CN112063712A

CN112063712A - Specific primer pair and kit for detecting septin9 gene methylation based on high-resolution melting curve

Info

Publication number: CN112063712A
Application number: CN201911029832.5A
Authority: CN
Inventors: 柳辉
Original assignee: Beijing Kangmei Tianhong Biotechnology Co ltd
Current assignee: Beijing Kangmei Tianhong Biotechnology Co ltd
Priority date: 2019-10-28
Filing date: 2019-10-28
Publication date: 2020-12-11

Abstract

The invention discloses a specific primer pair and a kit for detecting septin9 gene methylation based on a high-resolution melting curve. The invention firstly protects a specific primer pair consisting of SEQ ID NO: 1 and the single-stranded DNA molecule of SEQ ID NO: 2, and 2 is shown in the specification. The invention also protects the application of the specific primer pair in the preparation of the kit. The kit has the functions of diagnosing or assisting in diagnosing colorectal cancer, detecting methylation of a promoter region of a septin9 gene and detecting methylation of a CpG island of a promoter region of a septin9 gene. The primer pair provided by the invention is used for detecting the methylation of the septin9 gene by combining methylation specificity PCR with HRM curve analysis, and has the advantages of simple and convenient operation, good specificity, high sensitivity and low cost. The invention can be used for early screening or auxiliary diagnosis of colorectal cancer patients and has good prospect in health screening and clinical auxiliary diagnosis.

Description

Specific primer pair and kit for detecting septin9 gene methylation based on high-resolution melting curve

Technical Field

The invention belongs to the technical field of biology, and relates to a specific primer pair and a kit for detecting septin9 gene methylation based on a high-resolution melting curve.

Background

Colorectal cancer is a common malignant tumor of the digestive tract, and the incidence rate is second to gastric cancer and esophageal cancer. In the fatality rate of malignant tumors in our country, colorectal cancer patients account for the 5 th in men and the 6 th in women. The incidence rate of colorectal cancer is gradually increased in recent 20 years, more than 80% of patients in China have already developed to middle and late stages when diagnosed, the early diagnosis rate is only 10% -15%, and domestic investigation shows that the survival rate after early stage surgery is 90% -95%, and the survival rate at late stage is only 5%. The early colorectal cancer can be radically treated by the operation treatment, and the average survival time of the late colorectal cancer after the molecular targeted treatment combined with the chemoradiotherapy is still less than 30 months, so the early diagnosis of the colorectal cancer is very important and urgent.

The change of DNA methylation state is closely related to the occurrence and development of colorectal cancer. CpG islands of the septin9 gene were highly methylated in colorectal cancer patients. Therefore, CpG island methylation of septin9 gene can be used as a potential diagnostic marker of colorectal cancer. The Septin9 gene plays a role of cancer suppressor gene in colorectal cancer, and methylation can inhibit the normal expression of the gene, so that the cancer suppressor function is lost, and finally cell division and canceration are caused. Researches find that the Septin9 gene is highly expressed in tumor tissues and peripheral blood of colorectal cancer patients, is lowly expressed in healthy people or patients with other diseases, and has the highest sensitivity and specificity, thereby establishing the basic idea that the Septin9 gene expression level in blood plasma is the best candidate marker for colorectal cancer; subsequently, the Septin9 gene expression in plasma samples of colorectal cancer patients and healthy controls is analyzed through experiments, and the Septin9 gene is highly methylated in colorectal cancer tissues. The CpG island of Septin9 gene is methylated in colorectal cancer patient, while the CpG island of Septin9 gene is not methylated in normal person.

Gene methylation detection techniques can be broadly divided into two categories, site-specific methylation detection and genome-wide methylation analysis, the latter also known as methylation profiling. Methylation-specific PCR (MS-PCR) has the advantages of high sensitivity, no restriction by endonuclease, no need of special instrument, economy and practicality, and can be used for paraffin embedding samples. For methylation specific PCR, it is most important to design good quality primers. The bisulfite treatment combined sequencing method is a gold standard for DNA methylation analysis, is reliable and high in accuracy, can determine the methylation state of each CpG site in a target fragment, but needs a large amount of clone sequencing, and has a complex process.

The fluorescent quantitative method (Methylight) has the advantages of high flux and high sensitivity, and does not need operations such as electrophoresis and hybridization after PCR, thereby reducing pollution and operation errors. High-resolution melting curve analysis (HRM) is a novel molecular detection technology, is widely applied to the fields of gene mutation, gene methylation level detection, genotyping detection, HLA (human leukocyte antigen) typing and the like, has the characteristics of low cost, High flux, no pollution, quickness, simplicity and convenience in operation, good sensitivity and specificity, has the sensitivity of more than 1-5 percent, and does not need to perform subsequent operation on PCR products. The high-resolution melting technology is consistent with the principle of a common melting curve, and the combination condition of the double-stranded DNA fluorescent dye and a PCR amplification product in the temperature rising process is monitored in real time; the difference is mainly reflected in the rate of temperature rise and the dye used. It uses a new type of saturated dye (such as EVAGreen), the dye drops off when melting, the fluorescence signal is reduced, and the melting curve of the amplification product can be obtained by detecting the change of the fluorescence signal with the temperature. The dye applied by the traditional melting curve technology can inhibit PCR at high concentration, so that the sufficient concentration is difficult to achieve and the saturation combination with a PCR product is achieved, and the saturation dye applied by HRM does not influence PCR amplification in a quite large concentration range, so that the saturation combination with the PCR product can be achieved, and the DNA melting process can be reflected more accurately. The dye has stronger DNA binding capacity and very low inhibition effect, and is combined with the accurate temperature control capacity of a real-time fluorescence quantitative PCR instrument (such as Rotor-Gene Q) with a high-resolution melting analysis function. In addition, compared with the conventional melting instrument, the HRM melting instrument has higher data acquisition rate, fluorescence signal sensitivity and temperature accuracy, and ensures the consistency of the temperature among samples, thereby greatly improving the accuracy of data.

Disclosure of Invention

The invention aims to provide a specific primer pair and a kit for detecting septin9 gene methylation based on a high-resolution melting curve.

The invention firstly protects a specific primer pair consisting of SEQ ID NO: 1 and the single-stranded DNA molecule of SEQ ID NO: 2, and 2 is shown in the specification.

The function of the specific primer pair is to diagnose or assist in diagnosing the colorectal cancer.

The function of the specific primer pair is to detect methylation of a septin9 gene promoter region.

The specific primer pair has the function of detecting CpG island methylation of a promoter region of the septin9 gene.

The invention also protects the application of the specific primer pair in the preparation of the kit; the kit has the function of diagnosing or assisting in diagnosing colorectal cancer.

The invention also protects the application of the specific primer pair in the preparation of the kit; the kit has the function of detecting methylation of the promoter region of the septin9 gene.

The invention also protects the application of the specific primer pair in the preparation of the kit; the kit has the function of detecting CpG island methylation of the promoter region of the septin9 gene.

The invention also provides a kit, which comprises the specific primer pair; the kit has the function of diagnosing or assisting in diagnosing colorectal cancer.

The invention also provides a kit, which comprises the specific primer pair; the kit has the function of detecting methylation of the promoter region of the septin9 gene.

The invention also provides a reagent or a kit, which comprises the specific primer pair; the kit has the function of detecting CpG island methylation of the promoter region of the septin9 gene.

Any of the above kits further comprises a PCR amplification reagent and a fluorescent dye.

The fluorescent dye may in particular be the saturation dye EVAGreen.

The PCR amplification reagent specifically comprises: TaKaRa Ex Taq, 10 XEx Taq Buffer (Mg)²⁺free)、MgCl₂、dNTP Mixture。

Any of the kits above further comprising reagents for performing bisulfite treatment of DNA.

Any of the above kits further comprises a carrier carrying the following detection methods:

(1) extracting the total DNA of a sample to be detected, and then carrying out bisulfite treatment;

(2) PCR amplification and high resolution melting curve analysis were performed.

The reaction procedure for PCR amplification is detailed in Table 2.

The parameter setting of the high resolution melting curve analysis may specifically be: 65 ℃ for 1s, then from 65 ℃ to 95 ℃ at a rate of 0.02 ℃/s (25 fluorescences collected per second), and finally 40 ℃ for 30 s.

Any one of the above kits further comprises a vector carrying the following criteria: the presence of a melting peak at the 87 ℃. + -. 0.5 ℃ position indicates the presence of septin9 gene methylation in the sample (candidate colorectal cancer patient), and the absence of a melting peak at the 87 ℃. + -. 0.5 ℃ position indicates the absence of septin9 gene methylation in the sample (candidate non-colorectal cancer patient).

The invention also provides a preparation method of the kit, which comprises the step of independently packaging each primer.

Any one of the above-mentioned septin9 genes is a gene encoding human septin9 protein.

The human septin9 protein is specifically shown as a sequence 4 in a sequence table.

In the human cDNA, the open reading frame of the septin9 gene is specifically shown as the 309-2048 th site in the sequence 3 of the sequence table.

The CpG island of the promoter region of the septin9 gene can be specifically nucleotides 34-204 in the sequence 3 of the sequence table.

In order to solve the defects of complex operation, pollution risk, low sensitivity and poor specificity in the existing methylation detection method, the invention provides a pair of specific primers for detecting septin9 methylation, a CG locus is added into a septin9 gene detection primer, the primers are selectively combined with a methylated sequence in a complementary manner, the PCR bias caused by the tendency of an unmethylated sequence is compensated, the sensitivity of septin9 gene detection is effectively increased, and the sensitivity of the method is better than that of methylation fluorescence quantitative PCR.

The primer pair provided by the invention is used for detecting the methylation of the septin9 gene by combining methylation specificity PCR with HRM curve analysis, and has the advantages of simple and convenient operation, good specificity, high sensitivity and low cost. The invention can be used for early screening or auxiliary diagnosis of colorectal cancer patients and has good prospect in health screening and clinical auxiliary diagnosis.

Drawings

FIG. 1 is a graph showing the change in fluorescence of HRM curve when primer pair A was used in example 3.

FIG. 2 is a peak diagram of a melting curve when the primer set A is used in example 3 (100% methylated sample).

FIG. 3 is a peak diagram of a melting curve when the primer set A is used in example 3 (75% methylated specimen).

FIG. 4 is a peak diagram of a melting curve when the primer set A is used in example 3 (50% methylated specimen).

FIG. 5 shows the melting curve peak (25% methylated specimen) when the primer set A was used in example 3.

FIG. 6 is a peak diagram of a melting curve when the primer set A is used in example 3 (10% methylated sample).

FIG. 7 is a peak diagram of a melting curve when the primer set A is used in example 3 (5% methylated sample).

FIG. 8 is a peak diagram of a melting curve when the primer set A is used in example 3 (2% methylated specimen).

FIG. 9 shows the melting curve peak (1% methylated specimen) when the primer set A was used in example 3.

FIG. 10 is a peak diagram of a melting curve when the primer set A is used in example 3 (0% methylation sample).

FIG. 11 is a peak diagram of a melting curve when the primer set A is used in example 3 (overlay of each sample).

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. Unless otherwise specified, in the DNA molecules of the examples, A's each represent adenine deoxyribonucleotide, T's each represent thymine deoxyribonucleotide, C's each represent cytosine deoxyribonucleotide, and G's each represent guanine deoxyribonucleotide. pMD20-T vector: baozi medicine technology (Beijing) Inc., cat # 6028.

Example 1 design and preparation of primers and preparation of Standard plasmids

Design and preparation of primers

A plurality of primer pairs are designed based on the CpG island of the septin9 gene, the effect of the primer pairs is verified through a pre-experiment respectively, and 3 primer pairs are obtained through preliminary screening.

The primer pair A consists of a primer F1 and a primer R1.

F1(SEQ ID NO：1)：5’-GTTAGTGCGAGATAGGGAGGTC-3’；

R1(SEQ ID NO：2)：5’-CAATACAAAACTAACCGCCG-3’。

Primer pair B consists of primer F2 and primer R2.

F2：5’-AGAAATTTTAGGATTTGGGCG-3’；

R2：5’-GACGTTCTCCACCTACTTAAACG-3’。

Primer pair C consists of primer F3 and primer R3.

F3：5’-GTGTTGGGTTATAAGACGTTAGAATC-3’；

R3：5’-TAACAAAAACAATAAACACCTCGAA-3’。

Each of the above primers was synthesized separately.

Second, preparation of Standard plasmid

Preparation of Standard plasmid I (non-methylated plasmid): DNA molecules (C all represent cytosine deoxyribonucleotide) shown in a sequence 3 of a sequence table are inserted between enzyme cutting sites of pMD20-T vector Hind III and Kpn I to obtain a standard plasmid I.

Preparation of Standard plasmid II (methylated plasmid): the DNA molecule shown in the sequence 3 of the sequence table (all C in CG in three specific intervals represent 5-methylcytosine deoxyribonucleotide, the other C represent cytosine deoxyribonucleotide; the three specific intervals are 34-204 th site, 469 th site and 654 th site and 696 th site of 828 th site in the sequence 3) is inserted between Hind III and Kpn I enzyme cutting sites of pMD20-T vector to obtain the standard plasmid II.

Example 2 establishment of a method for detecting methylation by methylation specific PCR in combination with HRM Curve analysis

1. Bisulfite treatment

Mu.l of sample solution is taken and treated with bisulfite to obtain 10. mu.l of product solution, namely the template solution. The kit used for the bisulfite treatment was: EZ-96DNA Methylation-Gold MagPrep (ZYMO, cat # D5042); the operation is carried out according to the kit instructions.

2. Performing PCR amplification and high resolution melting curve analysis

The reaction system is shown in Table 1.

The reaction is carried out by the following apparatus

480II (Roche).

The amplification reaction procedure is shown in Table 2.

High-resolution melting (HRM) curve analysis is performed after amplification. Parameters for HRM curve analysis were set as: 65 ℃ for 1s, then from 65 ℃ to 95 ℃ at a rate of 0.02 ℃/s (25 fluorescences collected per second), and finally 40 ℃ for 30 s.

TABLE 1

Components	Volume of addition
		TaKaRa Ex Taq	0.4μL
10×Ex Taq Buffer(Mg²⁺free)	3μL
		25mM MgCl₂Solutions of	3μL
Primer solution	2μL
		dNTP Mixture	3μL
Saturated dye EVAGreen	1.5μL
		Template solution	5μL
DNase-free water	Make up to 30. mu.L

TaKaRa Ex Taq: takara, cat No. RR01AM, product size 5U/. mu.l; and (3) product website: https:// www.takarabiomed.com.cn/productshow. aspx? m 20141215102854153148& productID 20141223192454673006.

10×Ex Taq Buffer(Mg²⁺free) and 25mM MgCl₂Solution: takara, cat number 9152 AM; and (3) product website: https:// www.takarabiomed.com.cn/productshow. aspx? m-20141215102854153148&productID＝20141223195903453090。

The primer solution provides effective components of a primer F and a primer R. In the primer solution, the concentration of each of the primers F and R was 10. mu.M.

The effective components provided by dNTP mix are dATP, dTTP, dCTP and dGTP. dNTP mix: biometrics (Shanghai) Inc., cat # B500056-0005; and (3) product website: https:// www.sangon.com/product detail? Code ═ B500056.

Saturated dye EVAGreen: beijing Boleidekko technology development Limited (Biotium, manufacturer U.S.); and (3) product website: http:// www.bjbiolead.com/biolead 2013-ParentList-859509/.

TABLE 2

3. Result judgment

Quality control standard: taking the methylated plasmid solution as a sample solution, sequentially performing the step 1 and the step 2, wherein the melting curve is normal, and a melting peak appears only at the position of 87 +/-0.5 ℃; the non-methylated plasmid solution is used as a sample solution to sequentially carry out the step 1 and the step 2, the melting curve is normal, and the melting peak appears only at the position of 82 +/-0.5 ℃.

And (4) judging a result standard: and (2) sequentially performing step 1 and step 2 on a sample solution (namely the solution obtained by extracting the genome DNA of the biological sample to be detected), wherein the existence of septin9 gene methylation in the sample (the sample is a candidate colorectal cancer patient) is indicated by the occurrence of a melting peak at the position of 87 +/-0.5 ℃, and the absence of septin9 gene methylation in the sample (the sample is a candidate non-colorectal cancer patient).

Example 3 sensitivity detection

The sample solution, also called 100% methylated sample, was prepared with methylated plasmid and TE buffer. A sample solution (the mass percentage of the methylated plasmids to the total plasmids is 75%) also called a 75% methylated sample is prepared by using the methylated plasmids, the unmethylated plasmids and a TE buffer solution. A sample solution (the mass percentage of the methylated plasmids to the total plasmids is 50%) also called 50% methylated sample is prepared by using the methylated plasmids, the unmethylated plasmids and TE buffer. A sample solution (the mass percentage of the methylated plasmids to the total plasmids is 25%) also called 25% methylated sample is prepared by using the methylated plasmids, the unmethylated plasmids and TE buffer. A sample solution (the mass percentage of the methylated plasmids to the total plasmids is 10%) also called 10% methylated sample is prepared by using the methylated plasmids, the unmethylated plasmids and TE buffer. A sample solution (the mass percentage of the methylated plasmids to the total plasmids is 5%) also called a 5% methylated sample is prepared by using the methylated plasmids, the unmethylated plasmids and a TE buffer solution. A sample solution (the mass percentage of the methylated plasmids to the total plasmids is 2%) also called 2% methylated sample is prepared by using the methylated plasmids, the unmethylated plasmids and TE buffer. A sample solution (the mass percentage of the methylated plasmids to the total plasmids is 1%) also called a 1% methylated sample is prepared by using the methylated plasmids, the unmethylated plasmids and a TE buffer solution. The sample solution, also called 0% methylated sample, was prepared with unmethylated plasmid and TE buffer. The DNA content was 100ng per 5. mu.L of the sample solution.

Detection was performed in the order of step 1 and step 2 of example 2. Three primer pairs of example 1 were used, respectively.

Methylation was detected from 2% methylated to 100% methylated samples using three primer pairs. In 1% methylated samples, only primer pair A could detect methylation, and neither primer pair B nor primer pair C could detect methylation. 0% methylated samples, no methylation was detected with all three primer pairs. That is, the sensitivity of the primer pair A for detecting methylation was 1%, the sensitivity of the primer pair B and the primer pair C for detecting methylation was 2%, and the sensitivity of the primer pair A was 2 times that of the other two primer pairs.

The change in fluorescence of the primer pair A when analyzed by HRM curve is shown in FIG. 1. The melting curve peaks when primer pairs were used for A are shown in FIGS. 2 to 11. Fig. 2 to 10 correspond in sequence to 100% methylated samples to 0% methylated samples, and fig. 11 is an overlay of fig. 2 to 10.

Example 4 reproducibility test

The sample solution was prepared with methylated plasmid, unmethylated plasmid and TE buffer. The DNA content was 100ng per 5. mu.L of the sample solution. In the sample solution 1, the methylated plasmids accounted for 10% by mass of the total plasmids. In the sample solution 2, the mass percentage of the methylated plasmids to the total plasmids was 1%.

Detection was performed in the order of step 1 and step 2 of example 2. Primer set A of example 1 was used.

20 replicates were performed. The results are shown in Table 3. The mixed template with two concentrations is repeatedly detected for 20 times, and the result can normally detect methylation, which shows that the method has good repeatability.

TABLE 3

Example 5 clinical sample testing

Paraffin sections of colorectal tissue were obtained from a hospital from 10 volunteers. Wherein clinical sample 2 and clinical sample 8 are both obtained from hospital confirmed colorectal cancer patients. The remaining 8 clinical specimens were obtained from hospital confirmed non-colorectal cancer patients.

The paraffin sections were taken and total DNA was extracted as a sample solution.

The results are shown in Table 4.

Clinical samples

2 and 8 were able to detect Septin9 gene methylation.

TABLE 4

Clinical sample numbering	Clinical sample 1	Clinical sample 2	Clinical sample 3	Clinical sample 4	Clinical sample 5
						Results	Non-methylated	Methylation of	Non-methylated	Non-methylated	Non-methylated
Clinical sample numbering	Clinical sample 6	Clinical sample 7	Clinical sample 8	Clinical sample 9	Clinical sample 10
						Results	Non-methylated	Non-methylated	Methylation of	Non-methylated	Non-methylated

SEQUENCE LISTING

<110> Beijing kang Mei Tianhong Biotech Co., Ltd

<120> specific primer pair and kit for detecting septin9 gene methylation based on high-resolution melting curve

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Met Ser Asp Pro Ala Val Asn Ala Gln Leu Asp Gly Ile Ile Ser Asp

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Phe Glu Ala Leu Lys Arg Ser Phe Glu Val Glu Glu Val Glu Thr Pro

20 25 30

Asn Ser Thr Pro Pro Arg Arg Val Gln Thr Pro Leu Leu Arg Ala Thr

35 40 45

Val Ala Ser Ser Thr Gln Lys Phe Gln Asp Leu Gly Val Lys Asn Ser

50 55 60

Glu Pro Ser Ala Arg His Val Asp Ser Leu Ser Gln Arg Ser Pro Lys

65 70 75 80

Ala Ser Leu Arg Arg Val Glu Leu Ser Gly Pro Lys Ala Ala Glu Pro

85 90 95

Val Ser Arg Arg Thr Glu Leu Ser Ile Asp Ile Ser Ser Lys Gln Val

100 105 110

Glu Asn Ala Gly Ala Ile Gly Pro Ser Arg Phe Gly Leu Lys Arg Ala

115 120 125

Glu Val Leu Gly His Lys Thr Pro Glu Pro Ala Pro Arg Arg Thr Glu

130 135 140

Ile Thr Ile Val Lys Pro Gln Glu Ser Ala His Arg Arg Met Glu Pro

145 150 155 160

Pro Ala Ser Lys Val Pro Glu Val Pro Thr Ala Pro Ala Thr Asp Ala

165 170 175

Ala Pro Lys Arg Val Glu Ile Gln Met Pro Lys Pro Ala Glu Ala Pro

180 185 190

Thr Ala Pro Ser Pro Ala Gln Thr Leu Glu Asn Ser Glu Pro Ala Pro

195 200 205

Val Ser Gln Leu Gln Ser Arg Leu Glu Pro Lys Pro Gln Pro Pro Val

210 215 220

Ala Glu Ala Thr Pro Arg Ser Gln Glu Ala Thr Glu Ala Ala Pro Ser

225 230 235 240

Cys Val Gly Asp Met Ala Asp Thr Pro Arg Asp Ala Gly Leu Lys Gln

245 250 255

Ala Pro Ala Ser Arg Asn Glu Lys Ala Pro Val Asp Phe Gly Tyr Val

260 265 270

Gly Ile Asp Ser Ile Leu Glu Gln Met Arg Arg Lys Ala Met Lys Gln

275 280 285

Gly Phe Glu Phe Asn Ile Met Val Val Gly Gln Ser Gly Leu Gly Lys

290 295 300

Ser Thr Leu Ile Asn Thr Leu Phe Lys Ser Lys Ile Ser Arg Lys Ser

305 310 315 320

Val Gln Pro Thr Ser Glu Glu Arg Ile Pro Lys Thr Ile Glu Ile Lys

325 330 335

Ser Ile Thr His Asp Ile Glu Glu Lys Gly Val Arg Met Lys Leu Thr

340 345 350

Val Ile Asp Thr Pro Gly Phe Gly Asp His Ile Asn Asn Glu Asn Cys

355 360 365

Trp Gln Pro Ile Met Lys Phe Ile Asn Asp Gln Tyr Glu Lys Tyr Leu

370 375 380

Gln Glu Glu Val Asn Ile Asn Arg Lys Lys Arg Ile Pro Asp Thr Arg

385 390 395 400

Val His Cys Cys Leu Tyr Phe Ile Pro Ala Thr Gly His Ser Leu Arg

405 410 415

Pro Leu Asp Ile Glu Phe Met Lys Arg Leu Ser Lys Val Val Asn Ile

420 425 430

Val Pro Val Ile Ala Lys Ala Asp Thr Leu Thr Leu Glu Glu Arg Val

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His Phe Lys Gln Arg Ile Thr Ala Asp Leu Leu Ser Asn Gly Ile Asp

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Val Tyr Pro Gln Lys Glu Phe Asp Glu Asp Ser Glu Asp Arg Leu Val

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Asn Glu Lys Phe Arg Glu Met Ile Pro Phe Ala Val Val Gly Ser Asp

485 490 495

His Glu Tyr Gln Val Asn Gly Lys Arg Ile Leu Gly Arg Lys Thr Lys

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Trp Gly Thr Ile Glu Val Glu Asn Thr Thr His Cys Glu Phe Ala Tyr

515 520 525

Leu Arg Asp Leu Leu Ile Arg Thr His Met Gln Asn Ile Lys Asp Ile

530 535 540

Thr Ser Ser Ile His Phe Glu Ala Tyr Arg Val Lys Arg Leu Asn Glu

545 550 555 560

Gly Ser Ser Ala Met Ala Asn Gly Met Glu Glu Lys Glu Pro Glu Ala

565 570 575

Pro Glu Met

Claims

1. A specific primer pair consisting of SEQ ID NO: 1 and the single-stranded DNA molecule of SEQ ID NO: 2, and 2 is shown in the specification.

2. The specific primer pair of claim 1, wherein: the function of the specific primer pair is to diagnose or assist in diagnosing the colorectal cancer.

3. The specific primer pair of claim 1, wherein: the function of the specific primer pair is to detect methylation of a septin9 gene promoter region.

4. The use of the specific primer pair of claim 1 in the preparation of a kit; the kit has the function of diagnosing or assisting in diagnosing colorectal cancer.

5. The use of the specific primer pair of claim 1 in the preparation of a kit; the kit has the function of detecting methylation of the promoter region of the septin9 gene.

6. The use of the specific primer pair of claim 1 in the preparation of a kit; the kit has the function of detecting CpG island methylation of the promoter region of the septin9 gene.

7. A kit comprising a specific primer pair of claim 1; the kit has the function of diagnosing or assisting in diagnosing colorectal cancer.

8. A kit comprising a specific primer pair of claim 1; the kit has the function of detecting methylation of the promoter region of the septin9 gene.

9. The kit of claim 7 or 8, wherein: the kit also has PCR amplification reagents and a fluorescent dye.

10. A method for preparing a kit according to claim 7 or 8, comprising the step of packaging each primer separately.