JPWO2019223517A5

JPWO2019223517A5 -

Info

Publication number: JPWO2019223517A5
Application number: JP2020565479A
Authority: JP
Publication date: 2022-07-20
Anticipated expiration: 2039-05-05

Description

本発明のいくつかの具体的な実施形態において、ステップａ）では、磁気ビーズ捕捉法による被検サンプルのＤＮＡの抽出は、
被検サンプルを取り、保護液中で混合し、粉砕し、遠心分離した後、上清を取るステップと、
上清を再度遠心分離し、上清を取り、溶解液及び特定の相補的なオリゴヌクレオチド捕捉配列を有する磁気ビーズを上清液に入れ、インキュベートするステップと、
一部の上清を捨て、磁気ビーズを洗浄し、清潔な遠沈管に移し、洗浄液を加え、室温、１００－２０００ｒｐｍで０．５－５ｍｉｎインキュベートし、磁気スタンドに置き、上清を吸って除去し、３回繰り返すステップと、
緩衝液で標的遺伝子ＤＮＡを溶出するステップと、
を含む。
In some specific embodiments of the present invention, in step a), extraction of the DNA of the test sample by the magnetic bead capture method is performed.
The step of taking the test sample, mixing it in a protective solution, pulverizing it, centrifuging it, and then taking the supernatant.
The steps of centrifuging the supernatant again, removing the supernatant, placing the lysate and magnetic beads with specific complementary oligonucleotide capture sequences in the supernatant and incubating.
Discard some supernatant, wash the magnetic beads, transfer to a clean centrifuge tube, add wash solution, incubate for 0.5-5 min at room temperature, 100-2000 rpm, place on magnetic stand, suck and remove supernatant And the step that repeats 3 times,
The step of eluting the target gene DNA with a buffer solution,
including.

本発明のいくつかの具体的な実施形態において、前記結果出力機構は、被験体が腫瘍に罹患する確率又は可能性を出力するために使用される。
In some specific embodiments of the invention, the result output mechanism is used to output the probability or likelihood that a subject will develop a tumor.

以下、実施例により本発明をさらに説明する。
実施例１
１６３例の糞便検体（８０例の結腸直腸がん、８３例の正常検体；全て大腸内視鏡検査又は病理的検査により確診された）を粉砕して遠心分離し、１００ｕｌ捕捉磁気ビーズ（ＣＯＬ４Ａ２遺伝子の捕捉配列を含む）を加え、以下の手順に従って操作し、Ｂｉｓｕｌｆｉｔｅで転換された後のＤＮＡを１５ｕｌ得た。次に、ｑＭＳＰによりＣＯＬ４Ａ２のメチル化のレベルを検出した。
Hereinafter, the present invention will be further described with reference to Examples.
Example 1
163 fecal specimens (80 colorectal cancers, 83 normal specimens; all confirmed by colonoscopy or pathological examination) were crushed and centrifuged to capture 100 ul magnetic beads (COL4A2 gene). (Containing the capture sequence of) was added and operated according to the following procedure to obtain 15 ul of DNA after conversion by colonoscopy. Next, the level of methylation of COL4A2 was detected by qMSP.

手順は以下の通りである。
１）大腸内視鏡結果を有する健常者及び結腸直腸腫瘍患者の糞便検体を収集し、１ｇ糞便：４ｍＬ保護液で混合して粉砕した後、５０００ｒｐｍで１０ｍｉｎ遠心分離し、上清を取り、沈殿を捨てた。
２）１０ｍＬ上清を取り、再度遠心分離し、上清３．２ｍＬを取り、２ｍＬ溶解液及び１００ｕｌ捕捉磁気ビーズＭ１を加え、９２℃で１０ｍｉｎインキュベートし、その後、室温で１時間放置した。
３）磁気スタンドに置き、一部の上清を捨てた後、磁気ビーズを洗浄し、２ｍＬ遠沈管に移し、８００ｕｌ洗浄液Ｗ１を加え、室温、１３００ｒｐｍで１ｍｉｎインキュベートし、磁気スタンドに置き、上清を吸って除去し、３回繰り返した。
４）５５ｕｌ溶出液を加え、９２℃、１３００ｒｐｍで１０ｍｉｎインキュベートし、磁気スタンドに置き、３ｍｉｎ内で５０ｕｌ溶出液を新しいＥＰ管に移した。
５）ＥＺＤＮＡＭｅｔｈｙｌａｔｉｏｎＫｉｔ（ＺｙｍｏＲｅｓｅａｒｃｈ）により前のステップにおけるＤＮＡ断片をメチル化処理し、最後の溶出液１５ｕｌをｑＭＳＰ検出に使用した。
The procedure is as follows.
1) Collect stool samples from healthy subjects with colonoscopy results and patients with colorectal tumors, mix with 1 g stool: 4 mL protective solution, crush , centrifuge at 5000 rpm for 10 minutes, remove the supernatant, and precipitate. Was thrown away.
2) 10 mL supernatant was taken, centrifuged again, 3.2 mL of supernatant was taken, 2 mL lysate and 100 ul capture magnetic beads M1 were added, incubated at 92 ° C. for 10 min, and then left at room temperature for 1 hour.
3) Place on a magnetic stand, discard a part of the supernatant, wash the magnetic beads, transfer to a 2 mL centrifuge tube, add 800 ul cleaning solution W1, incubate for 1 min at room temperature, 1300 rpm, place on the magnetic stand, and place the supernatant. Was sucked and removed, and repeated 3 times.
4) 55 ul eluate was added, incubated at 92 ° C. and 1300 rpm for 10 min, placed on a magnetic stand, and within 3 min, 50 ul eluate was transferred to a new EP tube.
5) The DNA fragment in the previous step was methylated with EZ DNA Methylation Kit (Zymo Research), and the final eluate 15 ul was used for qMSP detection.

Claims

The use of a COL4A2 gene methylation detection reagent in the manufacture of a tumor detection reagent or kit, wherein the COL4A2 gene methylation detection reagent comprises a primer pair, the primer pair is set forth in SEQ ID NO: 2 and SEQ ID NO: 3.
The tumor is a colorectal tumor
The test sample to be detected reagent is intestinal tissue or feces, use.

The use according to claim 1, wherein the tumor is colorectal cancer or adenoma.

The use according to claim 1, wherein the methylation detection reagent for the COL4A2 gene further comprises a capture sequence shown in SEQ ID NO: 1 or in a complementary sequence of the sequence shown in SEQ ID NO: 1.

The use according to claim 1, wherein the COL4A2 gene methylation detection reagent further comprises a probe shown in SEQ ID NO: 4 or in a complementary sequence of the sequence shown in SEQ ID NO: 4.

In step (1) of detecting the methylation level of the COL4A2 gene of the subject,
The step (2) of comparing the methylation level of the COL4A2 gene of the subject with the methylation level of the normal control sample, and
If the level of the COL4A2 gene of the subject is higher than the methylation level of the normal control sample, it indicates that the subject has a tumor or is at risk of developing a tumor, and the normal sample and the tumor sample are selected. Step (3) to distinguish and
The methylation level was detected by the primer pairs set forth in SEQ ID NO: 2 and SEQ ID NO: 3, and the tumor was a colorectal tumor.
The test sample is intestinal tissue or feces,
How to detect tumor risk in vitro.

The method of claim 5 , wherein the tumor is colorectal cancer or adenoma.

The method according to claim 5 , wherein the normal sample and the tumor sample are distinguished by detecting the methylation level of the COL4A2 gene promoter region and the region close to the promoter region.

The method of claim 5 , wherein the methylation level is detected by methylation-specific PCR or methylation-specific quantitative PCR.

The method of claim 5 , wherein the methylation level is detected by methylation-specific quantitative PCR.

In step (1), the detection of the methylation level of the COL4A2 gene of the subject is
Step a) to extract the DNA of the test sample by the magnetic bead capture method,
Step b) in which the DNA of the test sample is converted with hydrogen sulfite, sodium bisulfite or hydrazine salt, and
Step c) detected by methylation-specific quantitative PCR,
5. The method of claim 5 .

In step a), the DNA extraction of the test sample by the magnetic bead capture method is performed.
The step of taking the test sample, mixing it in a protective solution, pulverizing it, centrifuging it, and then taking the supernatant.
The steps of centrifuging the supernatant again, removing the supernatant, placing the lysate and magnetic beads with specific complementary oligonucleotide capture sequences in the supernatant and incubating.
Discard some supernatant, wash the magnetic beads, transfer to a clean centrifuge tube, add wash solution, incubate for 0.5-5 min at room temperature, 100-2000 rpm, place on magnetic stand, suck supernatant The step of removing and repeating 3 times,
The step of eluting the target gene DNA with a buffer solution,
10. The method of claim 10 .

The detection criteria are as follows,
Judge tumor specimens and normal specimens based on the limit value,
In the stool sample, the limit value of the Ct value is 32 to 42, and when the Ct value of the stool sample is equal to or less than the limit value of the Ct value, it is determined as a tumor sample, and the Ct value of the stool sample is the Ct value of the stool sample. If it is larger than the limit value, it is judged as a normal sample, and it is judged as a normal sample.
In the tissue sample, the limit value of the methylation level value is 1 to 10, and when the methylation level value of the tissue sample is equal to or more than the limit value of the methylation level value, it is judged as a tumor sample and the methyl of the tissue sample is determined. The method according to claim 5 , wherein when the methylation level value is smaller than the limit value of the methylation level value, it is determined as a normal sample.

(1) COL4A2 gene methylation detection mechanism and
(2) Data processing mechanism and
(3) Result output mechanism and
Is a tumor detection system that includes
The methylation detection mechanism further comprises the primer pairs set forth in SEQ ID NO: 2 and SEQ ID NO: 3, and the tumor is a colorectal tumor.
The test sample is intestinal tissue or feces, a system.

13. The system of claim 13 , wherein the tumor is colorectal cancer or adenoma.

The system according to claim 13 , wherein the methylation detection mechanism includes one or more of a fluorescence quantitative PCR device, a PCR device, and a sequencer.

The data processing mechanism
a. Received test data of test sample and normal control sample,
b. Memorize the test data of the test sample and the normal control sample,
c. Compare the test data of the same type of test sample and normal control sample,
d. 13. The system of claim 13 , which is arranged to determine the probability or likelihood that a subject will develop a tumor based on the comparison results.

13. The system of claim 13 , wherein the result output mechanism is used to output the probability or likelihood that a subject will develop a tumor.

The judgment criteria of the data processing mechanism are as follows.
Judge tumor specimens and normal specimens based on the limit value,
In the stool sample, the limit value of the Ct value is 32 to 42, and if the Ct value of the stool sample is smaller than the limit value of the Ct value, it is judged as a tumor sample, and the Ct value of the stool sample is the limit of the Ct value. If it is above the value, it is judged as a normal sample, and it is judged as a normal sample.
The limit value of the methylation level value in the tissue sample is 1 to 10, and when the methylation level value of the tissue sample is larger than the limit value of the methylation level value, it is judged as a tumor sample.
The system according to claim 13 , wherein when the methylation level value of the tissue sample is equal to or less than the limit value of the methylation level value, it is determined as a normal sample.