Molecular and Cellular Probes (1992) 6, 89-92
Detection of Chlamydiae trachomatis by use of polymerase chain reaction Gerard Lucotte, 1,2 * Marie-Christine Petit,' Marie-Helene Francois' and Stephane Reveilleau 2 'Laboratoire d'Anthropologie Physique, College de France, 11, Place Marcelin Berthelot, Paris 5e , France, and 2Laboratoire Eurobio, ZA de Courtaboeuf, 7 Avenue de Scandinavie, Les Ulis, 91 France (Received 29 March 1991, Accepted 22 July 1991)
A polymerase chain reaction (PCR) assay was developed for detection of Chlamydia trachomatis DNA . One primer set was used, from the published sequence of the common C . trachomatis plasmid . Detection of amplified sequences was carried out by agarose gel electrophoresis . Analysis of 106 clinical samples tested by cell culture and PCR showed a sensitivity of 100% when PCR was compared with cell culture .
KEYWORDS: Chlamydia trachomatis, DNA plasmid, PCR, detection
INTRODUCTION highly conserved cryptic 7.5 kb plasmid:' restriction mapping of plasmids from 15 serotypes and direct sequence analysis of plasmids from serotypes B, 6 L1' and L28 have shown this plasmid to be highly conserved within the species C . trachomatis . (The entire plasmid sequence is known .) We, and others, 9'' 0 have thus chosen this plasmid as a target sequence for DNA amplification, and this study compares cell culture and PCR sensitivity of a large number of clinical specimens.
Chlamydia trachomatis, an obligate intracellular parasite, is a common agent of sexually transmitted disease and the causative agent of several human clinical conditions including urethritis, cervicitis, salpingitis, female infertility, lymphogranoluma venereum and trachoma . The diagnosis of C. trachomatis as a pathogen is currently difficult and expensive, because laboratory diagnosis is achieved by isolation in cell culture, a technique which is both cumbersome and time-consuming. The polymerase chain reaction (PCR), an efficient in vitro method of DNA amplification, is a powerful means for detecting specific sequences .' This technique has been used to detect and differentiate C . trachomatis and Chlamydia psittaci in laboratory samples of infected McCoy cells .' PCR has also been used to detect major outer membrane protein gene sequences from the three species of Chlamydia,; and in serovars of C . trachomatis ." We adapted PCR to detect C. trachomatis in clinical samples . All C . trachomatis strains possess a
MATERIALS AND METHODS Samples A total of 106 endocervical specimens (obtained from patients in Paris) were studied . Clinical samples were cultured" and stained at 48-h with fluorescent-antibody culture confirmation reagent; a direct immunofluorescence test (DFA) was also performed using a
*Author to whom correspondence should be addressed at Laboratoires Eurobio, ZA Courtaboeuf, 7, Avenue de Scandinavie, F 91953 Les Ulis Cedex B ., France.
0890-8508/92/020089 + 04 $03 .00/0
89
° 1992 Academic Press Limited
G. Lucotte et al .
90 commercially
available
assay
(Chlamydiazyme,
1 mgl - ') ; 5µl of each of primers A and B (2µM per
µp;
Abbott) . Clinical samples were taken with plastic
10
swabs,
dTTP (100/mM) ; 1 unit of Taq polymerase (Cetus Corp .) . The samples were subjected to 29 cycles of
put
into
transport
medium
(sacharose
68 . 46 gl - ', K,HPO 4 2 gl - ', KH Z PO, 1 gl - ') and conserved at -80 ° C . A volume, 100µI, of each was of lysis buffer (10 mm Tris-HCI, pH mixed with 200
8 gl of the four dNTPs mix : dATP, dGTP, dCTP,
amplification in a thermal cycler as follows : 95 ° C for
µl
15 s, 55 ° C for 1 min, 72°C for 1 min (and kept for the
7 . 5, 1 mm EDTA, 0 . 1 % SDS), 5 .tl of proteinase K
last 30th cycle at 72 ° C for 10 min to complete the
(10 mg ml - ') and shaken at 42°C overnight (and then
extension) . PCR products were resolved in a 2 . 5%
heated to 95 ° C for 5 min).
agarose gel stained with ethidium bromide (Fig . 1) and DNA fragments were detected by ultra-violet light exposure . Southern transfer and hybridization in 3 x SSC were performed on Hybond N (Amersham) .
Oligo One pair of primers which defines one segment of 201
by
was selected : 10
primer
RESULTS
A, 5'-
TTCCCCTTGTAATTCGTTGC-3' and primer B, 5'-TAGTAACTGCCACTTCATCA-3', between the Eco RI and
In this survey, which took into account 106 samples,
Cla I sites in the corresponding plasmid sequence,
PCR was compared with cell culture and direct
centered on the unique Eco RV site.
immunofluorescence for C . trachomatis detection . Among the specimens processed (Table 1), 10 were positive for PCR; seven of these were positive by cell
PCR
culture, and one was positive by the direct immunofluorescence test . The sensitivity of cell culture tech-
A volume, 22 pl, of each of the prepared samples was used in a 50 PCR reaction : 5 .tl of PCR buffer (10 mm
nique is not precisely known, but is less than 100% ;"
Tris-HCI, pH 8 . 3, 1 .5 MM MgCl2, 50 mm KCI, gelatin
mens positive by culture and positive by direct
µl
P
2
3
4
if we use a standard of positivity the sum of speci-
5
6
7
8
9
10
12
201 by --t
Fig. 1 . Detection of amplification product by means of electrophoresis of C . trachomatis plasmid obtained by PCR (the 201 by specific band) . M, DNA marker; P, a positive sample ; 1-12, 12 samples tested, 1 and 2 being positive . After hybridization of the corresponding blot with 12 P-labelled plasmid probe," the PCR product was revealed .
91
PCR Detection of Chlamydia trachomatis Table 1 . Summary of the results obtained concerning 106 samples studied during 15 experiments, where PCR, culture and/or direct examination are simultaneously involved Experiment no . 1 2
3 4 5 6 7 8 9 10 11
No . analysed
PCR positive
Culture positive
5
1
1
3 11
3
2
2
2
16 7 6 6 5 7 11 7
1
2
1
12 13
5
14 15
10 4
1
1
106
10
7
Total
FA positive
3
immunofluorescence, then PCR diagnosed all positive samples detected in this study. There were two specimens which were culture negative and PCR positive . One of these samples was obtained from a person undergoing antibiotic treatment .
DISCUSSION Detection of genital chlamydial infection is currently based on the cell culture technique, and enzymelinked immunasorbents and immunofluorescence essays ; none of these tests display optimal sensitivity . In this report we describe the development of a method to detect Chlamydia trachomatis by amplifying fragments of the sequenced common plasmid present in all serovars of C . trachomatis . PCR was performed directly on crude cell lysates, so the need for purification of DNA14 was eliminated . Furthermore, our target DNA is located on a plasmid of which about 10 copies were found in every elementary body .' The C . trachomatis plasmid has an A + T content of 64 . 7%, and there are multiple A-Trich areas throughout the plasmid (this facilitates the selection of efficient primers) . On the basis of results presented here, we conclude that our PCR technique is a valuable tool for diagnosis of genital Chlamydia trachomatis infections, because of its high sensitivity and specificity . This PCR method is not time-consuming ; specimen manipulation can be reduced to a few incubation
1
steps; sample evaluation can be judged directly from the presence/absence of one DNA band in agarose gel . However, some authors" have recently reported a plasmid negative isolate of C . trachomatis which would give a false-negative in this PCR procedure . The sensitivity and specificity of this PCR procedure should thus be evaluated with large numbers of specimens .
ACKNOWLEDGEMENTS Thanks are due to Dr R . Griffais, Pasteur Institute of Paris, for helpful discussion, and Laboratory A . Burckel, from Paris, for Chlamydia cell cultures .
REFERENCES Saiki, R . K ., Gelfand, D . H . & Stoffel, S . (1988) . Primerdirected enzymatic amplification of DNA with a thermostable DNA polymerase . Science 239, 487-91 . 2. Pollard, D . R ., Tyler, S. D., Ng, C . W. & Rozee, K . R . (1989) . A polymerase chain reaction (PCR) protocol for the specific detection of Chlamydia spp . Molecular and Cellular Probes 3, 383-9 . 3. Holland, S . M., Gaydos, C . A . & Quinn, T. C . (1990) . Detection and differentiation of Chlamydia trachomatis, C. psitacci, and C . pneumoniae by DNA amplification . Journal of Infectious Diseases 162, 984-7. 4 . Dutilh, B ., Bebear, C ., Rodriguez et a!. (1989) . Specific amplification of a DNA sequence common to all Chlamydia trachomatis serovars using the polymerase 1.
92
G . Lucotte et al.
chain reaction . Research in Microbiology 140, 7-16. 5 . Palmer, L . & Falkow, S . (1986). A common plasmid of the genus Chlamydia trachomatis . Plasmid 16, 52-62 . 6. Sriprakash, K . S . & Mac Avoy, E . S . (1987) . Characterisation and sequence of a plasmid from the trachoma biovar of Chlamydia trachomatis . Plasmid 18, 205-14 . 7 . Hatt, C ., Ward, M . E . & Clarke, I . N . (1988) . Analysis of the entire nucleotide sequence of the cryptic plasmid of Chlamydia trachomatis serovar Li evidence for involvement in DNA replication . Nucleic Acids Research 16, 4053-67 . 8 . Comanducci, M., Ricci, S. & Ratti, G . (1988) . The structure of a plasmid of Chlamydia trachomatis believed to be required for growth within mammalian cells. Molecular Microbiology 2, 531-8 . 9 . Ostergaard, L., Birkelund, S . & Christiansen, G . (1990) . Use of polymerase chain reaction for detection of Chlamydia trachomatis . Journal of Clinical Microbiology 28, 1254-60 . 10. Griffais, R . & Thibon, M . (1989) . Detection of Chlamydia trachomatis by the polymerase chain reaction . Research in Microbiology 140, 139-41 .
11 . Ripa, K . V. & Mardh, P . A . (1977) . Cultivation of Chlamydia trachomatis in cyclohexamide-treated McCoy cells. journal of Clinical Microbiology 6, 32931 . 12 . Griffais, R ., Andre, P. M . & Thibon, M . (1990) . Synthesis of digoxygenin-labelled DNA probe by polymerase chain reaction : application to Epstein-Barr virus and Chlamydia trachomatis . Research in Virology 141, 3315. 13 . Schachter, J . & Martin, D . H . (1987). Failure of multiples passages to increase chlamydial recovery . journal of Clinical Microbiology 25, 1851-3 . 14 . Solbrig, M . V., Wong, M . L . & Stephens, R . S . (1990) . Developmental-stage-plasmid supercoiling in Chlamydia trachomatis. Molecular Microbiology 4, 1535-41 . 15 . Petersen, E . M ., Markoff, B . A ., Schachter, J . & De la Maza, L . M . (1990) . The 7-5 kb plasmid present in Chlamydia trachomatis is not essential for the growth of this organism . Plasmid 23, 144-8.
Detection of Chlamydiae trachomatis by use of polymerase chain reaction Gerard Lucotte, 1,2 * Marie-Christine Petit,' Marie-Helene Francois' and Stephane Reveilleau 2 'Laboratoire d'Anthropologie Physique, College de France, 11, Place Marcelin Berthelot, Paris 5e , France, and 2Laboratoire Eurobio, ZA de Courtaboeuf, 7 Avenue de Scandinavie, Les Ulis, 91 France (Received 29 March 1991, Accepted 22 July 1991)
A polymerase chain reaction (PCR) assay was developed for detection of Chlamydia trachomatis DNA . One primer set was used, from the published sequence of the common C . trachomatis plasmid . Detection of amplified sequences was carried out by agarose gel electrophoresis . Analysis of 106 clinical samples tested by cell culture and PCR showed a sensitivity of 100% when PCR was compared with cell culture .
KEYWORDS: Chlamydia trachomatis, DNA plasmid, PCR, detection
INTRODUCTION highly conserved cryptic 7.5 kb plasmid:' restriction mapping of plasmids from 15 serotypes and direct sequence analysis of plasmids from serotypes B, 6 L1' and L28 have shown this plasmid to be highly conserved within the species C . trachomatis . (The entire plasmid sequence is known .) We, and others, 9'' 0 have thus chosen this plasmid as a target sequence for DNA amplification, and this study compares cell culture and PCR sensitivity of a large number of clinical specimens.
Chlamydia trachomatis, an obligate intracellular parasite, is a common agent of sexually transmitted disease and the causative agent of several human clinical conditions including urethritis, cervicitis, salpingitis, female infertility, lymphogranoluma venereum and trachoma . The diagnosis of C. trachomatis as a pathogen is currently difficult and expensive, because laboratory diagnosis is achieved by isolation in cell culture, a technique which is both cumbersome and time-consuming. The polymerase chain reaction (PCR), an efficient in vitro method of DNA amplification, is a powerful means for detecting specific sequences .' This technique has been used to detect and differentiate C . trachomatis and Chlamydia psittaci in laboratory samples of infected McCoy cells .' PCR has also been used to detect major outer membrane protein gene sequences from the three species of Chlamydia,; and in serovars of C . trachomatis ." We adapted PCR to detect C. trachomatis in clinical samples . All C . trachomatis strains possess a
MATERIALS AND METHODS Samples A total of 106 endocervical specimens (obtained from patients in Paris) were studied . Clinical samples were cultured" and stained at 48-h with fluorescent-antibody culture confirmation reagent; a direct immunofluorescence test (DFA) was also performed using a
*Author to whom correspondence should be addressed at Laboratoires Eurobio, ZA Courtaboeuf, 7, Avenue de Scandinavie, F 91953 Les Ulis Cedex B ., France.
0890-8508/92/020089 + 04 $03 .00/0
89
° 1992 Academic Press Limited
G. Lucotte et al .
90 commercially
available
assay
(Chlamydiazyme,
1 mgl - ') ; 5µl of each of primers A and B (2µM per
µp;
Abbott) . Clinical samples were taken with plastic
10
swabs,
dTTP (100/mM) ; 1 unit of Taq polymerase (Cetus Corp .) . The samples were subjected to 29 cycles of
put
into
transport
medium
(sacharose
68 . 46 gl - ', K,HPO 4 2 gl - ', KH Z PO, 1 gl - ') and conserved at -80 ° C . A volume, 100µI, of each was of lysis buffer (10 mm Tris-HCI, pH mixed with 200
8 gl of the four dNTPs mix : dATP, dGTP, dCTP,
amplification in a thermal cycler as follows : 95 ° C for
µl
15 s, 55 ° C for 1 min, 72°C for 1 min (and kept for the
7 . 5, 1 mm EDTA, 0 . 1 % SDS), 5 .tl of proteinase K
last 30th cycle at 72 ° C for 10 min to complete the
(10 mg ml - ') and shaken at 42°C overnight (and then
extension) . PCR products were resolved in a 2 . 5%
heated to 95 ° C for 5 min).
agarose gel stained with ethidium bromide (Fig . 1) and DNA fragments were detected by ultra-violet light exposure . Southern transfer and hybridization in 3 x SSC were performed on Hybond N (Amersham) .
Oligo One pair of primers which defines one segment of 201
by
was selected : 10
primer
RESULTS
A, 5'-
TTCCCCTTGTAATTCGTTGC-3' and primer B, 5'-TAGTAACTGCCACTTCATCA-3', between the Eco RI and
In this survey, which took into account 106 samples,
Cla I sites in the corresponding plasmid sequence,
PCR was compared with cell culture and direct
centered on the unique Eco RV site.
immunofluorescence for C . trachomatis detection . Among the specimens processed (Table 1), 10 were positive for PCR; seven of these were positive by cell
PCR
culture, and one was positive by the direct immunofluorescence test . The sensitivity of cell culture tech-
A volume, 22 pl, of each of the prepared samples was used in a 50 PCR reaction : 5 .tl of PCR buffer (10 mm
nique is not precisely known, but is less than 100% ;"
Tris-HCI, pH 8 . 3, 1 .5 MM MgCl2, 50 mm KCI, gelatin
mens positive by culture and positive by direct
µl
P
2
3
4
if we use a standard of positivity the sum of speci-
5
6
7
8
9
10
12
201 by --t
Fig. 1 . Detection of amplification product by means of electrophoresis of C . trachomatis plasmid obtained by PCR (the 201 by specific band) . M, DNA marker; P, a positive sample ; 1-12, 12 samples tested, 1 and 2 being positive . After hybridization of the corresponding blot with 12 P-labelled plasmid probe," the PCR product was revealed .
91
PCR Detection of Chlamydia trachomatis Table 1 . Summary of the results obtained concerning 106 samples studied during 15 experiments, where PCR, culture and/or direct examination are simultaneously involved Experiment no . 1 2
3 4 5 6 7 8 9 10 11
No . analysed
PCR positive
Culture positive
5
1
1
3 11
3
2
2
2
16 7 6 6 5 7 11 7
1
2
1
12 13
5
14 15
10 4
1
1
106
10
7
Total
FA positive
3
immunofluorescence, then PCR diagnosed all positive samples detected in this study. There were two specimens which were culture negative and PCR positive . One of these samples was obtained from a person undergoing antibiotic treatment .
DISCUSSION Detection of genital chlamydial infection is currently based on the cell culture technique, and enzymelinked immunasorbents and immunofluorescence essays ; none of these tests display optimal sensitivity . In this report we describe the development of a method to detect Chlamydia trachomatis by amplifying fragments of the sequenced common plasmid present in all serovars of C . trachomatis . PCR was performed directly on crude cell lysates, so the need for purification of DNA14 was eliminated . Furthermore, our target DNA is located on a plasmid of which about 10 copies were found in every elementary body .' The C . trachomatis plasmid has an A + T content of 64 . 7%, and there are multiple A-Trich areas throughout the plasmid (this facilitates the selection of efficient primers) . On the basis of results presented here, we conclude that our PCR technique is a valuable tool for diagnosis of genital Chlamydia trachomatis infections, because of its high sensitivity and specificity . This PCR method is not time-consuming ; specimen manipulation can be reduced to a few incubation
1
steps; sample evaluation can be judged directly from the presence/absence of one DNA band in agarose gel . However, some authors" have recently reported a plasmid negative isolate of C . trachomatis which would give a false-negative in this PCR procedure . The sensitivity and specificity of this PCR procedure should thus be evaluated with large numbers of specimens .
ACKNOWLEDGEMENTS Thanks are due to Dr R . Griffais, Pasteur Institute of Paris, for helpful discussion, and Laboratory A . Burckel, from Paris, for Chlamydia cell cultures .
REFERENCES Saiki, R . K ., Gelfand, D . H . & Stoffel, S . (1988) . Primerdirected enzymatic amplification of DNA with a thermostable DNA polymerase . Science 239, 487-91 . 2. Pollard, D . R ., Tyler, S. D., Ng, C . W. & Rozee, K . R . (1989) . A polymerase chain reaction (PCR) protocol for the specific detection of Chlamydia spp . Molecular and Cellular Probes 3, 383-9 . 3. Holland, S . M., Gaydos, C . A . & Quinn, T. C . (1990) . Detection and differentiation of Chlamydia trachomatis, C. psitacci, and C . pneumoniae by DNA amplification . Journal of Infectious Diseases 162, 984-7. 4 . Dutilh, B ., Bebear, C ., Rodriguez et a!. (1989) . Specific amplification of a DNA sequence common to all Chlamydia trachomatis serovars using the polymerase 1.
92
G . Lucotte et al.
chain reaction . Research in Microbiology 140, 7-16. 5 . Palmer, L . & Falkow, S . (1986). A common plasmid of the genus Chlamydia trachomatis . Plasmid 16, 52-62 . 6. Sriprakash, K . S . & Mac Avoy, E . S . (1987) . Characterisation and sequence of a plasmid from the trachoma biovar of Chlamydia trachomatis . Plasmid 18, 205-14 . 7 . Hatt, C ., Ward, M . E . & Clarke, I . N . (1988) . Analysis of the entire nucleotide sequence of the cryptic plasmid of Chlamydia trachomatis serovar Li evidence for involvement in DNA replication . Nucleic Acids Research 16, 4053-67 . 8 . Comanducci, M., Ricci, S. & Ratti, G . (1988) . The structure of a plasmid of Chlamydia trachomatis believed to be required for growth within mammalian cells. Molecular Microbiology 2, 531-8 . 9 . Ostergaard, L., Birkelund, S . & Christiansen, G . (1990) . Use of polymerase chain reaction for detection of Chlamydia trachomatis . Journal of Clinical Microbiology 28, 1254-60 . 10. Griffais, R . & Thibon, M . (1989) . Detection of Chlamydia trachomatis by the polymerase chain reaction . Research in Microbiology 140, 139-41 .
11 . Ripa, K . V. & Mardh, P . A . (1977) . Cultivation of Chlamydia trachomatis in cyclohexamide-treated McCoy cells. journal of Clinical Microbiology 6, 32931 . 12 . Griffais, R ., Andre, P. M . & Thibon, M . (1990) . Synthesis of digoxygenin-labelled DNA probe by polymerase chain reaction : application to Epstein-Barr virus and Chlamydia trachomatis . Research in Virology 141, 3315. 13 . Schachter, J . & Martin, D . H . (1987). Failure of multiples passages to increase chlamydial recovery . journal of Clinical Microbiology 25, 1851-3 . 14 . Solbrig, M . V., Wong, M . L . & Stephens, R . S . (1990) . Developmental-stage-plasmid supercoiling in Chlamydia trachomatis. Molecular Microbiology 4, 1535-41 . 15 . Petersen, E . M ., Markoff, B . A ., Schachter, J . & De la Maza, L . M . (1990) . The 7-5 kb plasmid present in Chlamydia trachomatis is not essential for the growth of this organism . Plasmid 23, 144-8.
