Use of Polymerase Chain Reaction for Detection of Chlamydia ...

JOURNAL

OF

CLINICAL MICROBIOLOGY, June 1990,

p.

Vol. 28, No. 6

1254-1260

0095-1137/90/061254-07$02.00/0 Copyright © 1990, American Society for Microbiology

Use of Polymerase Chain Reaction for Detection of Chlamydia trachomatis LARS 0STERGAARD,* SVEND BIRKELUND, AND GUNNA CHRISTIANSEN Institute of Medical Microbiology, University of Aarhus, DK-8000 Aarhus C., Denmark Received 29 September 1989/Accepted 12 February 1990

A polymerase chain reaction (PCR) assay was developed for detection of Chlamydia trachomatis DNA. From the published sequence of the common C. trachomatis plasmid, two primer sets were selected. Detection of amplified sequences was done by agarose gel electrophoresis of cleaved or uncleaved amplified sequences, Southern hybridization, or dot blot analysis. The PCR assay was optimized and, after 40 cycles of amplification with primer set Il, demonstrated a sensitivity of 10i- g of DNA, which corresponds to the detection of one copy of the plasmid. Because of the high sensitivity, we developed a closed system in which airborne contamination was minimized. Analysis of 228 clinical samples tested by cell culture, IDEIA enzyme immunosorbent assay (Medico-Nobel, Boots-Celltech Ltd., Berkshire, United Kingdom), and PCR showed a sensitivity of 100%, a specificity of 93% when PCR was compared with cell culture, and a corrected specificity of 99% when PCR was compared with cell culture or IDEIA.

TCGAGTATGCGTTGTTAGG-3', from bases 1607 to 1638 (primer IIA) (24), and 5'-GCGTCGCGATCTCCGGCCAG3', from bases 2079 to 2060 (primer IIB) (24). The primer sets were constructed such that a common TaqI site was positioned asymmetrically in the sequence that was amplified. Primer IA was localized in the area amplified by primer set Il, and primer IIB was localized in the area amplified by primer set I. Thus, the sequence amplified by each primer set could be verified by using primer IA or IIB as a probe (Fig. 1). Reaction mixture. A total of 1 ,ug of each primer from either primer set I or II (5 ,ul) was mixed with 20 ,ul of a buffer consisting of 250 mM KCl, 50 mM Tris (pH 8.4), 12.5 mM MgCl2, and 0.5 mg of gelatin per ml. A total of 30 ,ul of a mixture of nucleotides, 200 ,uM of each of dATP, dTTP, dGTP, and dCTP, was then added. Two units (in 5 ,ul) of Taq DNA polymerase, which was supplied by either Cetus (Cetus Corp., Emeryville, Calif.) or Stratagene (Stratagene, San Diego, Calif.), was added and the reaction mixture was overlayed with 75 ktl of paraffin oil. The components were added to an Eppendorf tube with a set of unused pipettes (Finnpipette; Labsystems, Helsinki, Finland) in a laboratory that was never exposed to chlamydial DNA. A small hole was made in the tube with a hypodermic needle in the side of the lid, and the tube was closed. The tubes were frozen at -20°C until use. For optimization of the PCR assay and for sensitivity evaluation, 40 pl of purified total C. trachomatis serovar L2 DNA was added to the tubes in 10-fold dilutions from 10-1 to 10-18 g. For evaluation of samples from patients, 500 ,ul of the clinical sample in 2-SP solution (17) was vortexed, aspirated, and added to a microtube (1.5 ml; no. 72.694; Sarstedt, Numbrecht, Federal Republic of Germany). The tube was centrifuged at 20,000 x g for 20 min at 4°C. The supernatant was removed; and the pellet was suspended in 100 ,ul of a solution containing 200 ,ug of proteinase K per ml, 10 mM Tris, and 1 mM EDTA and was subsequently incubated at 37°C for 30 min and boiled for 10 min. A total of 40 ,ul was used for PCR. The tubes were opened just enough for the preformed holes to become visible, and the DNA or the treated clinical specimens were injected by using a disposable syringe. As a negative control, a reaction mixture

Development of the polymerase chain reaction (PCR) for detection of low-copy-number DNA sequences by amplification of a specific DNA fragment by use of a DNA polymerase was first described by Mullis and Faloona (13). Amplification is obtained by repeating a three-step reaction run at different temperatures. By repeating the reaction n times, the amount of DNA theoretically rises to 2' (13, 18-20). Initially, the Klenow fragment of Escherichia col DNA polymerase I was used for DNA amplification (10, 13, 18, 20). The discovery of a polymerase from Thermus aquaticus, the Taq DNA polymerase, which is stable at high temperatures (19), along with the introduction of automatic equipment for temperature changes, makes PCR relatively easy to perform. The PCR technique mainly has been used for diagnostic tests in genetics and virology (2, 4-9, 21, 22, 26). Detection systems that use PCR on bacterial genomic DNA have been developed for Salmonella typhimurium (23), Pseudomonas cepacia (25), enterotoxigenic E. coli (14), and Chlamydia trachomatis (3, 7). We describe here the development of a method to detect C. trachomatis by amplifying fragments of the sequenced common plasmid (24), which is present in all serovars of C. trachomatis (16). This plasmid has no known homology to other microorganisms (24). Use of this method for the detection of C. trachomatis in samples from patients was evaluated. High sensitivity and high specificity were obtained for chlamydial detection. MATERIALS AND METHODS Preparation of C. trachomatis L2 DNA. C. trachomatis L2 DNA was obtained from purified elementary bodies (1) by a previously described procedure (12). The concentration of L2 DNA was measured by spectrophotometry with a recording spectrophotometer (UV-260; Shimadzu, Kyoto, Japan). PCR technique. Two primer sets from the C. trachomatis plasmid (24) were constructed: Primer set I was 5'-GTTT AAGTGTTCCCATCATAAAAACATATTC-3', from bases 1698 to 1728 (primer IA) (24), and 5'-ATCCTTGTATCCT GTTGGGAAGCCATCAAAG-3', from bases 2200 to 2170 (primer IB) (24). Primer set Il was 5'-CGCATGCAAGATA *

Corresponding author. 1254

VOL. 28, 1990

was injected with 40 Fil of double-distilled H20 instead of DNA. The tubes were centrifuged for 3 min at 20,000 x g at 4°C to separate the aqueous phase from the oil. PCR was performed in a DNA Thermal Cycler (Perkin Elmer-Cetus, Norwalk, Conn.). Initially, the samples were treated at 94°C for 2 min to ensure full denaturation. Thereafter, the temperature was reduced for 2 min to 40, 42, 45, 47, or 50°C for primer set I and to 47, 50, 52, 55, 57, or 60°C for primer set II for annealing to optimize the annealing temperature. For extension of the DNA, the temperature was then raised to 70°C for 4 min. A total of 30 or 40 cycles were performed, and after the last cycle the temperature was kept at 70°C for 10 min to complete the extension. Primer set Il was used for analysis of the serotypes and for evaluation of clinical samples, using an annealing temperature of 57°C, and Taq DNA polymerase supplied by Stratagene was used. Preparation of urogenital serotypes of C. trachomatis. C. trachomatis serotypes D, E, F, G, H, J, and K were cultivated in cycloheximide-treated McCoy cells on cover slips as described by Ripa and Mardh (17). The cover slips were examined, and the total number of inclusions was determined to be approximately 50. The proteinase K treatment and DNA amplification by PCR was then performed as described above. Gel electrophoresis. A total of 20 ,ul of each amplified sample was electrophoresed on a 1.2% agarose gel at 120 V for 1.5 h. The DNA bands were visualized by ethidium bromide staining. Bacteriophage lambda DNA cleaved with HindIII was used as a molecular weight standard. The gels were examined under UV light and were photographed on Agfapan 400 film. Cleavage with restriction enzymes. A total of 10 ,ul of amplified samples was cleaved with the restriction enzyme TaqI (Boehringer GmbH, Mannheim, Federal Republic of Germany) in the buffer recommended by the supplier. The DNA fragments were separated by electrophoresis on 2% agarose gels that were run for 2 h at 150 V. pBR322 DNA cleaved with the restriction enzyme HaeIII was used as a molecular weight standard. DNA hybridization. To 10 ,uI of each sample amplified with either primer set I or primer set 11, 60 ,ul 0.3 M NaOH was added for 10 min at 0°C; then, 70 ,ul of 12x SSC (lx SSC is 0.15 M NaCI plus 0.015 M sodium citrate [pH 7.5]) was added and the solution was applied to a nylon filter (HybondN; Amersham, Little Chalfont, United Kingdom) with a slot blotter (Minifold II; Schleicher & Schuell, Inc., Keene, N.H.). Each slot was washed with 200 ,ul of 6x SSC, and the filters were cross-linked by using UV light (Stratalinker; Stratagene). The filters were prehybridized in 0.085 ml of 6x SSC per cm2-5 x Denhardt solution (l x Denhardt solution is 0.1% polyvinylpyrrolidone, 0.1% Ficoll [Pharmacia LKB Biotechnology, Uppsala, Sweden], and 0.1% bovine serum albumin)-0.5% sodium dodecyl sulfate-10 mg/100 ml of yeast RNA for 20 min at 45°C. Probe primer IA was labeled by phosphorylation at the 5' termini with [_y-32P]dATP (ICN Radiochemicals, Irvine, Calif.) and T4 polynucleotide kinase as described by Maniatis et al. (11) to an activity of 0.3 MBq/pmol. Hybridization was done for 4 h at 45°C. Filters were washed twice at 45°C for 2 h and 15 min in 2 x SSC-0.5% sodium dodecyl sulfate, and the dried filters were autoradiographed for 4 h. Clinical specimens. A total of 228 clinical specimens (40 from males, 180 from females, and 8 from newborns) were obtained over a 3-week period from patients consulting general practitioners in the county of Aarhus. All samples received during this period were analyzed. The specimens

PCR FOR DETECTION OF C. TRACHOMATIS

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were obtained by introducing and pivoting a cotton swab (type MW 142; E.N.T. Swab; Medical Wire Equipment Co. Ltd., Potley, Carsham, Wilshire, United Kingdom) in the urethra in males or the endocervix in females, or specimens were obtained from the conjunctiva of newborns. The swab was placed in 2 ml of 2-SP transport medium (17) and frozen at -20°C within 18 h. Cell culture was performed within 5 days. All specimens were randomized, and the results of culture were concealed until the results of PCR were obtained. Cell culture assay. All specimens were tested by cell culturing with 1 ml of the sample material. Culture and microscopy for C. trachomatis detection were performed at the Institute of Medical Microbiology, University of Aarhus. The specimens were cultivated in cycloheximide-treated McCoy cells as described by Ripa and Mardh (17) and stained with iodine, and the number of inclusions was counted. If one or more inclusions were observed by microscopy, the culture was considered positive. No blind passage to increase the sensitivity of the culture was performed. Verification of specimens. The specimens that were positive by PCR and negative by cell culturing were examined by an enzyme immunosorbent assay (EIA) (IDEIA; MedicoNobel, Boots-Celltech Ltd., Berkshire, United Kingdom). The sensitivity and specificity of this EIA compared with those of our culture procedure without blind passage were 90.8 and 95.4%, respectively (14a). A total of 50 ,ul of the specimens used for EIA was taken directly from the proteinase K-treated specimens, and the EIA was performed as described earlier (14a). All specimens that were false positive by PCR were tested in duplicate by EIA. Positive and negative controls were included. The microdilution plate was scanned in a single-beam multiphotometer (Immune Reader NJ-2000; Inter Med, Japan), and the extinction values were read at 490 nm and the cutoff value was set to a mean extinction value of 0.05. The sensitivity of the PCR technique was calculated as follows: the number of samples positive by PCR as well as positive by cell culture divided by the number of samples positive by cell culture. The specificity ofthe PCR technique was calculated as follows: the number of samples negative by PCR as well as negative by cell culture divided by the number of samples negative by cell culture. The corrected specificity was calculated as follows: the number of samples negative by PCR as well as negative by cell culture divided by the number of samples negative by cell culture as well as negative by IDEIA.

RESULTS Standardization of the PCR assay. The primers used for the PCR assay are shown in Fig. 1. To analyze whether there was any difference in the amplification of circular versus linear DNA, the C. trachomatis L2 DNA was cleaved with BamHI. No difference in sensitivity or in the occurrence of a smear was observed after cleavage (data not shown). The optimal annealing temperature was found to be 45°C for primer set t, and the optimal annealing temperature for primer set II was found to be 57°C. To determine the optimal concentration of the Taq DNA polymerase, 10-l' g of L2 DNA was amplified for 40 cycles at 57°C with primer set Il with 1, 1.5, 2, 2.5, 5, and 10 U of Taq DNA polymerase per sample. The most distinct band was found when 2 U per sample was used. With higher amounts, an overall smear was observed (data not shown). Tenfold dilutions of L2 DNA were performed for 40 and 30

0STERGAARD ET AL.

1256 1607

1638

1~

1

5 '-CGCATGCAAGATATCGAGTATGCGTIGTTAGG 3'

J. CLIN. MICROBIOL. 1698

1728 1899

~~~~~~~~~~~~~~~~~~~~~~~~

GTTAAGTGTTCCCATCATAAACATATTC--TCGA-AGCT-I

[-GCGTCGCGATCTCCGGCCAG 2060

Primer IIA

Primer IA and probe

2079 Primer IIB

3'

GAACTACCGAAGGGTTGTCCTATGTTCCTA--5' 2170

2200 Primer lB

FIG. 1. Positions and sequences of primer sets I and II. Numbers refer to the base number in the common C. trachomatis plasmid (24). The TaqI restriction site at base 1899 is indicated.

PCR cycles by using primer set II, an annealing temperature of 57°C, and Taq DNA polymerase. The results are shown in Fig. 2. A sensitivity of 10-7 g of L2 DNA could be detected by performing the PCR technique for 40 cycles by using both gel electrophoresis and dot blot analysis as verification (Fig. 2A). This value corresponds to the detection of one copy of the plasmid. When PCR was performed for 30 cycles, a sensitivity of 10-16 g of L2 DNA was seen (Fig. 2B). This corresponded to 10 copies of the plasmid, which is the amount included in one to two elementary bodies. Influence of chemicals. To analyze how the Taq DNA A

log[DNA]

PCR

perforxned for 40 cvcles

10 il 12 13 '14 15 16 117 18 n

rt oses1| B

-log

[l)NA]

T>CR nnrfn-rnilfi nltr -30

R. .11. 1 .1 t

1-4 izn 1(

rln

I

l".I iggE

4 s1 FIG. 2. Tenfold dilutions of C. trachomatis L2 DNA performed by PCR for 40 (A) and 30 (B) cycles with primer set Il and verified by gel electrophoresis after ethidium bromide staining and dot blot analysis. Lambda DNA cleaved with HindIII was included in the unmarked lanes on the left in panels A and B.

polymerase was influenced by different chemicals, we amplified 2.5 x 10-8 g of L2 DNA in a reaction mixture to which the following chemicals were added: 0.4 and 4 mM EDTA; 0.04% azide; 0.04% Triton X-35; 0.04% Triton X-100; 0.04% Tween 20; 0.04% Tween 40; 0.04% Tween 80; and 0.008, 0.04, and 0.08% spermidine. The most distinct bands were seen with the addition of 0.4 mM EDTA and 0.04% azide. The most scanty band was seen with the addition of 0.04% Triton X-100. Concentrations of 4 mM EDTA and 0.04 and 0.08% spermidine resulted in no amplification (data not shown). Analysis of serotypes. The urogenital serotypes D, E, F, G, H, J, and K and serovar L2 cultivated in McCoy cells on cover slips were analyzed. After PCR for 40 cycles, all serotypes showed very distinct bands of either 503 or 473 base pairs (bp) (Fig. 3A), according to the primer set that was used. After TaqI cleavage of the 473-bp DNA amplified by use of primer set II, two distinct bands of 292 and 181 bp were observed for each serotype (Fig. 3B). The dot blot hybridizations showed similar activities for all serotypes tested (Fig. 3C). Contamination analysis. To analyze for airborne contamination, preparations of the reaction mixture consisting of primers, Taq DNA polymerase, deoxynucleoside triphosphates, and buffer were prepared in or outside a vertical-flow bench in a laboratory that handles chlamydial DNA or in a laboratory in which cervical biopsies were analyzed for the presence of human papillomavirus. An unused set of pipettes (Finnpipette; Labsystems) and new reagents for the reaction mixture were used in each laboratory. Preparation of controls in the laboratory for human papillomavirus research (Fig. 4, lanes 1 to 3) showed specific scanty bands in two of the three specimens. All six negative controls prepared in the laboratory handling chlamydial DNA were positive by Southern blot analysis (Fig. 4B). Gel electrophoresis of the amplified DNA fragments showed that samples prepared in the vertical-flow bench displayed distinct specific bands and multiple smaller and larger nonspecific bands (Fig. 4, lanes 4 to 6). Two of the three negative controls prepared outside the vertical-flow bench were negative by gel electrophoresis (Fig. 4, lanes 8 to 10), but Southern blot analysis showed specific bands for all these negative controls. Lane 7 in Fig. 4 is amplified total L2 DNA, which was used as a positive control. To avoid contamination, preparation of the reaction mixture was therefore done in a laboratory that had never been exposed to chlamydial DNA; and an unused set of pipettes (Finnpipette; Labsystems), new reagents for the reaction mixture, and the closed system described above were used. When dot blot hybridization was performed on negative controls prepared as described above, no contamination was seen (Fig. 3). Analysis of clinical specimens. A total of 220 genital specimens (40 from males and 180 from females) and 8 specimens obtained from the conjunctiva of newborns were tested both by culturing and PCR. Cell culture. Of the 228 specimens, 5 specimens analyzed by cell culture were contaminated with other bacteria and

PCR FOR DETECTION OF C. TRACHOMATIS

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1257

A. L

0 0) V E F G H I K

A

473 bp-

473bp-

B sid L.

1)

E

F

G

H

I

K

473 bp-

292bp' SlSbp-t.

b*

FIG. 4. (A) Gel electrophoresis of amplified negative controls by using primer set II. Lanes 1 to 3, negative controls prepared in a laboratory analyzing cervical biopsy specimens for human papillomavirus; lanes 4 to 6, negative controls prepared in a vertical-flow bench placed in a laboratory handling chlamydial DNA; lane 7, positive control (C. trachomatis L2 DNA); lanes 8 to 10, negative controls prepared outside the vertical-flow bench, without use of the closed system described in the text. As standards (std), lambda DNA cleaved with HindIII and pBR322 cleaved with HaeIII were used, as indicated in the two lanes on the left, respectively. (B) Southern blots of the gel described in panel A by using primer IA as a probe.

C

L-

D

E

F

G

H

I

K

specimens were negative. All positive samples showed a distinct band at 473 bp by gel electrophoresis. After cleavage with the TaqI restriction enzyme, distinctive bands of 181 and 292 bp were seen (data not shown). Comparison of the two diagnostic procedures. All 26 culture-positive specimens (14 specimens from PCR for 40 cycles and 12 specimens for PCR for 30 cycles) also were found to be positive by PCR, giving a total sensitivity of the PCR technique of 100% when either 30 or 40 cycles were used (Table 1). Specimens that were positive by PCR and negative by cell culture were tested by the IDEIA EIA. For PCR performed for 40 cycles, seven specimens (one from a male, five from females, and one from a newborn) were positive by PCR and negative by culture; this gave a specificity of 93% (94 of 101 specimens) (Table 1). All seven specimens were positive when tested by IDEIA EIA. For PCR performed for 30 cycles, seven specimens (four from males and three from females) were positive by PCR and negative by culture; this also gave a specificity of 93% (89 of 96 specimens) (Table 1). Five of the seven PCRpositive and culture-negative specimens gave a positive EIA result. The bands of those 14 specimens observed by gel electrophoresis were not more scanty than the bands observed for specimens with positive culture results. very

.

SI.

FIG. 3. (A) Amplified fragments of C. trachomatis L2 DNA and amplified fragments of the proteinase K-treated serotypes D, E, F, G, H, and K, using primer set Il and an annealing temperature of 57°C. PCR was performed for 40 cycles. The fragments were detected by ethidium bromide staining after agarose gel electrophoresis. Lanes marked 0 indicate negative controls. (B) The amplified fragments described for panel A cleaved with TaqI and detected by ethidium bromide staining after agarose gel electrophoresis. HaeIIIcleaved pBR322 was used as a standard (std). (C) Dot blot of amplified fragments from panel A. -y-32P-end-labeled primer IA was used as a probe for detection of amplified fragments. t,

excluded from analysis. Culture results were thus available for only 223 specimens. Of the 215 genital specimens, 24 (6 from males and 18 from females) were found to be positive by culturing. Two of the eight conjunctival specimens showed a positive result by culturing. The number of inclusion bodies in the culture-positive specimens varied from between 1 and more than 500. PCR technique. PCR was performed for 115 specimens for 40 cycles, and PCR was performed for 108 specimens for 30 cycles. Representative gels for 40 and 30 cycles are shown in Fig. SA and B, respectively. For PCR for 40 cycles, 21 specimens were positive and 94 specimens were negative. For PCR for 30 cycles, 19 specimens were positive and 89 were

DISCUSSION Detection of genital chlamydial infections is primarily based on the cell culture technique, enzyme-linked immuno-

0STERGAARD ET AL.

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J. CLIN. MICROBIOL.

TABLE 1. Number of positive and negative specimens tested by culture and PCR

A

No. of culture results

PCR result

40 Cycles Positive Negative Total 5 0:020

U

C,

0o~

30 Cycles Positive

Negative Total

Positive

Negative

Total

M

F

N

M

F

N

3 0

10 0 14

1 0

1 16

5 75 101

1 3

21 94 115

3 0

8 0 12

1 0

4 12

3 75 96

0 2

19 89 108

r-

a M, Specimens from males; F, specimens from females; N, conjunctival smears from newborns.

FIG.

Samples from patients verified by gel electrophoresis performed for 40 (A) and 30 (B) cycles. Numbers at the bottom of the gel refer to the amount of inclusion bodies found by culturing. Abbreviations: Sec., when analyzed by cell culture, this sample was contaminated with other bacteria and was excluded from analysis; pos., positive control; neg., negative control. The 5.

after PCR

was

unmarked lanes

on

the left contained lambda DNA cleaved with

HindIII.

sorbent assays,

or

based

on

tests

immunofluorescence assays. The last two

poly-

and monoclonal antibodies

against chlamydial membrane, either the lipopolysaccharide or the major outer membrane protein. None of these tests or the cell culture technique show an optimal sensitivity, and only cell culture has a specificity of 100%. We wanted to develop a more sensitive test system by using a PCR that also showed a high specificity. The efficiency of the PCR technique is influenced by different parameters, the most important of which seems to be the composition of primers, both in relation to each other are

components

that

are

part

of the

and in relation to the target DNA, and the temperature at

which

annealing of primers is performed. Compared with the percent G+C content of our target DNA (39%), primers A and B of primer set had relatively low percent G+C contents, 29 and 45%, respectively. Primers A and B in primer set Il had different lengths, but they had the

same

absolute G+C content, and the percent G+C

content for both

primers exceeded that of the target DNA. primers is important when the optimal annealing temperature is determined. When was primer set changed to primer set II, the optimal temperature of annealing changed from 45 to 570C. The bands became much more distinct, and a slight increase in sensitivity was seen. Therefore, each primer in a primer set should be as similar as possible to each other with regard to The percent G+C content of

their percent G+C contents. Furthermore, the percent G+C contents of primers should exceed that of target DNA to avoid reannealing of target DNA before annealing of primers. A higher sensitivity should be obtained under conditions of high annealing temperatures, since this reduces the formation of a secondary structure in the single-stranded DNA. When the number of cycles is raised, the efficiency per cycle decreases, probably because of reannealing of the amplified products (8). The concentration of Mg2` is important. It is required for the Taq DNA polymerase, but the presence of the cation in excess causes a decrease in the amplification efficiency (8). We determined that a concentration of 0.9 mM free Mg2+ gave the optimal result. The sensitivity of primer set I of 10-l' g of total L2 DNA corresponded well to the sensitivity of 10-l' g of hepatitis B virus DNA found by Kaneko et al. (9), when gel electrophoresis stained with ethidium bromide was used for verification. The higher sensitivity seen with the use of primer set Il was probably due to the higher G+C content of these primers in combination with the change to Stratagene Taq DNA polymerase. Since our sensitivity corresponds to detection of one to two chlamydial elementary bodies, and since one elementary body contains about 10 copies of the plasmid (16), we found it adequate that detection of C. trachomatis was done only by gel electrophoresis of uncleaved and restriction enzyme-cleaved amplified DNA fragments. PCR used for serotypes and clinical specimens was performed directly on crude cell lysates, as described previously (19) for the preparation of other sources of target DNA for PCR analysis. Therefore, the need for purification of DNA, which results in the loss of target DNA and a prolongation of preparation time, was eliminated. Recently, Dutilh et al. (7) described a PCR procedure for DNA amplification of a fragment of 129 bp from the major outer membrane protein gene in the genital serotypes of C. trachomatis. They used phenol-chloroform extraction, ethanol precipitation, and RNase treatment of their samples. All genital serotypes showed a positive reaction when portions of a cell culture containing 106 to 107 elementary bodies were used. The difference in sensitivity from our one to two elementary bodies could be due to inactivation of the Taq DNA polymerase or to loss of target DNA during phenolchloroform extraction or ethanol precipitation. Furthermore, our target DNA was placed on the plasmid, of which about 10 copies were found in every elementary body. For

VOL. 28, 1990

PCR, Dutilh et al. (7) used 0.75 U of Taq polymerase, which we found resulted in a lower sensitivity. Dean et al. (3) used PCR for amplifying and radiolabeling a probe that was generated from the plasmid of C. trachomatis serovar C. This probe was used to hybridize with conjunctival specimens from patients with trachoma. By this method, they found a sensitivity of 90% and a specificity of 94% when this method was compared with multiple tissue culture passages. Because of the extreme sensitivity of the PCR technique, contamination is probably the most extensive problem. Kaneko et al. (9) and Lo et al. (Y. M. D. Lo, W. Z. Mehal, and K. A. Fleming, Lancet ii:679, 1988) have described the problems with contaminated reagents, pipettes, and primers. Concerning our experiments in which negative controls were prepared in different places (Fig. 4), we conclude that airborne target DNA contamination is a factor of great importance. It is therefore crucial to handle reagents and target DNA in absolutely closed systems. By use of a system in which all reagents are mixed with unused pipettes (Finnpipettes; Labsystems) in a place that had never been exposed to the target DNA, and by ensuring that sample mixing is performed in a closed system with disposable syringes and containers, a very low risk of contamination is ensured. Negative controls should therefore be included in each processing step. The PCR technique used in the direct detection of C. trachomatis in urogenital specimens from patients has not been evaluated by others. Our sensitivity revealed a value of lo-17 g of DNA when PCR was performed for 40 cycles. This value corresponds to the detection of one copy of the plasmid. When PCR was performed for 30 cycles, the sensitivity corresponded to the detection of 10 copies of the plasmid, which corresponds to the number of copies found in one elementary body. Compared with cell culture, we found that the PCR technique had a sensitivity of 100%. The specificity of the PCR was 93% for either 40 or 30 cycles. The 14 false-positive specimens were tested with an EIA kit (IDEIA) after proteinase K treatment. Of the 14 falsepositive specimens, 12 were positive by this test system. Because the EIA had a sensitivity of 90.8% and a specificity of 95.4%, as we evaluated previously (14a), it seems reasonable to calculate an approximated corrected specificity of about 99% when PCR is performed for either 40 or 30 cycles. This is in agreement with the results reported by Mahony et al. (J. B. Mahony, K. E. Luinstra, and M. A. Chernesky, 8th Meet. Int. Soc. Sex. Transm. Dis. Res., abstr. no. 20, 1989). On the basis of the results presented here, we conclude that the PCR technique is a valuable tool for diagnosing genital C. trachomatis infections because of its high sensitivity and specificity. The PCR is not time-consuming. Specimen manipulation can be reduced to very few incubation steps before the sample from the patient is added to the reaction mixture, and sample evaluation can be judged directly from the presence of DNA bands in agarose gels. The importance of contamination must, however, be considered. Development of closed systems and the use of disposable utensils might eliminate this risk of contamination. ACKNOWLEDGMENT This work was supported by a grant from the Danish Biotechnological Center for Microbiology. LITERATURE CITED 1. Birkelund, S., A. G. Lundemose, and G. Christiansen. 1988. Chemical cross-linking of Chlamydia trachomatis. Infect. Im-

mun. 56:654-659.

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