JP2004121240A - Primer set for detecting human papilloma virus, method for detecting the same and dna array for detecting the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide primer sets capable of specifically detecting two or more types of human papilloma virus genes with high sensitivity. <P>SOLUTION: A kit for detecting human papilloma virus containing two or more sets of the primer sets expressed by 52 kinds of specified sequences and a method for specifically detecting the human papilloma virus in a sample by using the kit to conduct PCR reaction are disclosed in the specification, respectively. Further, a DNA array suitably used in the method is disclosed. <P>COPYRIGHT: (C)2004,JPO

Description

(4) The present invention relates to a specific primer set for detecting a human papillomavirus gene, and a method for detecting a human papillomavirus gene using the same.

５０ More than 50 types of human papillomavirus have been identified so far, of which more than 20 infect the genital tract. It is known that the probability of disease development and the degree of malignancy vary depending on the type of infected human papillomavirus. For example, type 11 is frequently detected in benign condyloma, types 6 and 11 are detected in relatively benign cervical cancer, and types 16 and 18 are detected in malignant cervical cancer. Therefore, determining the type of infected papillomavirus is very important in diagnosis.

As a diagnostic method for distinguishing papillomavirus types, a method of amplifying and detecting only a specific type of human papillomavirus gene using type-specific primers, and a method using random primers or non-type-specific primers have been used. A method has been known in which a human papillomavirus gene is non-specifically amplified and then detected by hybridization using a type-specific probe.

However, in the former method, since the amplification reaction must be performed in a separate tube for each type, the operation is complicated, and it is difficult to perform the reaction with a small amount of sample. In the latter method, high detection sensitivity could not be obtained due to low amplification efficiency.
Prior art document information related to the present invention is as follows.
JP 2001-197894 A JP-A-2001-231587 Special table 2001-502546

(4) An object of the present invention is to provide a primer set capable of detecting a plurality of types of human papillomavirus genes specifically and with high sensitivity.

The present invention provides a kit including a PCR primer set capable of specifically detecting 26 kinds of human papillomaviruses. The kit includes two or more primer sets selected from the group consisting of primer sets 1-26 shown in Table II below. More specifically, the kit of the present invention comprises a primer set 1 containing an oligonucleotide having a base sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and a base sequence represented by SEQ ID NO: 3 and SEQ ID NO: 4, respectively. A primer set 2 containing an oligonucleotide having a base sequence represented by SEQ ID NO: 5 and SEQ ID NO: 6, respectively, and an oligo having a base sequence represented by SEQ ID NO: 7 and SEQ ID NO: 8 respectively A primer set 4 containing nucleotides, a primer set 5 containing oligonucleotides having the nucleotide sequences represented by SEQ ID NOS: 9 and 10 respectively, and an oligonucleotide having the nucleotide sequences represented by SEQ ID NOs: 11 and 12 respectively. Primer set 6, including Primer set 7 containing oligonucleotides having the nucleotide sequences represented by SEQ ID NO: 13 and SEQ ID NO: 14, primer set 8 containing oligonucleotides having the nucleotide sequences represented by SEQ ID NO: 15 and SEQ ID NO: 16, respectively Primer set 9 including an oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 17 and SEQ ID NO: 18, a primer set 10 including an oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 19 and SEQ ID NO: 20, respectively, SEQ ID NO: 21 And a primer set 11 including an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 22, a primer set 12 including an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 23 and SEQ ID NO: 24, respectively, SEQ ID NO: 5 and a primer set 13 including an oligonucleotide having a base sequence represented by SEQ ID NO: 26, a primer set 14 including an oligonucleotide having a base sequence represented by SEQ ID NO: 27 and SEQ ID NO: 28, respectively, SEQ ID NO: 29 and A primer set 15 including an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 30, a primer set 16 including an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 31 and SEQ ID NO: 32, respectively, SEQ ID NO: 33 and SEQ ID NO: A primer set 17 including an oligonucleotide having a base sequence represented by SEQ ID NO: 34, a primer set 18 including an oligonucleotide having a base sequence represented by SEQ ID NO: 35 and SEQ ID NO: 36, respectively; And primer set 19 containing an oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 38 and SEQ ID NO: 38, a primer set 20 containing the oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 39 and SEQ ID NO: 40, respectively, SEQ ID NO: 41 and the sequence Primer set 21 including an oligonucleotide having a base sequence represented by SEQ ID NO: 42, Primer set 22 including an oligonucleotide having a base sequence represented by SEQ ID NO: 43 and SEQ ID NO: 44, respectively, SEQ ID NO: 45 and SEQ ID NO: 46 respectively A primer set 23 containing an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 47 and a primer set 24 containing an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 48, respectively, SEQ ID NO: 49 and a sequence respectively Or a primer set 25 comprising an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 50, a primer set 26 comprising an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 51 and SEQ ID NO: 52, respectively. Includes more primer sets.

According to the present invention, a specific type of human papillomavirus can be specifically detected even when each primer set is used as a mixture. As shown in the Examples below, since the primer set 1-26 of the present invention has high specificity for each target human papilloma virus, when the primer set 1-26 is mixed and subjected to a multiplex PCR reaction in one test tube, In addition, it is possible to specifically amplify a particular type of human papillomavirus gene present in a sample. This also applies to the case where any two primer sets, any three primer sets, any four primer sets, and the like are selected from the primer sets 1-26 and used as a mixture. It means that the corresponding target human papillomavirus is specifically amplified.

Preferably, the kit of the present invention comprises three or more primer sets, more preferably four or more primer sets selected from the group consisting of primer sets 1-26 shown in Table II below. More preferably, the kit of the present invention has a primer set 3 including an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 5 and SEQ ID NO: 6, respectively, having a nucleotide sequence represented by SEQ ID NO: 9 and SEQ ID NO: 10, respectively. Primer set 5 including oligonucleotides, Primer set 8 including oligonucleotides having nucleotide sequences represented by SEQ ID NOS: 15 and 16, respectively, oligonucleotide having nucleotide sequences represented by SEQ ID NOs: 17 and 18 respectively , A primer set 10 including an oligonucleotide having a base sequence represented by SEQ ID NO: 19 and SEQ ID NO: 20, respectively, including an oligonucleotide having a base sequence represented by SEQ ID NO: 23 and SEQ ID NO: 24, respectively ply Set 12, a primer set 13 including an oligonucleotide having a base sequence represented by SEQ ID NO: 25 and SEQ ID NO: 26, respectively, a primer set 16 including an oligonucleotide having a base sequence represented by SEQ ID NO: 31 and SEQ ID NO: 32, respectively A primer set 18 containing an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 35 and SEQ ID NO: 36, respectively, a primer set 19 containing an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 37 and SEQ ID NO: 38, respectively 3 or more primer sets, more preferably 3 or more primers selected from the group consisting of a primer set 23 comprising an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 45 and SEQ ID NO: 46 Including a set.

Also preferably, the present invention provides a human papilloma virus 16, 18, 31, and 8 comprising eight kinds of oligonucleotides each having the nucleotide sequence of SEQ ID NO: 5, 6, 9, 10, 17, 18, 31, and 32. A kit for detecting 45 is provided. Also preferably, the invention provides each of SEQ ID NOs: 5, 6, 9, 10, 17, 18, 19, 20, 23, 24, 25, 26, 31, 32, 35, 36, 37, 38, 45, Human papillomaviruses 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 comprising 24 types of oligonucleotides having the nucleotide sequences described in 46, 47, 48, 49 and 50. A kit for detection is provided. Also preferably, the invention provides each of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 9, 10, 17, 18, 19, 20, 23, 24, 25, 26, 29, 30, 31, Human papillomavirus comprising 36 kinds of oligonucleotides having the nucleotide sequences described in 32, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50 Kits for detecting 6, 11, 16, 18, 31, 33, 35, 39, 42, 45, 51, 52, 53, 54, 55, 56, 58 and 59 are provided. Most preferably, the kit of the present invention contains 52 types of oligonucleotides each having the nucleotide sequence of SEQ ID NOS: 1-52.

In another aspect, the present invention provides a method for detecting the presence of a human papillomavirus in a sample. The method includes performing a polymerase chain reaction using the above-described kit of the present invention with the DNA obtained from the sample as a template, and detecting the amplified human papillomavirus gene. In order to detect the expression of human papillomavirus in a sample, after preparing mRNA from the sample and synthesizing cDNA, the human papillomavirus gene can be amplified and detected in the same manner as described above.

In still another aspect, the present invention relates to a DNA for detecting human papilloma virus, wherein two or more DNA fragments selected from the group consisting of DNA fragments 1-26 shown in Table III are immobilized. Provide an array.

{Each oligonucleotide of the primer set of the present invention can be chemically synthesized using, for example, a general-purpose DNA synthesizer (for example, Model 394 manufactured by Applied Biosystems). Oligonucleotides may be synthesized using any of the other methods well known in the art.

In order to detect the presence of the human papillomavirus gene in a sample using the primer set of the present invention, DNA is extracted from the sample, and polymerase chain reaction is carried out using the primer set of the present invention as a template to obtain the human papillomavirus gene. To amplify. As a sample, a tissue sample, a biopsy, a smear, or the like obtained from a subject suspected of being infected with human papilloma virus can be used. Alternatively, cultured cells such as HeLa cells may be used.

方法 Methods for extracting DNA from a sample are well known in the art, and for example, extraction with phenol / chloroform can be used. Alternatively, a commercially available DNA extraction reagent may be used.

Furthermore, since the primer set of the present invention is selected so that the sequence in the coding region of the human papillomavirus gene is amplified, it can be used to detect the expression of the human papillomavirus gene in a sample. In order to detect the expression of the human papillomavirus gene, RNA is extracted from the sample to prepare a template cDNA, which is used as a template to perform a polymerase chain reaction using the primer set of the present invention, and is expressed in the sample. Amplify the human papillomavirus gene.

方法 Methods for extracting RNA from a sample are well known in the art, for example, guanidine-isothiocyanate and phenol / chloroform extraction can be used. Alternatively, a commercially available RNA extraction reagent may be used.

方法 Methods of synthesizing cDNA using RNA as a template are also well known in the art. Using a commercially available random primer as a primer, cDNA can be synthesized using reverse transcriptase in the presence of dNTPs. As the reverse transcriptase, for example, SuperScriptII (Invitrogen) can be used.

Next, polymerase chain reaction (PCR) is performed using the thus prepared DNA or cDNA as a template and a mixture of the primer set of the present invention as a primer. For example, Ex @ Taq (registered trademark) (Takara) can be used as the polymerase. Methods for selecting appropriate PCR reaction conditions based on the Tm of a primer are well known in the art, for example, at 94 ° C for 5 minutes, then at 94 ° C for 30 seconds, at 55 ° C for 30 seconds, at 72 ° C. For 1 minute for 25 cycles, then at 72 ° C. for 5 minutes. Next, the presence or absence of each human papilloma virus in a sample can be detected by hybridizing the amplification product with a detection array on which a gene specific to each human papilloma virus is spotted.

ＤＮＡ The DNA array for detection is a support on which a DNA fragment having a sequence capable of hybridizing with the sequence of the human papillomavirus gene to be detected is immobilized. The support is not limited, but a nylon membrane, a nitrocellulose membrane, glass, a silicon chip, or the like can be used. Preferably, the DNA fragments to be immobilized on the detection array are selected so as to hybridize only with the corresponding type of human papillomavirus gene and not with other types of human papillomavirus gene. Most preferably, the detection array used in the present invention has two or more of the DNA fragments shown in Table III immobilized thereon.

That is, in another aspect of the present invention, the present invention provides a method for detecting a human papillomavirus in which two or more DNA fragments selected from the group consisting of DNA fragments 1-26 shown in Table III are immobilized. To provide a DNA array.

キ ッ ト The kit of the present invention is characterized in that each human papillomavirus gene can be specifically amplified even when a mixture of primer sets corresponding to 26 kinds of human papillomaviruses is used. Therefore, since the presence or absence of 26 human papillomaviruses can be simultaneously examined in one test tube, compared with the prior art method of performing PCR in separate test tubes using type-specific primers, It has the advantage that the assay is simple and the time, cost and sample required for the assay are low. In addition, by performing a PCR reaction using the mixture of the primer set of the present invention and detecting it with the DNA array of the present invention, even when a plurality of types of human papillomaviruses are present in a sample, these can be detected simultaneously. It is.

Furthermore, by using the primer set and the DNA array of the present invention, a synergistic effect between the specificity of PCR amplification based on the specificity of the primer and the specificity of hybridization with the DNA array can be obtained. Therefore, it is compared with the prior art method in which various human papillomavirus genes are non-specifically amplified using a random primer or a type non-specific primer (common primer), and then detected by hybridization using a type-specific probe. As a result, detection with higher specificity becomes possible.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Design of primers The gene sequence of human papillomavirus was obtained from a public database. Table I shows the GenBank accession numbers of the gene sequences used in the present invention.
Table I

Based on the sequence of each human papillomavirus gene, a primer set meeting the following criteria was designed: the sequence of the amplification product was included in the coding region; the melting temperature was in the range of 58-63 ° C; Has no significant homology with other types of human papillomaviruses; does not form secondary structures in the molecule; primers do not hybridize. Thus, a primer set specific to each human papilloma virus was designed.

Table II

オ リ ゴ Based on the sequences of the primer set shown in Table II, oligonucleotide DNA was synthesized by a DNA synthesizer and the sequence was confirmed.

Example 2 Confirmation of primer specificity HPV06, HPV11, HPV16, HPV17, HPV18, HPV20, HPV21, HPV30, HPV31, HPV33, HPV34, HPV35, HPV39, HPV40, HPV42, HPV45, HPV47, HPV51, HPV52, HPV53, HPV54 , HPV55, HPV56, HPV58, HPV59 and HPV61 were each introduced into plasmid pUC18 to prepare a plasmid for template.

Total RNA was extracted from HeLa cell cultures known to contain the HPV18 sequence and cDNA was synthesized using random primers. In addition, cDNA was similarly synthesized from SW480 cell culture. Furthermore, genomic DNA was prepared by extracting DNA from human tissues. A test template mixture was prepared by mixing each of these template plasmids with a 100-fold amount of HeLa / cDNA, SW480 / cDNA and genomic DNA.

用いたＰＣＲの反応条件は次のとおりであった：９４℃で５分間、次に９４℃で３０秒間、５５℃で３０秒間、７２℃で１分間を３０サイクル、次に７２℃で５分間の後に４℃で保存。得られたＰＣＲ産物をゲル電気泳動により分離した。図１に示されるように、いずれのプライマーセットについても、予測される長さの増幅産物が得られ、非特異的ハイブリダイズに起因する夾雑増幅物がないことが確認された。 Using this test template mixture as a template, a PCR reaction was performed using each primer set prepared in Example 1. The composition of the reaction was as follows:

The PCR reaction conditions used were as follows: 94 ° C for 5 minutes, then 30 cycles of 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 1 minute, then 72 ° C for 5 minutes. After storage at 4 ° C. The obtained PCR products were separated by gel electrophoresis. As shown in FIG. 1, for each of the primer sets, an amplification product of an expected length was obtained, and it was confirmed that there was no contaminant amplification product due to non-specific hybridization.

用いたＰＣＲの反応条件は次のとおりであった：９４℃で５分間、次に９４℃で３０秒間、５５℃で３０秒間、７２℃で１分間を３０サイクル、次に７２℃で５分間の後に４℃で保存。増幅産物を水で１００倍に希釈し、同じ条件で２回目のＰＣＲ反応を行った。得られたＰＣＲ産物の量および純度はゲル電気泳動により確認した。次に、ナイロンフィルタに図２に示す配置で各ＰＣＲ産物を４ｎｇずつスポットして、検出用アレイを作成した。表ＩＩＩにスポットしたフラグメントを示す。 Example 3 Preparation of Array for Detection Using the plasmid for each template prepared in Example 2 as a template, a PCR reaction was performed using the corresponding F primer and R primer (the sequences are shown in Table II above), The spot DNA fragment was amplified. The composition of the reaction was as follows:

The PCR reaction conditions used were as follows: 94 ° C for 5 minutes, then 30 cycles of 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 1 minute, then 72 ° C for 5 minutes. After storage at 4 ° C. The amplification product was diluted 100-fold with water, and a second PCR reaction was performed under the same conditions. The amount and purity of the obtained PCR product were confirmed by gel electrophoresis. Next, 4 ng of each PCR product was spotted on a nylon filter in the arrangement shown in FIG. 2 to prepare a detection array. Table III shows the spotted fragments.

Table III

用いたＰＣＲの反応条件は次のとおりであった：９４℃で５分間、次に９４℃で３０秒間、５５℃で３０秒間、７２℃で１分間を２５サイクル、次に７２℃で５分間の後に４℃で保存。
（ｂ）　テンプレート：ＨｅＬａ／ｃＤＮＡ、ＳＷ４８０／ｃＤＮＡ、ゲノムＤＮＡの混合物
プライマー：プライマーセット混合物
反応条件は、ＰＣＲのサイクル数を３０サイクルにしたことを除き、上述の（ａ）と同じ。
（ｃ）　テンプレート：ＨｅＬａ／ｃＤＮＡ、ＳＷ４８０／ｃＤＮＡの混合物；
プライマー：ランダムプライマー
反応液の組成は以下のとおりであった：

用いたＰＣＲの反応条件は次のとおりであった：９８℃で３分間、次に９８℃で２０秒間、５５℃で１０秒間、７４℃で６分間を２５サイクル、次に４℃で保存。
（ｄ）　テンプレート：ゲノムＤＮＡ
プライマー：ランダムプライマー
反応条件は上述の（ｃ）と同じ。 Example 4 Detection of human papilloma virus using a primer mixture HeLa / cDNA, SW480 / cDNA and genomic DNA described in Example 2 or a mixture thereof were used as a template, and a mixture of the primer set of the present invention was used as a primer to prepare the following: The PCR reaction was performed under the reaction conditions.
(A) Template: mixture of HeLa / cDNA, SW480 / cDNA, genomic DNA Primer: The composition of the primer set mixture reaction solution was as follows:

The PCR reaction conditions used were as follows: 94 ° C. for 5 minutes, then 25 cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 1 minute, and then at 72 ° C. for 5 minutes. After storage at 4 ° C.
(B) Mixture of template: HeLa / cDNA, SW480 / cDNA, genomic DNA Primer: Primer set mixture Reaction conditions were the same as in (a) above, except that the number of PCR cycles was 30.
(C) template: a mixture of HeLa / cDNA, SW480 / cDNA;
Primer: The composition of the random primer reaction was as follows:

The PCR reaction conditions used were as follows: 98 ° C. for 3 minutes, then 25 cycles of 98 ° C. for 20 seconds, 55 ° C. for 10 seconds, 74 ° C. for 6 minutes, then stored at 4 ° C.
(D) Template: Genomic DNA
Primer: Random primer reaction conditions are the same as in (c) above.

Next, these PCR products were analyzed using the array prepared in Example 3. Hybridization was performed at 72 ° C. using 10.0 μl of the probe. The results are shown in FIG. As shown in FIG. 3A, when a mixture of cDNA and genomic DNA was used as a sample and the mixture of the primer set of the present invention was used as a primer, the sequence of HPV18 in the sample was specifically detected. (B) shows that increasing the number of cycles results in non-specific amplification. (C) shows that no detectable signal was obtained for HPV18 when a random primer was used. (D) is a control reaction. It should be noted that HPV55 signal was observed in all filters, because the signal due to non-specific hybridization of DNA fragments spotted on the DNA array was strong, and the level of this background signal was HPV55. Is clearly distinguishable from the level of the positive signal in the presence of this gene.

These results indicate that the primer set of the present invention can specifically detect human papillomavirus in a sample even when used in combination.

FIG. 1 is an electrophoretic photograph of an amplification product showing the specificity of the primer of the present invention. FIG. 2 shows the arrangement of spots of each array on the array used in the method of the present invention. FIG. 3 shows the results of array hybridization analysis of PCR products amplified using the primer mixture of the present invention.

Claims (9)

A kit for detecting human papillomavirus, comprising two or more primer sets selected from the group consisting of primer sets 1-26 shown in Table II. A kit for detecting human papilloma virus, comprising three or more primer sets selected from the group consisting of primer sets 1-26 shown in Table II. Detect human papillomavirus comprising three or more primer sets selected from the group consisting of primer sets 3, 5, 8, 9, 10, 12, 13, 16, 18, 19 and 23 shown in Table II Kit for. A kit for detecting human papillomaviruses 16, 18, 31, and 45, each comprising eight types of oligonucleotides having the nucleotide sequences of SEQ ID NOs: 5, 6, 9, 10, 17, 18, 31, and 32. SEQ ID NOs: 5, 6, 9, 10, 17, 18, 19, 20, 23, 24, 25, 26, 31, 32, 35, 36, 37, 38, 45, 46, 47, 48, 49 and A kit for detecting human papilloma virus 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59, comprising 24 kinds of oligonucleotides having the nucleotide sequence of 50. SEQ ID NOs: 1, 2, 3, 4, 5, 6, 9, 10, 17, 18, 19, 20, 23, 24, 25, 26, 29, 30, 31, 32, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50, comprising 36 kinds of oligonucleotides having the nucleotide sequences described in human papillomavirus 6, 11, 16, 18, A kit for detecting 31, 33, 35, 39, 42, 45, 51, 52, 53, 54, 55, 56, 58 and 59. A kit for detecting human papilloma virus, comprising 52 types of oligonucleotides each having the nucleotide sequence of SEQ ID NOS: 1-52. A method for detecting the presence or expression of a human papilloma virus in a sample, wherein the DNA obtained from the sample or cDNA prepared by reverse transcription from mRNA obtained from the sample is used as a template as a template. A method comprising performing a polymerase chain reaction using the kit according to 1 above, and detecting the amplified human papillomavirus gene. A DNA array for detecting human papilloma virus, wherein two or more DNA fragments selected from the group consisting of DNA fragments 1-26 shown in Table III are immobilized.

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