reaction primer sets for diagnosis of Lyme disease and for species-specific identification of Lyme disease isolates by 16S rRNA signature nucleotide analysis.
J. Clin. Microbiol.
30:2830-2834[Abstract/Free Full Text].
|
| 19.
|
Marconi, R. T.,
L. Lubke,
W. Hauglum, and C. F. Garon.
1992.
Species-specific identification of and distinction between Borrelia burgdorferi genomic groups by using 16S rRNA-directed oligonucleotide probes.
J. Clin. Microbiol.
30:628-632[Abstract/Free Full Text].
|
| 20.
|
Marconi, R. T.,
D. Liveris, and I. Schwartz.
1995.
Identification of novel insertion elements, restriction fragment length polymorphism patterns, and discontinuous 23S rRNA in Lyme disease spirochetes: phylogenetic analyses of rRNA genes and their intergenic spacers in Borrelia japonica sp. nov. and genomic group 21038 (Borrelia andersonii sp. nov.) isolates.
J. Clin. Microbiol.
33:2427-2434[Abstract].
|
| 20a.
| Misonne, M. C., et al. Unpublished data.
|
| 21.
|
Misonne, M. C.,
M. Schuttler,
J. M. Dernelle,
M. De Kesel, and P. Hoet.
1997.
Cloning and sequencing of a species-specific nucleotide fragment of Borrelia burgdorferi sensu stricto, which is repeated in several plasmids of the species.
FEMS Microbiol. Lett.
150:157-164[Medline].
|
| 22.
|
Persing, D. H.,
S. R. Telford III,
A. Spielman, and S. W. Barthold.
1990.
Detection of Borrelia burgdorferi infection in Ixodes dammini ticks with the polymerase chain reaction.
J. Clin. Microbiol.
28:566-572[Abstract/Free Full Text].
|
| 23.
|
Pichon, B.,
E. Godfroid,
B. Hoyois,
A. Bollen,
F. Rodhain, and C. Pérez-Eid.
1995.
Simultaneous infection of Ixodes ricinus nymphs by two Borrelia burgdorferi sensu lato species: possible implications for clinical manifestations.
Emerg. Infect. Dis.
1:89.
|
| 24.
|
Picken, R. P.
1992.
Polymerase chain reaction primers and probes derived from flagellin gene sequences for specific detection of the agents of Lyme disease and North American relapsing fever.
J. Clin. Microbiol.
30:99-114[Abstract/Free Full Text].
|
| 25.
|
Postic, D.,
J. Belfaiza,
E. Isogal,
I. Saint Girons,
P. A. D. Grimont, and G. Baranton.
1993.
A new genomic species in B. burgdorferi sensu lato isolated from Japanese ticks.
Res. Microbiol.
144:467-473[Medline].
|
| 26.
|
Rijpkema, S. G. T.,
M. J. C. H. Molkenboer,
L. M. Schouls,
F. Jongejan, and J. F. P. Schellekens.
1995.
Simultaneous detection and genotyping of three genomic groups of Borrelia burgdorferi sensu lato in Dutch Ixodes ricinus ticks by characterization of the amplified intergenic spacer region between 5S and 23S rRNA genes.
J. Clin. Microbiol.
33:3091-3095[Abstract].
|
| 27.
|
Rosa, P. A.,
D. Hogan, and T. G. Schwan.
1991.
Polymerase chain reaction analyses identify two distinct classes of Borrelia burgdorferi.
J. Clin. Microbiol.
29:524-532[Abstract/Free Full Text].
|
| 28.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 29.
|
Sanger, F.,
S. Nicklen, and A. R. Coulson.
1977.
DNA sequencing with chain-terminating inhibitors.
Proc. Natl. Acad. Sci. USA
74:5463-5467[Abstract/Free Full Text].
|
| 30.
|
Simpson, W. J.,
C. F. Garon, and T. G. Schwan.
1990.
Borrelia burgdorferi contains repeated DNA sequences that are species specific and plasmid associated.
Infect. Immun.
58:847-853[Abstract/Free Full Text].
|
| 31.
|
Xu, Y., and R. C. Johnson.
1995.
Analysis and comparison of plasmid profiles of Borrelia burgdorferi sensu lato strains.
J. Clin. Microbiol.
33:2679-2685[Abstract].
|
| 32.
|
Zingg, B. C.,
R. N. Brown,
R. S. Lane, and R. B. LeFebvre.
1993.
Genetic diversity among Borrelia burgdorferi isolates from wood rats and kangaroo rats in California.
J. Clin. Microbiol.
31:3109-3114[Abstract/Free Full Text].
|
| 33.
|
Zückert, W. R., and J. Meyer.
1996.
Circular and linear plasmids of Lyme disease spirochetes have extensive homology: characterization of a repeated DNA element.
J. Bacteriol.
178:2287-2298[Abstract/Free Full Text].
|
Journal of Clinical Microbiology, January 1998, p. 269-272, Vol. 36, No. 1
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Ruzic-Sabljic, E., Arnez, M., Logar, M., Maraspin, V., Lotric-Furlan, S., Cimperman, J., Strle, F.
(2005). Comparison of Borrelia burgdorferi Sensu Lato Strains Isolated from Specimens Obtained Simultaneously from Two Different Sites of Infection in Individual Patients. J. Clin. Microbiol.
43: 2194-2200
[Abstract]
[Full Text]
-
Ranka, R., Bormane, A., Salmina, K., Baumanis, V.
(2004). Identification of Three Clinically Relevant Borrelia burgdorferi Sensu Lato Genospecies by PCR-Restriction Fragment Length Polymorphism Analysis of 16S-23S Ribosomal DNA Spacer Amplicons. J. Clin. Microbiol.
42: 1444-1449
[Abstract]
[Full Text]
-
Pietilä, J., He, Q., Oksi, J., Viljanen, M. K.
(2000). Rapid Differentiation of Borrelia garinii from Borrelia afzelii and Borrelia burgdorferi Sensu Stricto by LightCycler Fluorescence Melting Curve Analysis of a PCR Product of the recA Gene. J. Clin. Microbiol.
38: 2756-2759
[Abstract]
[Full Text]
-
Wang, G., van Dam, A. P., Schwartz, I., Dankert, J.
(1999). Molecular Typing of Borrelia burgdorferi Sensu Lato: Taxonomic, Epidemiological, and Clinical Implications. Clin. Microbiol. Rev.
12: 633-653
[Abstract]
[Full Text]
-
Misonne, M.-C., Van Impe, G., Hoet, P. P.
(1998). Genetic Heterogeneity of Borrelia burgdorferi Sensu Lato in Ixodes ricinus Ticks Collected in Belgium. J. Clin. Microbiol.
36: 3352-3354
[Abstract]
[Full Text]
Top
Abstract
Text
