Molecular and Cellular Probes (1991) 5, 2 8 1 -284
Specific detection of the toxic shock syndrome toxin-1 gene using the polymerase chain reaction B. Jaulhac,* G. Prevost and Y. Piemont Institut de Bacteriologie, Faculte de Medecine, 3, rue Koeberle, 67000 Strasbourg, France (Received 16 November 1990, Accepted 19 February 1991)
A rapid and specific assay for toxic shock syndrome toxin-1 gene (tst gene) detection in Staphylococcus aureus was developed using the polymerase chain reaction . A two-primer set and an oligonucleotide detection probe were synthesized . After 40 cycles of amplification, detection of a 160-bp amplified DNA fragment was carried out by agarose gel electrophoresis and Southern blot hybridization . This assay was sensitive since it was able to detect 1-10 bacteria . It was also specific since no amplification was documented with DNAs from enterotoxigenic S . aureus or Gram-negative bacteria devoid of the tst gene .
KEYWORDS : Staphylococcus aureus, TSST-1, polymerase chain reaction
INTRODUCTION using the S . aureus reference strain FRI 1169 which produced TSST-1 . We also tested eight clinical TSST1-producing S. aureus strains . The specificity of the method was assessed with
The toxic shock syndrome toxin-1 (TSST-1) from Staphylococcus aureus is the major cause of toxic shock syndrome (TSS), characterized mainly by fever, hypotension, cutaneous rash followed by desquamation and multi-system involvement . This severe disease is lethal in 10% of cases .' TSS clinical diagnosis may be difficult . Moreover, the identification of S . aureus strains producing TSST-
strains that did not produce TSST-1 (reference S . aureus strains FRI 722, S6, FRI 137, FRI 361, FRI 1230, FRI 1151M, FRI 326, which respectively produced enterotoxins SEA, SEB, SEC„ SEC 2 , SEC S , SED, SEE), and eight other strains isolated from clinical specimens (Escherichia coli, Proteus vulgaris, Pseudomonas maltophilia, Neisseria gonorrhoeae, Flavobacterium sp ., Streptococcus agalactiae, Bacteroides fragilis, Haemophilus influenzae) . We tested one strain for each species .
1 is time-consuming because of the three steps required : isolation of the S . aureus strain, culture of the strain for toxin production, and phenotypic assay . It would be advantageous to reduce this delay in order to obtain a rapid detection of the TSST-1 . We developed a method to detect low amounts of tst gene-harbouring staphylococci by in vitro amplification of a tst gene fragment using the polymerase chain reaction (PCR) . This method can detect a single copy of target DNA in cultured staphylococci .
Processing of staphylococcal samples All strains were grown in tryptase yeast extract medium (Institut Pasteur Production) . After 18 h of
MATERIALS AND METHODS
culture, the micro-organisms were pelleted by centrifugation and resuspended in 20 tl TE buffer (10 mm Tris-HCI, 1 mm EDTA pH 8) with lysostaphin (Sigma) (10 i .u . ml -1 ) and lysozyme (Sigma) (1 mg ml -1 ) . After
Bacterial strains The in vitro gene amplification was developed by * Author to whom correspondence should be addressed .
0890-8508/91/040281 + 04 $03 .00/0
281
© 1991 Academic Press Limited
282
B. Jaulhac et al .
incubation for 40 min at 37 ° C, 180 pl of lysis buffer (150 mm sodium acetate, 1 mm EDTA, 4% sarcosyl pH 5) were added and samples were thoroughly mixed .
three different rooms and DNA solutions were transferred to Eppendorf microtubes with positive deplacement pipettes (Microman ; Gilson) .
Nucleic acids were twice extracted with phenol and once with phenol-chloroform-isoamyl alcohol (5048-2) . The DNA was then precipitated with ethanol at -80 ° C for 30 min according to the method pre-
Gel electrophoresis
viously described by Maniatis et a!., 2 and pelleted by centrifugation at 10,000 X g for 10 min at +4° C and resuspended in 20 pl TE buffer.
Six microlitres of each sample containing amplified DNA were electrophoresed in 0 . 5 X TEB buffer (44 . 5 mm Tris-HCI, 44 . 5 mm boric acid, 1 . 25 mm EDTA pH 8. 3) on a 1 % SeaKem GTG-3% Nu Sieve agarose
DNA preparation from other bacteria
gel (FMC Products) at 120 V for 1 . 5 h . After staining with ethidium bromide, gel was examined under u .v. light .
Bacteria were incubated in TE buffer with 2 mg ml - ' lysozyme, 0 . 5% SDS and 1 mg ml - ' proteinase K (Sigma) for 60 min at 37° C. DNA was then purified by phenol-chloroform-isoamyl alcohol extraction and ethanol precipation as described above.
Synthetic oligonucleotides Two primers from the tst gene sequence 3 were chosen and synthesized . Primers 1 and 2 were 30 mers coding for amino-acids -37 to -28 and +15 to +24 . Primer 1 was 5'-TTCACTATTTGTAAAAGTGTCAGACCCACT-3' and primer 2 was 5'TACTAATGAATTTTTTTATCGTAAGCCCTT-3' . A detection probe coding for amino-acids -17 to -10 was also synthesized . Its sequence was 5'-GATTTTACCCCTGTTCCCTTATCAT-3' .
Southern blot hybridization For Southern blotting, the DNA was transferred to Immobilon-P membrane (Millipore) using NaOH 0 .5 nn and fixed onto the membrane by heating for 2 h at 80 ° C . The hybridization probe was 5'-labelled with (y 32 P)-ATP (> 30000 mmol - ') as described by Maniatis et al.' The membrane was prehybridized for 0. 5 h at 55°C in 6 X SSPE buffer' containing 0 . 1 % SDS, 0. 02% bovine serum albumin, 0 . 02% Ficoll 400 and 0. 02% polyvinylpyrrolidone. The filters were transferred into fresh prehybridization buffer and 0 . 5 pmole of labelled probe per ml was added . Hybridization was performed for 2 h at 55 ° C, followed by two washes in 2 X SSPE buffer with 0 . 1 % SDS . After airdrying, membranes were exposed to Agfa Curix X-ray film with intensifying screens at -70 ° C for 2-12 h .
DNA sequence amplification by PCR Sensitivity of tst gene detection by PCR Amplification was performed as described by Saiki et al.' with some modifications . Briefly, total DNA was incubated within 100 pl volume containing 0 . 5 to 2. 5 units of Taq polymerase (Beckman), 0. 1 to 1 µnn of each primer, 0 . 2 gm of each of the four deoxynucleotides, 10 mm Tris-HCI pH 8 . 4, 50 mm KCI, 0 . 5 to 2 . 5 mm MgCl 2 and 0 . 2 mg ml - ' gelatin . Before amplification, reaction volumes were overlaid with 100 pl mineral oil (Perkin Elmer-Cetus) and DNA was denatured by heating for 5 min at 95 ° C . Each amplification cycle was performed as follows : 1 min for annealing at 55 °C, 1 min for primer extension at 72 ° C, 1 . 5 min for denaturation at 94 ° C. After 40 cycles, all samples were incubated for 5 min at 72 ° C to ensure that the final extension step was complete . Negative controls containing all reagents except template DNA were performed in each amplification set . To avoid contamination, sample preparation, PCR amplification and electrophoresis were performed in
The lowest detection threshold of the PCR method for the tst gene was assessed as follows . After overnight cultivation, the TSST-1-producing strain FRI 1169 was aseptically sonicated (W-375, Heat Systems-Ultronics, Inc .) for 20 s to dissociate staphylococcal clusters. The number of colony-forming units was determined by serial dilutions of the cultures on blood agar plates . Fifty microlitre culture samples were serially 10-fold diluted in phosphate-buffered saline and the staphylococci contained in each 50 gl diluted sample were processed as described above for tst gene amplification .
RESULTS
Optimization of PCR conditions The DNA denaturation must be performed for at least
Detection of TSST-1 gene 1 . 5 min at 94 ° C or more for efficient amplification . A 1 min period of time was sufficient for the annealing and extension steps . Under these conditions, the 40 cycles of PCR were performed in less than 3 h . We also tested PCR efficiency for Taq polymerase concentrations ranging from 0. 5 to 2 . 5 units for 100 µl reaction volume . The best results were obtained with 1 unit or more for 100 µl . We then decided to use 1 . 5 unit Taq polymerase . The optimum primers concentration was also determined with primer concentration ranging from 0 . 05 to 1 µM . For concentrations above 0 . 1 µM we did not observe any increase of the amplification efficiency . We therefore employed 0 . 1 µM concentration of each primer. We also tested different MgCI Z concentrations ranging from 0 . 5 to 2. 5 mm . Concentrations lower than 1 . 2 mm reduced PCR efficiency and concentrations between 1 . 2 mm and 2 . 5 mm gave the optimum
28 3
ence strains and eight strains from other bacterial species . After PCR, all TSST-1-producing S . aureus strains were tested and only these staphylococcal strains exhibited the expected 160 by band . None of the other species gave any specific amplification product as observed by ethidium bromidestaining of the agarose gel [Fig . 1(a)] or by hybridization of the membrane-transferred to DNA with the detection probe [Fig. 1(b)] .
Sensitivity After 40 cycles of amplification, genomic DNA, from as little as 100 TSST-1-producing S . aureus, was detected by visualization of the ethidium bromidestained gel [Fig . 2(a)] . Southern blot hybridization with the tst detection probe lowered the minimal detection threshold to 10 organisms [Fig. 2(b)].
level of efficiency .
Specificity DISCUSSION To determine the specificity of the method, we used In this study, we developed a specific and sensitive test for tst gene detection using DNA amplification . The complete PCR test from the sample preparation
the genomes from various bacterial species as templates : eight clinical TSST-1-producing S . aureus strains, seven TSST-1-non-producing S . aureus refer-
(
(a)
b) 1 2 3 4 5 6 7 8 9 10 11 12 13 14
1 2 3 4 5 6 7 8 9 1011 121314
Fig. 1 . (b) 4
5 6
7 8
1 2 3 4 5 6 7 8 9
9 10
Fig. 2 .
10 1I
284
B. Jaulhac et al .
to the film detection was carried out within 36 h . Preparation of 20 samples could be carried out within 3 h because no complete DNA purification was required . PCR amplification and gel electrophoresis were achieved in 5 h, transfer was performed overnight . After a 2 h hybridization, the membranes were exposed to X-ray film for 2-12 h . For templates containing low DNA concentrations (less than 106 copies of the tst gene), a small fragment of about 50 by was routinely visualized on the agarose gel . Southern blot hybridizations using either of the two primers as a probe gave positive signals for the 160 by and for the 50 by fragments . The latter band might result from an undesirable annealing between nine nucleotides of the 5' ends of the two primers resulting in a primer dimer . The high sensitivity of the test was partly due to the method of sample preparation. Cell lysis of Grampositive cocci is often difficult and the techniques generally described for Gram-negative bacteria" 0 are not suitable . For example, in our experience thermic lysis procedures", " . " allowed the detection of only 105 S . aureus or more. This low detection threshold requires the extensive processing of staphylococcal samples . To reduce the risk of cross-contamination of samples by foreign DNAs, the following precautions were taken . At each step, to avoid airborne contamination, each sample was uncovered carefully and separately . As pointed out by several authors, 13 ' 14 gloves were changed between each step of sample processing ; in the final step where DNA contained in microtubes was added to the reaction mixture, gloves were changed between each sample . Several negative controls were included : one containing the reaction mixture without any DNA and another containing purified DNA from an S. aureus strain devoid of the tst gene .
ACKNOWLEDGEMENTS This work was supported in part by grant No . 861018 from the Institut National de la Sante et de la Recherche Medicale .
REFERENCES 1 . Todd, J . K . (1988) . Toxic shock syndrome . Clinical Microbiology Reviews 1, 432-46. 2 . Maniatis, T ., Fritsch, E . F . & Sambrook, J . (1982) . Molecular cloning. A laboratory manual . New York : Cold Spring Harbor Laboratory . 3 . Blomster-Hautamaa, D . A ., Kreiswirth, B . N ., Kornblum, J . S ., Novick, R . P . & Schlievert, P . M . (1986) . The nucleotide and partial amino acid sequence of toxic shock syndrome toxin-1 . Journal of Biological Chemistry 261, 15783-6 . 4 . Saiki, R . K ., Gelfand, D . H ., Stoffel, S . et al . (1988) . Primerdirected enzymatic amplification of DNA with a thermostable DNA polymerase . Science 239, 487-91 . 5 . Bernet, C ., Garret, M., de Barbeyrac, B ., Bebear, C. & Bonnet, J . (1989) . Detection of Mycoplasma pneumoniae using the polymerase chain reaction . Journal of Clinical Microbiology 27, 2492-6. 6 . Karch, H . & Meyer, T . (1989) . Single primer pair for amplifying segments of distinct shiga-like-toxin genes by polymerase chain reaction . Journal of Clinical Microbiology 27, 2751-7 . 7 . Olive, D . M . (1989) . Detection of enterotoxigenic Escherichia coli after polymerase chain reaction amplification with a thermostable DNA polymerase. Journal of Clinical Microbiology 27, 261-5 . 8 . 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. 9 . Van Eys, G . J . J . M., Grayekamp, C ., Gerritsen, M . J . et al . (1989) . Detection of leptospires in urine by polymerase chain reaction . Journal of Clinical Microbiology 27, 2258-62 . 10 . Webb, L., Carl, M ., Malloy, D . C ., Dasch, G . A. & Azad, A . F. (1990). Detection of murine typhus infection in fleas using the polymerase chain reaction . Journal of Clinical Microbiology 28, 530-4. 11 . Persing, D . H ., Telford, S . R., Spielman, A. & Barthold, S . W. (1990) . Detection of Borrelia burgdorferi infection in Ixodes dammini ticks with the polymerase chain reaction . Journal of Clinical Microbiology 28, 566-72 . 12 . Starnbach, M. N ., Falkow, S . & Tompkins, L . S . (1989) . Species-specific detection of Legionella pneumophila in water by DNA amplification and hybridization . Journal of Clinical Microbiology 27, 1257-61 . 13 . Kitchin, P . A ., Szotyori, Z ., Fromholc, C . & Almond, N . (1990) . Avoidance of false positive. Nature 344, 201 . 14 . Kwok, S . & Higuchi, R . (1989) . Avoiding false positives with PCR . Nature 339, 237-9.
Specific detection of the toxic shock syndrome toxin-1 gene using the polymerase chain reaction B. Jaulhac,* G. Prevost and Y. Piemont Institut de Bacteriologie, Faculte de Medecine, 3, rue Koeberle, 67000 Strasbourg, France (Received 16 November 1990, Accepted 19 February 1991)
A rapid and specific assay for toxic shock syndrome toxin-1 gene (tst gene) detection in Staphylococcus aureus was developed using the polymerase chain reaction . A two-primer set and an oligonucleotide detection probe were synthesized . After 40 cycles of amplification, detection of a 160-bp amplified DNA fragment was carried out by agarose gel electrophoresis and Southern blot hybridization . This assay was sensitive since it was able to detect 1-10 bacteria . It was also specific since no amplification was documented with DNAs from enterotoxigenic S . aureus or Gram-negative bacteria devoid of the tst gene .
KEYWORDS : Staphylococcus aureus, TSST-1, polymerase chain reaction
INTRODUCTION using the S . aureus reference strain FRI 1169 which produced TSST-1 . We also tested eight clinical TSST1-producing S. aureus strains . The specificity of the method was assessed with
The toxic shock syndrome toxin-1 (TSST-1) from Staphylococcus aureus is the major cause of toxic shock syndrome (TSS), characterized mainly by fever, hypotension, cutaneous rash followed by desquamation and multi-system involvement . This severe disease is lethal in 10% of cases .' TSS clinical diagnosis may be difficult . Moreover, the identification of S . aureus strains producing TSST-
strains that did not produce TSST-1 (reference S . aureus strains FRI 722, S6, FRI 137, FRI 361, FRI 1230, FRI 1151M, FRI 326, which respectively produced enterotoxins SEA, SEB, SEC„ SEC 2 , SEC S , SED, SEE), and eight other strains isolated from clinical specimens (Escherichia coli, Proteus vulgaris, Pseudomonas maltophilia, Neisseria gonorrhoeae, Flavobacterium sp ., Streptococcus agalactiae, Bacteroides fragilis, Haemophilus influenzae) . We tested one strain for each species .
1 is time-consuming because of the three steps required : isolation of the S . aureus strain, culture of the strain for toxin production, and phenotypic assay . It would be advantageous to reduce this delay in order to obtain a rapid detection of the TSST-1 . We developed a method to detect low amounts of tst gene-harbouring staphylococci by in vitro amplification of a tst gene fragment using the polymerase chain reaction (PCR) . This method can detect a single copy of target DNA in cultured staphylococci .
Processing of staphylococcal samples All strains were grown in tryptase yeast extract medium (Institut Pasteur Production) . After 18 h of
MATERIALS AND METHODS
culture, the micro-organisms were pelleted by centrifugation and resuspended in 20 tl TE buffer (10 mm Tris-HCI, 1 mm EDTA pH 8) with lysostaphin (Sigma) (10 i .u . ml -1 ) and lysozyme (Sigma) (1 mg ml -1 ) . After
Bacterial strains The in vitro gene amplification was developed by * Author to whom correspondence should be addressed .
0890-8508/91/040281 + 04 $03 .00/0
281
© 1991 Academic Press Limited
282
B. Jaulhac et al .
incubation for 40 min at 37 ° C, 180 pl of lysis buffer (150 mm sodium acetate, 1 mm EDTA, 4% sarcosyl pH 5) were added and samples were thoroughly mixed .
three different rooms and DNA solutions were transferred to Eppendorf microtubes with positive deplacement pipettes (Microman ; Gilson) .
Nucleic acids were twice extracted with phenol and once with phenol-chloroform-isoamyl alcohol (5048-2) . The DNA was then precipitated with ethanol at -80 ° C for 30 min according to the method pre-
Gel electrophoresis
viously described by Maniatis et a!., 2 and pelleted by centrifugation at 10,000 X g for 10 min at +4° C and resuspended in 20 pl TE buffer.
Six microlitres of each sample containing amplified DNA were electrophoresed in 0 . 5 X TEB buffer (44 . 5 mm Tris-HCI, 44 . 5 mm boric acid, 1 . 25 mm EDTA pH 8. 3) on a 1 % SeaKem GTG-3% Nu Sieve agarose
DNA preparation from other bacteria
gel (FMC Products) at 120 V for 1 . 5 h . After staining with ethidium bromide, gel was examined under u .v. light .
Bacteria were incubated in TE buffer with 2 mg ml - ' lysozyme, 0 . 5% SDS and 1 mg ml - ' proteinase K (Sigma) for 60 min at 37° C. DNA was then purified by phenol-chloroform-isoamyl alcohol extraction and ethanol precipation as described above.
Synthetic oligonucleotides Two primers from the tst gene sequence 3 were chosen and synthesized . Primers 1 and 2 were 30 mers coding for amino-acids -37 to -28 and +15 to +24 . Primer 1 was 5'-TTCACTATTTGTAAAAGTGTCAGACCCACT-3' and primer 2 was 5'TACTAATGAATTTTTTTATCGTAAGCCCTT-3' . A detection probe coding for amino-acids -17 to -10 was also synthesized . Its sequence was 5'-GATTTTACCCCTGTTCCCTTATCAT-3' .
Southern blot hybridization For Southern blotting, the DNA was transferred to Immobilon-P membrane (Millipore) using NaOH 0 .5 nn and fixed onto the membrane by heating for 2 h at 80 ° C . The hybridization probe was 5'-labelled with (y 32 P)-ATP (> 30000 mmol - ') as described by Maniatis et al.' The membrane was prehybridized for 0. 5 h at 55°C in 6 X SSPE buffer' containing 0 . 1 % SDS, 0. 02% bovine serum albumin, 0 . 02% Ficoll 400 and 0. 02% polyvinylpyrrolidone. The filters were transferred into fresh prehybridization buffer and 0 . 5 pmole of labelled probe per ml was added . Hybridization was performed for 2 h at 55 ° C, followed by two washes in 2 X SSPE buffer with 0 . 1 % SDS . After airdrying, membranes were exposed to Agfa Curix X-ray film with intensifying screens at -70 ° C for 2-12 h .
DNA sequence amplification by PCR Sensitivity of tst gene detection by PCR Amplification was performed as described by Saiki et al.' with some modifications . Briefly, total DNA was incubated within 100 pl volume containing 0 . 5 to 2. 5 units of Taq polymerase (Beckman), 0. 1 to 1 µnn of each primer, 0 . 2 gm of each of the four deoxynucleotides, 10 mm Tris-HCI pH 8 . 4, 50 mm KCI, 0 . 5 to 2 . 5 mm MgCl 2 and 0 . 2 mg ml - ' gelatin . Before amplification, reaction volumes were overlaid with 100 pl mineral oil (Perkin Elmer-Cetus) and DNA was denatured by heating for 5 min at 95 ° C . Each amplification cycle was performed as follows : 1 min for annealing at 55 °C, 1 min for primer extension at 72 ° C, 1 . 5 min for denaturation at 94 ° C. After 40 cycles, all samples were incubated for 5 min at 72 ° C to ensure that the final extension step was complete . Negative controls containing all reagents except template DNA were performed in each amplification set . To avoid contamination, sample preparation, PCR amplification and electrophoresis were performed in
The lowest detection threshold of the PCR method for the tst gene was assessed as follows . After overnight cultivation, the TSST-1-producing strain FRI 1169 was aseptically sonicated (W-375, Heat Systems-Ultronics, Inc .) for 20 s to dissociate staphylococcal clusters. The number of colony-forming units was determined by serial dilutions of the cultures on blood agar plates . Fifty microlitre culture samples were serially 10-fold diluted in phosphate-buffered saline and the staphylococci contained in each 50 gl diluted sample were processed as described above for tst gene amplification .
RESULTS
Optimization of PCR conditions The DNA denaturation must be performed for at least
Detection of TSST-1 gene 1 . 5 min at 94 ° C or more for efficient amplification . A 1 min period of time was sufficient for the annealing and extension steps . Under these conditions, the 40 cycles of PCR were performed in less than 3 h . We also tested PCR efficiency for Taq polymerase concentrations ranging from 0. 5 to 2 . 5 units for 100 µl reaction volume . The best results were obtained with 1 unit or more for 100 µl . We then decided to use 1 . 5 unit Taq polymerase . The optimum primers concentration was also determined with primer concentration ranging from 0 . 05 to 1 µM . For concentrations above 0 . 1 µM we did not observe any increase of the amplification efficiency . We therefore employed 0 . 1 µM concentration of each primer. We also tested different MgCI Z concentrations ranging from 0 . 5 to 2. 5 mm . Concentrations lower than 1 . 2 mm reduced PCR efficiency and concentrations between 1 . 2 mm and 2 . 5 mm gave the optimum
28 3
ence strains and eight strains from other bacterial species . After PCR, all TSST-1-producing S . aureus strains were tested and only these staphylococcal strains exhibited the expected 160 by band . None of the other species gave any specific amplification product as observed by ethidium bromidestaining of the agarose gel [Fig . 1(a)] or by hybridization of the membrane-transferred to DNA with the detection probe [Fig. 1(b)] .
Sensitivity After 40 cycles of amplification, genomic DNA, from as little as 100 TSST-1-producing S . aureus, was detected by visualization of the ethidium bromidestained gel [Fig . 2(a)] . Southern blot hybridization with the tst detection probe lowered the minimal detection threshold to 10 organisms [Fig. 2(b)].
level of efficiency .
Specificity DISCUSSION To determine the specificity of the method, we used In this study, we developed a specific and sensitive test for tst gene detection using DNA amplification . The complete PCR test from the sample preparation
the genomes from various bacterial species as templates : eight clinical TSST-1-producing S . aureus strains, seven TSST-1-non-producing S . aureus refer-
(
(a)
b) 1 2 3 4 5 6 7 8 9 10 11 12 13 14
1 2 3 4 5 6 7 8 9 1011 121314
Fig. 1 . (b) 4
5 6
7 8
1 2 3 4 5 6 7 8 9
9 10
Fig. 2 .
10 1I
284
B. Jaulhac et al .
to the film detection was carried out within 36 h . Preparation of 20 samples could be carried out within 3 h because no complete DNA purification was required . PCR amplification and gel electrophoresis were achieved in 5 h, transfer was performed overnight . After a 2 h hybridization, the membranes were exposed to X-ray film for 2-12 h . For templates containing low DNA concentrations (less than 106 copies of the tst gene), a small fragment of about 50 by was routinely visualized on the agarose gel . Southern blot hybridizations using either of the two primers as a probe gave positive signals for the 160 by and for the 50 by fragments . The latter band might result from an undesirable annealing between nine nucleotides of the 5' ends of the two primers resulting in a primer dimer . The high sensitivity of the test was partly due to the method of sample preparation. Cell lysis of Grampositive cocci is often difficult and the techniques generally described for Gram-negative bacteria" 0 are not suitable . For example, in our experience thermic lysis procedures", " . " allowed the detection of only 105 S . aureus or more. This low detection threshold requires the extensive processing of staphylococcal samples . To reduce the risk of cross-contamination of samples by foreign DNAs, the following precautions were taken . At each step, to avoid airborne contamination, each sample was uncovered carefully and separately . As pointed out by several authors, 13 ' 14 gloves were changed between each step of sample processing ; in the final step where DNA contained in microtubes was added to the reaction mixture, gloves were changed between each sample . Several negative controls were included : one containing the reaction mixture without any DNA and another containing purified DNA from an S. aureus strain devoid of the tst gene .
ACKNOWLEDGEMENTS This work was supported in part by grant No . 861018 from the Institut National de la Sante et de la Recherche Medicale .
REFERENCES 1 . Todd, J . K . (1988) . Toxic shock syndrome . Clinical Microbiology Reviews 1, 432-46. 2 . Maniatis, T ., Fritsch, E . F . & Sambrook, J . (1982) . Molecular cloning. A laboratory manual . New York : Cold Spring Harbor Laboratory . 3 . Blomster-Hautamaa, D . A ., Kreiswirth, B . N ., Kornblum, J . S ., Novick, R . P . & Schlievert, P . M . (1986) . The nucleotide and partial amino acid sequence of toxic shock syndrome toxin-1 . Journal of Biological Chemistry 261, 15783-6 . 4 . Saiki, R . K ., Gelfand, D . H ., Stoffel, S . et al . (1988) . Primerdirected enzymatic amplification of DNA with a thermostable DNA polymerase . Science 239, 487-91 . 5 . Bernet, C ., Garret, M., de Barbeyrac, B ., Bebear, C. & Bonnet, J . (1989) . Detection of Mycoplasma pneumoniae using the polymerase chain reaction . Journal of Clinical Microbiology 27, 2492-6. 6 . Karch, H . & Meyer, T . (1989) . Single primer pair for amplifying segments of distinct shiga-like-toxin genes by polymerase chain reaction . Journal of Clinical Microbiology 27, 2751-7 . 7 . Olive, D . M . (1989) . Detection of enterotoxigenic Escherichia coli after polymerase chain reaction amplification with a thermostable DNA polymerase. Journal of Clinical Microbiology 27, 261-5 . 8 . 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. 9 . Van Eys, G . J . J . M., Grayekamp, C ., Gerritsen, M . J . et al . (1989) . Detection of leptospires in urine by polymerase chain reaction . Journal of Clinical Microbiology 27, 2258-62 . 10 . Webb, L., Carl, M ., Malloy, D . C ., Dasch, G . A. & Azad, A . F. (1990). Detection of murine typhus infection in fleas using the polymerase chain reaction . Journal of Clinical Microbiology 28, 530-4. 11 . Persing, D . H ., Telford, S . R., Spielman, A. & Barthold, S . W. (1990) . Detection of Borrelia burgdorferi infection in Ixodes dammini ticks with the polymerase chain reaction . Journal of Clinical Microbiology 28, 566-72 . 12 . Starnbach, M. N ., Falkow, S . & Tompkins, L . S . (1989) . Species-specific detection of Legionella pneumophila in water by DNA amplification and hybridization . Journal of Clinical Microbiology 27, 1257-61 . 13 . Kitchin, P . A ., Szotyori, Z ., Fromholc, C . & Almond, N . (1990) . Avoidance of false positive. Nature 344, 201 . 14 . Kwok, S . & Higuchi, R . (1989) . Avoiding false positives with PCR . Nature 339, 237-9.
