Method of detecting chlamydia trachomatis and kit therefor

Technical Field

0001The present invention relates to a method for detecting <i>Chlamydia trachomatis</i> and a kit therefore.

Background Art

0002<i>Chlamydia trachomatis</i> is one of nongonococcal urethritis pathogens, and as a method of detecting it, a method of amplifying a partial sequence of a cryptic plasmid existing in <i>Chlamydia trachomatis</i> cells in a multiple copy number by a gene amplification method and detecting the amplification product is known (Japanese Patent Nos. <patcit id="pcit0001" dnum="JP2719225B"><text>2719225</text></patcit> and <patcit id="pcit0002" dnum="JP3127135B"><text>3127135</text></patcit>). <patcit id="pcit0003" dnum="WO0173129A"><text>WO01/73129</text></patcit> relates to the use of "LOCI" technology to monitor amplification reactions, especially polymerase chain reactions. <patcit id="pcit0004" dnum="US6004826A"><text>US6,004,826</text></patcit> relates to a process for amplifying and detecting any desired specific nucleic acid sequence that exists in a nucleic acid or mixture thereof. <patcit id="pcit0005" dnum="US5232829A"><text>US5,232,829</text></patcit> relates to the synthesis of amplified biotin - labelled DNA target sequences of <i>Chlamydia trachomatis</i> by polymerase chain reaction techniques. <patcit id="pcit0006" dnum="EP0812921A2"><text>EP0812921 A2</text></patcit> relates to amplification primers that are useful in assays for species-specific detection of a target sequence in the cryptic plasmid of C. <i>trachomatis.</i>

Disclosure of the Invention

0003An object of the present invention is to provide a method for quickly detecting <i>Chlamydia trachomatis</i> with superior sensitivity and specificity and a kit therefor.

0004The inventors of the present invention found that, if PCR was performed with primers designed for a specific region in the cryptic plasmid pLGV440 of <i>Chlamydia trachomatis, Chlamydia trachomatis</i> could be quickly detected, and thus accomplished the present invention.

0005The present invention provides a method for detecting <i>Chlamydia trachomatis</i> comprising performing PCR using DNA obtained from a sample as a template and detecting an amplification product, wherein a primer pair used for PCR is designed on the basis of the nucleotide sequences of the regions corresponding to the nucleotide numbers 5157 to 5201 and 5245 to 5276 in the nucleotide sequence of SEQ ID NO: 1 so that the nucleotide sequence between the two regions can be amplified and wherein performing PCR comprises annealing at a temperature of 64° to 68.2°C (detection method of the present invention).

0006The present invention also provides a kit used for the method of the present invention, that is, a kit for detection of <i>Chlamydia trachomatis</i> by performing PCR using DNA obtained from a sample as a template, which comprises a primer pair designed on the basis of the nucleotide sequences of the regions corresponding to the nucleotide numbers 5157 to 5201 and 5245 to 5276 in the nucleotide sequence of SEQ ID NO: 1 so that the nucleotide sequence between the two regions can be amplified and wherein performing PCR comprises annealing at a temperature of 64° to 68.2°C(detection kit of the present invention).

0007In the present invention, the primer pair preferably consists of an oligonucleotide having an nucleotide sequence of SEQ ID NO: 2, 3 or 4 and an oligonucleotide having a nucleotide sequence of SEQ ID NO: 5 or 6, more preferably an oligonucleotide having the nucleotide sequence of SEQ ID NO: 3 and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 5, or an oligonucleotide having the nucleotide sequence of SEQ ID NO: 4 and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 6.

0008The present invention also provides a hybridization probe containing an oligonucleotide designed on the basis of the nucleotide sequence of the region corresponding to the nucleotide numbers 5210 to 5245 in the nucleotide sequence of SEQ ID NO: 1 and a label.

0009The nucleotide sequence of SEQ ID NO: 1 is the nucleotide sequence of the cryptic plasmid pLGV440 of the <i>Chlamydia trachomatis</i> (GenBank accession number X06707), and those skilled in the art can easily identify and recognize the region specified by the nucleotide numbers of the nucleotide sequence of SEQ ID NO: 1 also in a strain having a mutation in pLGV440 by taking difference in the nucleotide sequence that may exist depending on individuals or the like into consideration.

Brief Description of the Drawing

0010<figref idref="f0001">Fig. 1</figref> shows a production time course of an amplification product (detection results of real-time PCR): a: no copy, b: 2 copies, c: 20 copies, d: 200 copies, and e: 2000 copies.

Best Mode for Carrying out the Invention

<1> Detection method of the present invention

0011The detection method of the present invention is a method for detecting <i>Chlamydia trachomatis</i> comprising performing PCR using DNA obtained from a sample as a template and detecting an amplification product, wherein a primer pair used for PCR is designed on the basis of the nucleotide sequences of the regions corresponding to the nucleotide numbers 5157 to 5201 and 5245 to 5276 in the nucleotide sequence of SEQ ID NO: 1 so that the nucleotide sequence between the two regions can be amplified.

0012The sample is not particularly limited so long as it contains or possibly contains <i>Chlamydia trachomatis</i>. Examples thereof include urine, urethra swab, cervical swab and so forth. From these samples, DNA can be obtained by a usual method under conditions where DNA of the cryptic plasmid of <i>Chlamydia trachomatis</i> can be prepared.

0013PCR in the detection method of the present invention can be performed according to a usual PCR procedure except that DNA obtained from the sample is used as a template, and that the specific primer pair is used.

0014The primer pair used in the present invention is designed on the basis of a nucleotide sequence of a region corresponding to the nucleotide numbers 5157 to 5201 in the nucleotide sequence of SEQ ID NO: 1 (first region) and a nucleotide sequence of a region corresponding to the nucleotide numbers 5245 to 5276 (second region) so that the nucleotide sequence between the two regions can be amplified.

0015The length of the primers is usually 10 to 40 nucleotides. Further, the position in each region and the length of the primers are preferably set so that the Tm value should become 55 to 70°C, and thus the annealing temperature used in PCR can be set to be relatively high. The Tm value used herein is a value calculated by the nearest neighbor base pair analysis. The primers constituting the primer pair are preferably designed to have substantially the same Tm values.

0016Specific examples of the primer designed on the basis of the first region include a primer having the nucleotide sequence of SEQ ID NO: 2 (corresponding to the nucleotide numbers 5157 to 5185 in the nucleotide sequence of SEQ ID NO: 1) or a nucleotide sequence complementary thereto, a primer having the nucleotide sequence of SEQ ID NO: 3 (corresponding to the nucleotide numbers 5171 to 5201 in the nucleotide sequence of SEQ ID NO: 1) or a nucleotide sequence complementary thereto, and a primer having the nucleotide sequence of SEQ ID NO: 4 (corresponding to the nucleotide numbers 5171 to 5196 in the nucleotide sequence of SEQ ID NO: 1) or a nucleotide sequence complementary thereto. Examples of the primer designed on the basis of the second region include a primer having the nucleotide sequence of SEQ ID NO:5 (corresponding to the nucleotide numbers 5276 to 5252 in the nucleotide sequence of SEQ ID NO:1) or a nucleotide sequence complementary thereto, and a primer having the nucleotide sequence of SEQ ID NO:6 (corresponding to the nucleotide numbers 5276 to 5246 in the nucleotide sequence of SEQ ID NO: 1) or a nucleotide sequence complementary thereto.

0017The primer pair is designed so that the nucleotide sequence between the two regions can be amplified, that is, one primer should be a sense primer and the other primer should be an antisense primer. Because the cryptic plasmid is a cyclic plasmid, it has two nucleotide sequences between the two regions. However, the primers are usually designed so that a shorter nucleotide sequence should be amplified.

0018Preferred examples of the primer pair include a combination of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 2, 3 or 4 and an oligonucleotide having a nucleotide sequence of SEQ ID NO: 5 or 6. Further, more preferred examples of the primer pair include a combination of an oligonucleotide having the nucleotide sequence of SEQ ID NO: 3 and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 5 and a combination of an oligonucleotide having the nucleotide sequence of SEQ ID NO: 4 and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 6.

0019The primer pair for the aforementioned specific regions can be designed by a method known to those skilled in the art taking PCR conditions into consideration. The primer pair can be designed by using a computer program for designing primers.

0020The primers may be designed as a mixing primer obtained by mixing two or more kinds of primers for one or both of the sense primer and the antisense primer. When a mutation of a nucleotide exists in the region for which the primer is designed, detection efficiency can be improved by using a mixed primer.

0021Although the PCR conditions may be determined according to a usual PCR method, the annealing temperature should be set to be relatively high due to the use of the aforementioned specific primer pair. The annealing temperature is usually 50 to 70°C and comprises 64° to 68.2°C. Because the annealing temperature is relatively high, the conditions for two-step PCR in which annealing and elongation are performed under the same conditions may be determined.

0022A typical example of composition of the PCR reaction mixture is as follows. <dl id="dl0001" compact="compact"><dt>DNA fragments</dt><dd>1 molecule or more</dd><dt>Primers</dt><dd>100 to 2000 nM</dd><dt>Nucleotides</dt><dd>100 to 500 µM for each</dd><dt>DNA polymerase</dt><dd>0.25 to 1.25 Units/µl</dd><dt>Tris-HCl (pH 7 to 9)</dt><dd>1 to 5 mM</dd><dt>MgCl₂</dt><dd>1.5 to 5 mM</dd><dt>Surfactant or gelatin</dt><dd>0 to 25%</dd></dl>

(Final volume: 25 to 100 µl)

0023Further, a typical example of the temperature cycle is as follows, and such a temperature cycle is usually repeated 30 to 60 times. <ol id="ol0001" compact="compact"><li>(1) Denaturation: 90 to 95°C, 1 to 60 seconds</li><li>(2) Annealing: 55 to 70°C, 6 to 60 seconds</li><li>(3) Elongation: 72 to 75°C, 6 to 60 seconds</li></ol>

0024A typical example of the temperature cycle for the two-step PCR is as follows, and this temperature cycle is usually repeated 30 to 60 times. <ol id="ol0002" compact="compact"><li>(1) Denaturation: 90 to 95°C, 1 to 60 seconds</li><li>(2) Annealing and elongation: 55 to 70°C, 6 to 60 seconds</li></ol>

0025In the detection method of the present invention, the amplification product can be detected by a usual method for detecting amplification products. For example, the amplification product may be detected by agarose gel electrophoresis, or by real time PCR, in which PCR is performed in the presence of a substance of which fluorescence changes when it binds to the amplification product (for example, a fluorescent dye of which fluorescence intensity changes when it binds to a doublestranded DNA, a hybridization probe designed so that it should hybridize with a single-stranded DNA, and fluorescence intensity thereof should change due to fluorescence resonance energy transfer (FRET) upon hybridization, and so forth), and then fluorescence is measured.

0026According to the detection method of the present invention, primers having a relatively high Tm value can be designed for specified regions, and therefore the annealing temperature used in PCR can be made relatively high. Accordingly, the time required to lower temperature from the denaturation temperature to the annealing temperature is reduced. Further, non-specific amplification is reduced due to the high annealing temperature. Furthermore, two-step PCR in which annealing and elongation are performed under the same conditions is also possible. As a result, quicker gene amplification is enabled.

0027The present invention also provides a hybridization probe suitable for detection by hybridization. Examples of such a hybridization probe include a probe comprising an oligonucleotide designed on the basis of a nucleotide sequence of a region corresponding to the nucleotide numbers 5210 to 5245 in the nucleotide sequence of SEQ ID NO: 1 and a label. This oligonucleotide may be complementary to a sense chain or complementary to an antisense chain.

0028The chain length and Tm value of the oligonucleotide of the hybridization probe are suitably determined depending on the hybridization conditions. The length of this oligonucleotide is usually 25 to 45 nucleotides, and the Tm value is usually 50 to 70°C. When the hybridization probe is used for real-time PCR, the Tm value of the oligonucleotide is preferably set to be higher than the Tm value of the primer by 2 to 5°C so that the probe should hybridize with a target sequence before hybridization of the primer. Examples of such an oligonucleotide include an oligonucleotide having the nucleotide sequence of SEQ ID NO: 7.

0029The hybridization probe can be labeled in such a manner that the hybridization should not be inhibited under the hybridization conditions. The hybridization probe for real-time PCR is preferably labeled in such a manner that fluorescence intensity should change when it hybridizes with a single-stranded DNA.

0030In the detection method of the present invention, the amplification product is preferably detected by a detection method using the hybridization probe of the present invention.

<2> Detection kit of the present invention

0031The detection kit of the present invention is a kit usable for the detection method of the present invention, that is, a kit for detection of <i>Chlamydia trachomatis</i> by performing PCR using DNA obtained from a sample as a template, which is characterized by including a primer pair designed on the basis of the nucleotide sequences of the regions corresponding to the nucleotide numbers 5157 to 5201 and 5245 to 5276 in the nucleotide sequence of SEQ ID NO: 1 so that the nucleotide sequence between the two regions can be amplified, and wherein performing PCR comprises annealing at a temperature of 64° to 68.2°C.

0032The primer pair is as described above with regard to the detection method of the present invention.

0033In the kit of the present invention, the primers of the primer pair may be included as a mixture or included separately.

0034The kit of the present invention may further include reagents required for PCR and/or detection of an amplification product in addition to the primer pair. Examples of such reagents include the aforementioned hybridization primer.

Examples

0035Hereafter, the present invention will be explained more specifically with reference to the following examples.

Example 1: Detection by PCR

0036From the first region (nucleotide numbers 5157 to 5201) and the second region (nucleotide number 5245 to 5276) of the nucleotide sequence of the cryptic plasmid of <i>Chlamydia trachomatis</i> (SEQ ID NO: 1, GenBank accession number X06707), nucleotide sequences having a high GC content (48 to 55%) and high Tm value (63 to 65°C (calculated by the nearest neighbor base pair method)) were selected. That is, 3 types of upstream primers (SEQ ID NOS: 2, 3 and 4) and 2 types of downstream primers (SEQ ID NOS: 5 and 6) were selected as target binding sequences. Then, oligonucleotides having these target binding sequences were synthesized as primers. <tables id="tabl0001" num="0001"><table frame="none"><title>Table 1</title><tgroup cols="1" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="97mm" /><thead><row><entry valign="top">Upstream (forward) primers:</entry></row></thead><tbody><row><entry>CT-F5157-29: cag tca cac cca aaa gct ctg gga gca tg (SEQ ID NO: 2)</entry></row><row><entry>CT-F5171-31: agc tct ggg agc atg ttc tta gtc tca gca g (SEQ ID NO: 3)</entry></row><row><entry>CT-F5171-26: agc tct ggg agc atg ttc tta gtc tc (SEQ ID NO: 4)</entry></row></tbody></tgroup><tgroup cols="1" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="97mm" /><thead><row><entry valign="top">Downstream (reverse) primers:</entry></row></thead><tbody><row><entry>CT-R5276-25: tcg cgt agg gct tag aat cac ctt c (SEQ ID NO: 5)</entry></row><row><entry>CT-R5276-31: tcg cgt agg gct tag aat cac ctt ctc gta c (SEQ ID NO: 6)</entry></row></tbody></tgroup></table></tables>

0037Three kinds of pairs each consisting of a combination of the aforementioned upstream and downstream amplification primers (SEQ ID NOS: 2-5, 3-5 and 4-6) were used for PCR. The reaction mixture (25 µL) contained 1 x Gene Taq buffer, 0.625 U of Gene Taq polymerase (Nippon Gene), 0.2 mM each of dGTP, dCTP, dATP and dUTP, 0.5 µM each of the upstream and downstream primers, and 2 to 2000 copies of <i>Chlamydia trachomatis</i> cell genomic DNA (ATCC strain CT-VR878). To examine appropriate reaction temperature, PCR was performed by using a temperature gradient for the annealing temperature for the amplification reaction in iCycler (BIO-RAD). The reaction temperatures were as follows: 95°C for 4 minutes, (95°C for 30 seconds, gradient of 62 to 72°C for 30 seconds, 72°C for 30 seconds) x 50 cycles, and 72°C for 7 minutes. Occurrence of amplification was confirmed by applying the product to 3% agarose gel electrophoresis.

0038Whichever primer pair was used, 2 copies of <i>Chlamydia trachomatis</i> cell genomic DNA could be detected with a relatively high annealing temperature condition of 64.0 to 68.2°C.

0039The above results showed that the aforementioned primer sequences had superior sensitivity and specificity and were effective for reducing the reaction time.

Example 2: Detection using two-step PCR

0040Among the primer pairs used in Example 1, the primer pairs of SEQ ID NOS: 3-5 and 4-6 were used for two-step PCR. The reaction mixture (25 µL) contained 1 x ΔTth buffer, 0.625 U of ΔTth (TOYOBO), 0.2 mM each of dGTP, dCTP, dATP and dUTP, 0.5 µM each of the upstream and downstream primers, and 2 to 2000 copies of <i>Chlamydia trachomatis</i> cell genomic DNA (ATCC strain CT-VR878). The reaction temperatures were as follows: 95°C for 1 minute, (95°C for 15 seconds, 65 or 68°C for 20 seconds) x 2 cycles, and (90°C for 15 seconds, 65 or 68°C for 20 seconds) x 58 cycles. The reaction was performed by using a thermal cycler (TaKaRa). Occurrence of amplification was confirmed by applying the product to 3% agarose gel electrophoresis.

0041When the primer pair of SEQ ID NOS: 3-5 was used, 2 copies of <i>Chlamydia trachomatis</i> cell genomic DNA could be detected with an annealing temperature of 68°C. When the primer pair of SEQ ID NOS: 4-6 was used, 2 copies of <i>Chlamydia trachomatis</i> cell genomic DNA could be detected with an annealing temperature of 65°C.

0042The above results showed that it was possible to perform two-step PCR in which the reaction time could be further reduced.

Example 3: Detection using real-time PCR

0043Among the primer pairs used in Example 1, the primer pair of SEQ ID NOS: 3-5 was used for two-step PCR. The reaction mixture (25 µL) contained 1 x ΔTth buffer, 0.625 U of ΔTth (TOYOBO), 0.2 mM each of dGTP, dCTP, dATP and dUTP, 1 µM of the upstream primer, 0.5 µM of the downstream primer, 0.2 µM of a probe for real-time detection (5FL-CT-5210-36: 5'-caa agc tag aac aac gcc gcc ttc cat tct tga tgc-3' (SEQ ID NO: 7), c at the 5' end was labeled with a fluorescent dye, type of the marker: BODIPY-FL (Molecular Probe)), and 2 to 2000 copies of <i>Chlamydia trachomatis</i> cell genomic DNA (ATCC strain CT-VR878). The reaction temperatures were as follows: 95°C for 1 minute, (95°C for 15 seconds, 65°C for 20 seconds) x 2 cycles, (90°C for 15 seconds, 65°C for 20 seconds) x 58 cycles. The reaction was performed in iCycler (BIO-RAD). The real-time detection procedure was according to Japanese Patent Laid-open Publication (Kokai) No. <patcit id="pcit0007" dnum="JP2001286300A"><text>2001-286300</text></patcit>.

0044The results are shown in <figref idref="f0001">Fig. 1</figref>. With the primer pair used in this example, 2 copies of <i>Chlamydia trachomatis</i> cell genomic DNA could be detected in real time with an annealing temperature of 65°C.

Industrial Applicability

0045The present invention provides a method for quickly detecting <i>Chlamydia trachomatis</i> with superior sensitivity and specificity.

Sequence Listing

0046<ul id="ul0001" list-style="none"><li><110> Arkray, Inc.</li><li><120> Method for detecting Chlamydia trachomatis and kit therefor</li><li><130> G849-0PC4043</li><li><150> <patcit id="pcit0008" dnum="JP2003050662A"><text>JP 2003-50662</text></patcit> <151> 2003-02-27</li><li><160> 7</li><li><210> 1 <211> 7501 <212> DNA <213> Chlamydia trachomatis</li><li><400> 1 <img file="EP1598431B1_D0001.tif" /><img file="EP1598431B1_D0002.tif" /><img file="EP1598431B1_D0003.tif" /><img file="EP1598431B1_D0004.tif" /></li><li><210> 2 <211> 29 <212> DNA <213> Artificial Sequence</li><li><220> <223> primer</li><li><400> 2 cagtcacacc caaaagctct gggagcatg    29</li><li><210> 3 <211> 31 <212> DNA <213> Artificial Sequence</li><li><220> <223> primer</li><li><400> 3 agctctggga gcatgttctt agtctcagca g    31</li><li><210> 4 <211> 26 <212> DNA <213> Artificial Sequence</li><li><220> <223> primer</li><li><400> 4 26 agctctggga gcatgttctt agtctc    26</li><li><210> 5 <211> 25 <212> DNA <213> Artificial Sequence</li><li><220> <223> primer</li><li><400> 5 25 tcgcgtaggg cttagaatca ccttc    25</li><li><210> 6 <211> 31 <212> DNA <213> Artificial Sequence</li><li><220> <223> primer</li><li><400> 6 31 tcgcgtaggg cttagaatca ccttctcgta c    31</li><li><210> 7 <211> 36 <212> DNA <213> Artificial Sequence</li><li><220> <223> probe</li><li><400> 7 36 caaagctaga acaacgccgc cttccattct tgatgc    36</li></ul>

Sequence Listing

0047<ul id="ul0002" list-style="none"><li><110> Arkray, Inc.</li><li><120> Method for detecting Chlamydia trachomatis and kit therefor</li><li><130> G849-0PC4043</li><li><150> <patcit id="pcit0009" dnum="JP2003050662A"><text>JP 2003-50662</text></patcit> <151> 2003-02-27</li><li><160> 7</li><li><210> 1 <211> 7501 <212> DNA <213> Chlamydia trachomatis</li><li><400> 1 <img file="EP1598431B1_D0005.tif" /><img file="EP1598431B1_D0006.tif" /><img file="EP1598431B1_D0007.tif" /><img file="EP1598431B1_D0008.tif" /></li><li><210> 2 <211> 29 <212> DNA <213> Artificial Sequence</li><li><220> <223> primer</li><li><400> 2 cagtcacacc caaaagctct gggagcatg    29</li><li><210> 3 <211> 31 <212> DNA <213> Artificial Sequence</li><li><220> <223> primer</li><li><400> 3 agctctggga gcatgttctt agtctcagca g    31</li><li><210> 4 <211> 26 <212> DNA <213> Artificial Sequence</li><li><220> <223> primer</li><li><400> 4 agctctggga gcatgttctt agtctc    26</li><li><210> 5 <211> 25 <212> DNA <213> Artificial Sequence</li><li><220> <223> primer</li><li><400> 5 tcgcgtaggg cttagaatca ccttc    25</li><li><210> 6 <211> 31 <212> DNA <213> Artificial Sequence</li><li><220> <223> primer</li><li><400> 6 tcgcgtaggg cttagaatca ccttctcgta c    31</li><li><210> 7 <211> 36 <212> DNA <213> Artificial Sequence</li><li><220> <223> probe</li><li><400> 7 caaagctaga acaacgccgc cttccattct tgatgc    36</li></ul>