RU2812023C2 - Combination for screening of rectal and colon cancer, as well as progressive adenoma and use of combination - Google Patents

Abstract

FIELD: molecular diagnostics.

SUBSTANCE: combinations of qPCR primers and probes that can be used to determine the status and methylation level of BMP3 and NDRG4 genes in a patient in need, providing remarkably high specificity and diagnostic sensitivity in determining the presence or absence of colorectal cancer (RPOC) and/or progressive adenoma (PA) in a patient in need of it. Compositions and methods of performing diagnostics are also presented.

EFFECT: combinations of preferred primers and probes according to the invention provide unexpectedly higher sensitivity and specificity in detecting tumour tissue.

64 cl, 42 dwg, 24 tbl, 5 ex

Description

Cross reference to related applications

[0001] This application claims the benefit of Chinese Patent Application No. 201810502359.7, filed on May 23, 2018, and Chinese Patent Application No. 201810502387.9, filed on May 23, 2018, each of which is incorporated herein in its entirety and for all purposes by reference.

Field of invention

[0002] The present invention relates to compositions and methods for screening for colorectal cancer and advanced adenoma, and other uses thereof.

Description of the text file presented in electronic format

[0003] The contents of the text file provided herein in electronic format are incorporated herein by reference in their entirety: Copy of the Sequence List in electronic format (file name: NEWH-017_01WO_SeqList_ST25.txt, filing date: May 22, 2019, file size: 22 kilobyte).

Prerequisites for creating the invention

[0004] Colorectal cancer (RCC) is the fourth most common cancer in the world, and its mortality rate is second only to that of lung cancer, liver cancer and stomach cancer. There are nearly 700,000 annual deaths from RPOC. OPOC is a “modernized” disease with a higher incidence in developed countries than in developing countries. In the United States, colorectal cancer remains the second leading cause of death (Clinical Interventions in Aging 2016;11:967-976). With the improvement of people's living standards in China, the morbidity and mortality rates of PCOS have been increasing since 2000 (CA CANCER J CLIN 2016;66:115-132). The 5-year survival rate for patients with early-stage localized disease (stages I and II) is close to 90%, while the survival rate for patients with advanced stage POCC is only 13.1%. Treatment of patients with advanced POC is often very expensive and may only alleviate symptoms of the disease (Clinical Interventions in Aging 2016; 11: 967-976).

[0005] The development of POC is a slow process that is usually asymptomatic and difficult to diagnose early until the tumor reaches several centimeters in size, which can block the passage of stool and lead to cramping, pain or visible bleeding. The development of PCOC goes through a multistage process involving a series of histological, morphological and genetic changes that accumulate over time, namely, from healthy state, hyperplasia, small polyps, large polyps and adenocarcinoma to cancer. Polyps are abnormal cells that grow or accumulate locally in the intestinal lining. Dividing polyp cells can accumulate genetic changes sufficient to penetrate the intestinal wall and ultimately become POC. However, only a small number of polyps develop into PTC after more than a decade of development. The two main types of potentially malignant polyps are adenomas and serrated nonpedunculated polyps (SSPs), which develop in PCOC with varying degrees of risk. The risk of an adenoma developing into a PTC depends on its size. Typically, the larger the size of the adenoma, the greater the likelihood that it will develop into a POC. Progressive adenomas (PA) are ≥1 cm in size or have ≥25% villous component or high grade dysplasia of any size. Although, in most cases, only approximately 10% of PAs become malignant, however, 60-70% of PTCs develop from adenomas, and the remaining 25-35% of PTCs develop from SSP (Clinical Interventions in Aging 2016; 11: 967-976). Therefore, early detection of POCA and PA and removal of lesions can effectively block the progression of POCA, and thereby save patients' lives, significantly improve patient survival for more than 5 years, and reduce the expensive costs of treating late-stage POCA, which will significantly reduce the economic burden on family and society.

[0006] Currently, there are several tests for detecting FOC, mainly including colonoscopy, sigmoidoscopy, CT colonography, fecal occult blood test (FOBT) and fecal immunochemical test (FIT).

[0007] The sensitivity of colonoscopy for detecting POC is >95%. The interval between screenings is 10 years. The advantage of colonoscopy is its high sensitivity, which allows it to examine the entire colon and simultaneously remove the lesion. However, its disadvantage is the invasive examination and bowel preparation, which causes discomfort in the patient, and therefore the patient needs rest after such a procedure. During a colonoscopy, there is a risk of intestinal perforation and bleeding. Because of these limitations, patients often do not consent to colonoscopic screening.

[0008] The sensitivity of sigmoidoscopy for detecting lesions in the distal colon exceeds 95%. The screening interval for RPOC using sigmoidoscopy is 5 years in combination with FOBT. The advantages of sigmoidoscopy screening for PCOS include high sensitivity, no need for systemic sedatives, and the ability to remove the lesion during the examination. The disadvantages of this procedure are the semi-invasive examination, which easily causes discomfort during the examination, and the high cost of such an examination.

[0009] CT colonography uses radiation to visualize the colon, has a sensitivity of >90%, and is performed every 5 years. The advantage of this method is that the sensitivity is so high that it allows observation of the entire colon and does not require the administration of sedatives. The disadvantage of this test is that it is a semi-invasive test and therefore patients may easily feel discomfort during the screening process. In addition, lesions cannot be removed during examination, and radiation safety must be considered.

[0010] In general, the above imaging-based tests for diagnosing POBC have high sensitivity, but they are expensive, and bowel preparation often causes discomfort and other side effects. As a result, the patient has poor compliance with such screening. In addition, these tests require professional equipment and require doctors with professional skills and extensive experience, which is not always possible. As a result, overall screening/diagnosis rates are low. In addition, some patients are not eligible for such tests. For example, in patients with diabetes, bowel preparation is less successful and this increases the risk of side effects (J Gastrointestin Liver Dis 2010; 19: 369-372, World J Gastrointest Endosc 2013; 5: 39-46).

[0011] FOBT and FIT can detect hemoglobin in the stool of patients using an enzymatic reaction and an immunochemical method, respectively, with a sensitivity of 33-75% and 60-85%, respectively, for the diagnosis of POC, and these tests are performed every year. Although FOBT and FIT are readily available, non-invasive and inexpensive tests, the detection rate of precancerous lesions is low (Clinical Interventions in Aging 2016; 11 967-976).

[0012] During the transformation of polyps into POC, mutations and methylation changes accumulate in some genes, such as APC, KRAS, p53, BRAF, NDRG4, BMP3, and the like. (Clinical Interventions in Aging 2016;11:967-976). Therefore, detection of these mutations or methylation changes helps in detecting PTC and precancerous lesions.

[0013] Zou et al. (Clinical Chemistry 2012; 58: 2375-383) performed methylation qPCR to determine the methylation levels of the BMP3, NDRG4, VIM and TFPI2 genes in tissue samples. A total of 37 samples of PCa tissue, 25 samples of adenoma tissue and 29 samples of healthy human tissue were tested. When the specificity was 95%, the sensitivity of detecting the BMP3, NDRG4, VIM and TFPI2 genes in diagnosing PCa was 84%, 92%, 86% and 92%, respectively, and their sensitivity in diagnosing adenoma was 68%, 76%, 76% and 88%, respectively. Detection of gene methylation in colon cancer tissues has been shown to have high sensitivity and specificity. However, the tissue sampling method has not yet been widely used because the sampling process causes certain damage to the patient's body. Therefore, it is not suitable for screening for POC and precancerous lesions in the general population.

[0014] Multi-target stool DNA (mt-sDNA) testing includes detection of methylation and mutations in exfoliated tumor cells and detection of hemoglobin in stool samples, and this testing, which is performed every 3 years, has the advantages of high sensitivity, non-invasiveness and accessibility to population (Clinical Interventions in Aging 2016; 11:967-976). As a screening method, mt-sDNA allows the detection of PCa and PA at an early stage, which significantly improves patient survival. Imperiale et al. (N. Eng.l J. Med. 2014; 370: 1287-97) developed a system based on mt-sDNA to detect methylation of the BMP3 and NDRG4 genes, to detect point mutations in the KRAS gene and to detect hemoglobin in stool, followed by risk assessment development of RPOC and PA using the logistic regression formula. The sensitivity of determining RPOC and PA is 92.3% and 42.4%, respectively, and the specificity is 86.6%.

[0015] mt-sDNA has been used to screen for sporadic POC and PA with the advantage of non-invasiveness compared to colonoscopy and greater sensitivity compared to FOBT and FIT, but the detection sensitivity of PA is still much lower than the detection sensitivity of POC (Clinical Interventions in Aging 2016; 11:967-976).

[0016] Currently, mt-sDNA-based products for diagnosing PCOS or PA, such as Cologuard®, are mainly being developed for the European and American populations. There are currently no available products for the determination of POC and PA in Asian populations. Specifically, according to the US Food and Drug Administration's Summary of Safety and Effectiveness Data (SSED) for Cologuard® (www.accessdata.fda.gov/cdrh_docs/pdf13/P130017b.pdf), the detection sensitivity of PA using Cologuard® in white and African American populations is 42.3% and 42.4%, respectively, but the detection sensitivity of PA using the same product in Asian populations is only 30.8%. Therefore, the need to develop an effective detection system for PTC and/or PA in Asian populations to combat the current increase in incidence and mortality from colorectal cancer in Asian countries remains urgent.

[0017] Although many studies have been carried out using methods to detect methylation of the BMP3 and NDRG4 genes in stool samples from patients with PCOS and PsA, however, there have been no thorough and comprehensive studies of hypermethylated CpG sites in the BMP3 and NDRG4 genes in Asian populations ( ONCOLOGY LETTERS 2014;8:1751-1756;ONCOLOGY LETTERS 2015;9:1383-1387). Additionally, due to limited sample size, previous studies on BMP3 and NDRG4 gene methylation in Asian patients have not been able to identify the methylation sites that are most relevant for POC and PA. Therefore, it is necessary to determine the precise localization of hypermethylated CpG sites of the BMP3 and NDRG4 genes in Asian populations, and to develop and optimize kits based on these methylated CpG sites to improve the sensitivity of PA detection.

The essence of the invention

[0018] The present invention relates to DNA sequences containing hypermethylated CpG sites in the promoter region of the BMP3 and NDRG4 genes.

[0019] The present invention also relates to preferred primers and probes for detecting methylation of the BMP3 or NDRG4 genes and combinations thereof for detecting methylation of the BMP3 and NDRG4 genes.

[0020] The present invention also relates to a kit for diagnosing POC and PA in Asian populations. DNA sequences containing hypermethylated CpG sites in the promoter region of the BMP3 and NDRG4 genes can be used as markers for the diagnosis of POC and/or PA in Asian populations.

[0021] Compared to other primers and probes, the preferred primer and probe pairs for detecting methylation levels of the BMP3 and/or NDRG4 genes have surprisingly higher sensitivity and specificity for tumor tissue, such as PTC and PA tissues, and especially PA. In addition, the combinations of these preferred primers and probes of the invention also provide unexpectedly higher sensitivity and specificity for the detection of tumor tissue such as PTC and PA tissue, and especially PA.

[0022] The present invention also provides a kit for diagnosing POC and PA in Asian populations based on the above preferred combinations of primers and probes.

[0023] In some embodiments, the kit includes: (1) a preferred combination of primer and probe pairs and corresponding qPCR reagents; (2) primers and probes for detecting seven mutations in the coding region of the KRAS gene and corresponding reagents for quantitative PCR; (3) reagents for the determination of hemoglobin in feces.

[0024] In some embodiments of the invention, the data obtained from the analysis using the set is adjusted and analyzed using a logistic regression formula. In some embodiments of the invention, this formula is used to calculate a value in determining the presence or absence of POC and/or PA. In some embodiments of the invention, the formula is P=e ^K /(1+e ^K ), where P is the general index, and K=a*ΔCt1+b*ΔCt2+c*ΔCt3+d*FIT+X, where e means natural constant, and a, b, c, d, X means clinical constants. In some embodiments, if the P value is equal to or greater than a predetermined threshold, then the result indicates the presence of POC and/or PA in the patient. In some embodiments, if the P value is less than a threshold value, then the result indicates that the patient does not have POC and/or PA, and the patient is determined to be healthy.

[0025] The present invention relates to a kit for determining the presence or absence of colorectal cancer (RCC) or advanced adenoma (PA) in a patient in need thereof. The patient in need of this is a patient suspected of developing POCR and/or PA, for example, a patient who has at least one sign of developing POCR and/or PA, or a patient at risk of developing POCR and/or PA, or an individual undergoing routine medical examination, but does not have any signs of the disease or the risk of developing it.

[0026] In some embodiments, the kit includes: (a) a first pair of primers and a first probe for detecting the methylation status or level of at least one CpG dinucleotide of the BMP3 gene in a biological sample taken from a patient. In some embodiments, each first primer pair and first probe includes a contiguous sequence of at least 16 nucleotides that is identical to and complementary to the sequence of SEQ ID NO. 1 or hybridizes to this sequence under stringent hybridization conditions.

[0027] In some embodiments, the kit includes: (b) a second pair of primers and a second probe for detecting the methylation status or level of at least one CpG dinucleotide of the NDRG4 gene in a biological sample taken from the patient. In some embodiments, every second pair of primers and second probe includes a contiguous sequence of at least 16 nucleotides that is identical to and complementary to, or hybridizes to, the sequence of SEQ ID NO: 2 under stringent hybridization conditions.

[0028] In some embodiments of the invention, the first pair of primers and the first probe are selected from the group consisting of:

[0029] i) a forward primer containing SEQ ID NO: 3, a reverse primer containing SEQ ID NO: 4, and a probe containing SEQ ID NO: 5;

[0030] ii) a forward primer containing SEQ ID NO: 9, a reverse primer containing SEQ ID NO: 10, and a probe containing SEQ ID NO: 11; And

[0031] iii) a forward primer containing SEQ ID NO: 15, a reverse primer containing SEQ ID NO: 16, and a probe containing SEQ ID NO: 17.

[0032] In some embodiments of the invention, the second pair of primers and the second probe are selected from the group consisting of:

[0033] iv) a forward primer containing SEQ ID NO: 6, a reverse primer containing SEQ ID NO: 7, and a probe containing SEQ ID NO: 8;

[0034] v) a forward primer containing SEQ ID NO: 12, a reverse primer containing SEQ ID NO: 13, and a probe containing SEQ ID NO: 14; b

[0035] vi) a forward primer containing SEQ ID NO: 18, a reverse primer containing SEQ ID NO: 19, and a probe containing SEQ ID NO: 20.

[0036] In some embodiments of the invention, the kit includes:

i) a forward primer containing SEQ ID NO: 3, a reverse primer containing SEQ ID NO: 4, and a probe containing SEQ ID NO: 5, for detecting the methylation state or level of at least one CpG dinucleotide of the BMP3 gene in a biological sample, taken from the patient, and

ii) a forward primer containing SEQ ID NO: 6, a reverse primer containing SEQ ID NO: 7, and a probe containing SEQ ID NO: 8, for detecting the methylation state or level of at least one CpG dinucleotide of the NDRG4 gene in a biological sample, taken from the patient.

[0037] In some embodiments of the invention, the kit includes:

i) a forward primer containing SEQ ID NO: 9, a reverse primer containing SEQ ID NO: 10, and a probe containing SEQ ID NO: 11, for detecting the methylation state or level of at least one CpG dinucleotide of the BMP3 gene in a biological sample, taken from the patient, and

ii) a forward primer containing SEQ ID NO: 12, a reverse primer containing SEQ ID NO: 13, and a probe containing SEQ ID NO: 14, for detecting the methylation state or level of at least one CpG dinucleotide of the NDRG4 gene in a biological sample, taken from the patient.

[0038] In some embodiments of the invention, the kit includes:

i) a forward primer containing SEQ ID NO: 15, a reverse primer containing SEQ ID NO: 16, and a probe containing SEQ ID NO: 17, for detecting the methylation state or level of at least one CpG dinucleotide of the BMP3 gene in a biological sample, taken from the patient, and

ii) a forward primer containing SEQ ID NO: 18, a reverse primer containing SEQ ID NO: 19, and a probe containing SEQ ID NO: 20, for detecting the methylation state or level of at least one CpG dinucleotide of the NDRG4 gene in a biological sample, taken from the patient.

[0039] In some embodiments of the invention, the first probe and the second probe comprise a fluorescent donor and an acceptor fluorophore.

[0040] In some embodiments of the invention, the first probe and the second probe are TAQMAN® probes.

[0041] In some embodiments of the invention, the kit further includes:

(1) means for detecting the presence or absence of at least one mutation in the KRAS gene in a patient; And

(2) a means for detecting the presence or absence of hemoglobin in a biological sample taken from a patient.

[0042] In some embodiments, means for detecting the presence or absence of at least one mutation in the KRAS gene in a patient includes at least one pair of primers capable of amplifying a region of exon 12 and/or exon 13 of the KRAS gene in a polymerase chain reaction ( PCR).

[0043] In some embodiments, the means for detecting the presence or absence of hemoglobin in a biological sample includes an anti-hemoglobin antibody.

[0044] In some embodiments, the primers are capable of amplifying a region of the KRAS gene containing at least one KRAS mutation selected from the group consisting of G12D, G12V, G12C, G13D, G12A, G12R, G12S, and G13C.

[0045] In some embodiments, the antibody is a colloidal gold conjugated antibody.

In some embodiments of the invention, the kit further includes means for amplifying an internal quality control gene. Internal controls can detect (1) inhibitory contamination introduced by the sample or extraction method, (2) instrument failure, (3) chemical factors (for example, an expired or spoiled kit or spoiled components or inappropriate combination of reagents), and (4) human factors. In some embodiments, the internal control gene is a positive control, such as a gene in a positive control sample that was found to be methylated. In some embodiments, the internal control gene is a negative control, such as a gene in a negative control sample that was found to be unmethylated.

[0046] In some embodiments of the invention, the kit further includes instructions for administering and/or interpreting the test result obtained with the kit.

[0047] In some embodiments, the kit further includes means for detecting a complex formed by an antibody and hemoglobin in a biological sample.

[0048] In some embodiments of the invention, the biological sample collected from the patient is a stool sample.

[0049] In some embodiments, the kit further includes a bisulfite reagent and a container suitable for mixing the bisulfite reagent and a patient biological sample or polynucleotides derived from the biological sample.

[0050] In some embodiments, the kit, instead of bisulfite, further includes a methylation-sensitive restriction enzyme reagent.

[0051] In some embodiments, the kit further includes: (1) a positive standard and a negative standard for detecting BMP3 methylation in a biological sample and (2) a positive standard and a negative standard for detecting NDRG4 methylation in a biological sample.

[0052] In some embodiments, the positive standard for detecting BMP3 methylation contains the polynucleotide sequence:

GTTAGTTTGGTCGGGTGTTTTTAAAAATAAAGCGAGGAGGGAAGGTATAGATAGATTTTGAAAATATTCGGGTTATATACGTCGCGATTTATAGTTTTTTTTTAGCGTTGGAGTGGAGACGGCGTTCGTAGCGTTTTGCGCGGGTGAGGTTCGCGTAGTTGTTGGGGAAGAGTTTATTTGTTAGGTTGCGTTGGGTTAGCGTAGTAAGTGGGGTTGGTCGTTATTTCGTTGTA TTCGGTCGCGTTTCGGGTTTCGTGCGTTTTCGTTTTAG (SEQ ID NO: 67).

[0053] In some embodiments, the negative standard for detecting BMP3 methylation contains the polynucleotide sequence:

GTTAGTTTGGTTGGGTGTTTTTAAAAATAAAGTGAGGAGGGAAGGTATAGATAGATTTTGAAAATATTTGGGTTATATATGTTGTGATTTATAGTTTTTTTTTAGTGTTGGAGTGGAGATGGTGTTTGTAGTGTTTTGTGTGGGTGAGGTTTGTGTAGTTGTTGGGGAAGAGTTTATTTGTTAGGTTGTGTTGGGTTAGTGTAGTAAGTGGGGTTGGTTGTTATTTTGTTG TATTTGGTTGTGTTTTGGGGTTTTGTGTGTTTTTGTTTTAG (SEQ ID NO: 68);

[0054] In some embodiments, the positive standard for detecting NDRG4 methylation contains the polynucleotide sequence:

TGAGAAGTCGGCGGGGGCGCGGATCGATCGGGGTGTTTTTTAGGTTTCGCGTCGCGGTTTTCGTTCGTTTTTTCGTTCGTTTATCGGGTATTTTAGTCGCGTAGAAGGCGGAAGTTACGCGCGAGGGATCGCGGTTCGTTCGGGATTAGTTTTAGGTTCGGTATCGTTTCGCGGGTCGAGCGTTTATATTCGTTAAATTTACGCGGGTACGTTTTTCGCGGCGTATCGTTTTTAG TT (SEQ ID NO: 69).

[0055] In some embodiments, the negative standard for detecting NDRG4 methylation contains the polynucleotide sequence:

TGAGAAGTTGGTGGGGGTGTGGATTGATTGGGGTGTTTTTTAGGTTTTGTGTTGTGGTTTTTGTTTGTTTTTTTGTTTGTTTATTGGGTATTTTAGTTGTGTAGAAGGTGGAAGTTATGTGTGAGGGATTGTGGTTTGTTTGGGATTAGTTTTAGGTTTGGTATTGTTTTGTGGGTTGAGTGTTTATATTTGTTAAATTTATGTGGGTATGTTTTTGTGGTGTATTGTTTT TAGTT (SEQ ID NO: 70).

[0056] The present invention also relates to a method for detecting the presence or absence of colorectal cancer (RCC) or advanced adenoma (PA) in a patient in need thereof.

[0057] In some embodiments, the method includes a) obtaining genomic DNA from a biological sample of the patient.

[0058] In some embodiments of the invention, the method further comprises b) treating genomic DNA(a) or a fragment thereof with one or more reagents to convert cytosine bases that are not methylated to uracil or another base that is markedly different from cytosine c from the point of view of hybridization properties.

[0059] In some embodiments, the method further includes c) contacting the processed genomic DNA or a processed fragment thereof with a first pair of primers to detect the presence or absence of methylation sites on a gene encoding bone morphogenesis protein 3 (BMP3) in the patient. In some embodiments, the method further includes contacting the processed genomic DNA or a fragment thereof with a second pair of primers to detect the presence or absence of methylation sites on a gene encoding a NDRG4 family member protein (NDRG4) in the patient.

[0060] In some embodiments, the first pair of primers contains a contiguous sequence of at least 9 nucleotides that is identical to or complementary to SEQ ID NO: 1 or hybridizes to this sequence under stringent hybridization conditions. In some embodiments, the second primer pair contains a contiguous sequence of at least 9 nucleotides that is identical to or complementary to SEQ ID NO: 2 or hybridizes to this sequence under stringent hybridization conditions.

[0061] In some embodiments of the invention, the processed genomic DNA or fragment thereof is either amplified to obtain at least one amplification using a first pair of primers or a second pair of primers, or is not amplified.

[0062] In some embodiments, the method further comprises d) determining the presence or absence of POC or PA in a patient based on the presence or absence of said amplification, status, or methylation level of at least one CpG dinucleotide of the BMP3 gene and the NDRG4 gene in the patient.

[0063] In some embodiments of the invention, quantitative PCR is performed to amplify the methylated BMP3 gene in the sample. In some embodiments, quantitative PCR is performed to amplify the methylated NDRG4 gene in the sample.

[0064] In some embodiments, the method also includes the use of primers to amplify a control gene (also known as a normalizer, housekeeping gene, or endogenous control). In some embodiments, quantitative PCR is performed to amplify a reference gene in the sample.

[0065] In some embodiments of the invention, the first pair of primers and the first probe are selected from the group consisting of:

i) a forward primer containing SEQ ID NO: 3, a reverse primer containing SEQ ID NO: 4, and a probe containing SEQ ID NO: 5;

ii) a forward primer containing SEQ ID NO: 9, a reverse primer containing SEQ ID NO: 10, and a probe containing SEQ ID NO: 11; And

iii) a forward primer containing SEQ ID NO: 15, a reverse primer containing SEQ ID NO: 16, and a probe containing SEQ ID NO: 17.

[0066] In some embodiments of the invention, the second first pair of primers and the second probe are selected from the group consisting of:

iv) a forward primer containing SEQ ID NO: 6, a reverse primer containing SEQ ID NO: 7, and a probe containing SEQ ID NO: 8;

v) a forward primer containing SEQ ID NO: 12, a reverse primer containing SEQ ID NO: 13, and a probe containing SEQ ID NO: 14; And

vi) a forward primer containing SEQ ID NO: 18, a reverse primer containing SEQ ID NO: 19, and a probe containing SEQ ID NO: 20.

[0067] In some embodiments of the invention, the method includes using:

i) a forward primer containing SEQ ID NO: 3, a reverse primer containing SEQ ID NO: 4, and a probe containing SEQ ID NO: 5, for detecting the methylation state or level of at least one CpG dinucleotide of the BMP3 gene in a biological sample, taken from the patient, and

ii) a forward primer containing SEQ ID NO: 6, a reverse primer containing SEQ ID NO: 7, and a probe containing SEQ ID NO: 8, for detecting the methylation state or level of at least one CpG dinucleotide of the NDRG4 gene in a biological sample, taken from the patient.

[0068] In some embodiments of the invention, the method includes using:

i) a forward primer containing SEQ ID NO: 9, a reverse primer containing SEQ ID NO: 10, and a probe containing SEQ ID NO: 11, for detecting the methylation state or level of at least one CpG dinucleotide of the BMP3 gene in a biological sample, taken from the patient, and

ii) a forward primer containing SEQ ID NO: 12, a reverse primer containing SEQ ID NO: 13, and a probe containing SEQ ID NO: 14, for detecting the methylation state or level of at least one CpG dinucleotide of the NDRG4 gene in a biological sample, taken from the patient.

[0069] In some embodiments of the invention, the method includes using:

i) a forward primer containing SEQ ID NO: 15, a reverse primer containing SEQ ID NO: 16, and a probe containing SEQ ID NO: 17, for detecting the methylation state or level of at least one CpG dinucleotide of the BMP3 gene in a biological sample, taken from the patient, and

ii) a forward primer containing SEQ ID NO: 18, a reverse primer containing SEQ ID NO: 19, and a probe containing SEQ ID NO: 20, for detecting the methylation state or level of at least one CpG dinucleotide of the NDRG4 gene in a biological sample, taken from the patient.

[0070] In some embodiments of the invention, the first probe and the second probe comprise a fluorescent donor and an acceptor fluorophore. In some embodiments, the first probe and the second probe are TAQMAN® probes.

[0071] In some embodiments, the method further includes the step of detecting the presence or absence of at least one mutation in the KRAS gene in a biological sample taken from the patient.

[0072] In some embodiments of the invention, the method further includes the step of detecting the presence or absence of hemoglobin in a biological sample taken from the patient. In some embodiments of the invention, the step of detecting the presence or absence of hemoglobin in a biological sample includes the use of an anti-hemoglobin antibody. In some embodiments, the antibody is a colloidal gold conjugated antibody.

[0073] In some embodiments, the step of detecting the presence or absence of at least one mutation in the KRAS gene in a patient includes using at least one pair of primers capable of amplifying a region of exon 12 and/or exon 13 of the KRAS gene in a polymerase chain reaction ( PCR). In some embodiments, the primers are capable of amplifying a region of the KRAS gene containing at least one KRAS mutation selected from the group consisting of G12D, G12V, G12C, G13D, G12A, G12R, G12S, and G13C.

[0074] In some embodiments, the mutant KRAS gene is amplified using one or more primer pairs selected from the group consisting of:

(1) forward primer G12D-F containing SEQ ID NO: 35 and reverse primer Kras-R containing SEQ ID NO: 42;

(2) forward primer G13D-F containing SEQ ID NO: 36 and reverse primer Kras-R containing SEQ ID NO: 42;

(3) forward primer G12V-F containing SEQ ID NO: 37 and reverse primer Kras-R containing SEQ ID NO: 42;

(4) forward primer G12C-F containing SEQ ID NO: 38 and reverse primer Kras-R containing SEQ ID NO: 42;

(5) forward primer G12S-F containing SEQ ID NO: 39 and reverse primer Kras-R containing SEQ ID NO: 42;

(6) forward primer G12A-F containing SEQ ID NO: 40 and reverse primer Kras-R containing SEQ ID NO: 42; And

(7) forward primer G12R-F containing SEQ ID NO: 41, and reverse primer Kras-R containing SEQ ID NO: 42.

[0075] In some embodiments, the KRAS probe for qPCR comprises SEQ ID NO: 46.

[0076] In some embodiments, the BMP3 gene is amplified using quantitative PCR (qPCR), and the method further includes amplifying a first reference gene (i.e., a first reference gene) to determine the Ct value of the BMP3 amplification as ΔCt1.

[0077] In some embodiments, the NDRG4 gene is amplified using quantitative PCR (qPCR), and the method further includes amplifying a second control gene (i.e., a second reference gene) to determine the Ct value of the NDRG4 amplification as ΔCt2.

[0078] In some embodiments, amplification of the mutant KRAS gene is performed using quantitative PCR (qPCR), and the method further includes amplifying a third reference gene (i.e., a third reference gene) to determine the Ct value of the mutant KRAS amplification as ΔCt3.

[0079] In some embodiments, the first and second control genes are the same. In some embodiments, said control gene is a B2M gene.

[0080] In some embodiments, the third control gene is an ACTB gene. In some embodiments, the qPCR primers for amplification of the ACTB gene contain SEQ ID NOs: 43 and 44, and the probe contains SEQ ID NO: 46.

[0081] In some embodiments, the method includes using (1) a positive standard and a negative standard to detect BMP3 methylation in a sample and (2) a positive standard and a negative standard to detect NDRG4 methylation in the sample.

[0082] In some embodiments, the positive standard for detecting BMP3 methylation comprises the polynucleotide sequence:

GTTAGTTTGGTCGGGTGTTTTTAAAAATAAAGCGAGGAGGGAAGGTATAGATAGATTTTGAAAATATTCGGGTTATATACGTCGCGATTTATAGTTTTTTTTTAGCGTTGGAGTGGAGACGGCGTTCGTAGCGTTTTGCGCGGGTGAGGTTCGCGTAGTTGTTGGGGAAGAGTTTATTTGTTAGGTTGCGTTGGGTTAGCGTAGTAAGTGGGGTTGGTCGTTATTTCGTTGTA TTCGGTCGCGTTTCGGGTTTCGTGCGTTTTCGTTTTAG (SEQ ID NO: 67);

[0083] In some embodiments, the negative standard for detecting BMP3 methylation comprises a polynucleotide sequence

GTTAGTTTGGTTGGGTGTTTTTAAAAATAAAGTGAGGAGGGAAGGTATAGATAGATTTTGAAAATATTTGGGTTATATATGTTGTGATTTATAGTTTTTTTTTAGTGTTGGAGTGGAGATGGTGTTTGTAGTGTTTTGTGTGGGTGAGGTTTGTGTAGTTGTTGGGGAAGAGTTTATTTGTTAGGTTGTGTTGGGTTAGTGTAGTAAGTGGGGTTGGTTGTTATTTTGTTG TATTTGGTTGTGTTTTGGGGTTTTGTGTGTTTTTGTTTTAG (SEQ ID NO: 68);

[0084] In some embodiments, the positive standard for detecting NDRG4 methylation comprises a polynucleotide sequence

TGAGAAGTCGGCGGGGGCGCGGATCGATCGGGGTGTTTTTTAGGTTTCGCGTCGCGGTTTTCGTTCGTTTTTTCGTTCGTTTATCGGGTATTTTAGTCGCGTAGAAGGCGGAAGTTACGCGCGAGGGATCGCGGTTCGTTCGGGATTAGTTTTAGGTTCGGTATCGTTTCGCGGGTCGAGCGTTTATATTCGTTAAATTTACGCGGGTACGTTTTTCGCGGCGTATCGTTTTTAG TT (SEQ ID NO: 69).

[0085] In some embodiments, the negative standard for detecting NDRG4 methylation comprises a polynucleotide sequence

TGAGAAGTTGGTGGGGGTGTGGATTGATTGGGGTGTTTTTTAGGTTTTGTGTTGTGGTTTTTGTTTGTTTTTTTGTTTGTTTATTGGGTATTTTAGTTGTGTAGAAGGTGGAAGTTATGTGTGAGGGATTGTGGTTTGTTTGGGATTAGTTTTAGGTTTGGTATTGTTTTGTGGGTTGAGTGTTTATATTTGTTAAATTTATGTGGGTATGTTTTTGTGGTGTATTGTTTT TAGTT (SEQ ID NO: 70).

[0086] In some embodiments of the invention, the method includes amplifying a quality control standard.

[0087] In some embodiments of the invention, a method of detecting the presence or absence of colorectal cancer (RCC) or advanced adenoma (PA) in a patient in need thereof involves using a kit according to the invention.

[0088] The present invention also relates to a method for detecting the presence or absence of colorectal cancer (RCC) or advanced adenoma (AA) in a patient in need thereof, wherein the method includes:

a) obtaining raw genomic DNA from a patient's stool sample;

b) treating the genomic DNA(s) or a fragment thereof with one or more reagents to convert cytosine bases that are unmethylated to uracil or another base that is markedly different from cytosine in terms of hybridization properties;

c) performing quantitative PCR (qPCR) using the processed genomic DNA (b) as a template and determining the Ct value of the BMP3 gene in the patient as ΔCt1;

d) performing qPCR using the processed genomic DNA (b) as a template and determining the Ct value of the NDRG4 gene in the patient as ΔCt2;

e) performing qPCR using raw genomic DNA as a template and determining the value of the mutant KRAS gene in the patient as ΔCt3;

f) performing a fecal immunochemical test for hemoglobin protein in the stool sample and assessing the result as FIT;

g) determination of the K value, where K=a*ΔCt1+b*ΔCt2+c*ΔCt3+d*FIT+X, where a, b, c, d, X=clinical constants; And

h) determination of the value of the general index P, where P=e ^K /(1+e ^K ), where e is a natural constant.

[0089] Clinical constants a, b, c, d and X can be determined by analyzing the distribution of clinical data in groups of patients.

[0090] In some embodiments of the invention, if P is equal to or greater than a predetermined threshold value, then the patient is determined to be a patient with POC and/or PA, and if P is less than a predetermined threshold value, then the patient is determined to be healthy.

[0091] In some embodiments of the invention, a predetermined threshold value is calculated from a distribution of clinical data, such as clinical data obtained from patients who were diagnosed with POCA and/or PA and patients who were not diagnosed with POCA and/or PA. PA.

[0092] In some embodiments of the invention, a PCR assay for amplifying the BMP3 gene includes a first pair of primers and a first probe, wherein the first pair of primers and the first probe are selected from the group consisting of:

i) a forward primer containing SEQ ID NO: 3, a reverse primer containing SEQ ID NO: 4, and a probe containing SEQ ID NO: 5;

ii) a forward primer containing SEQ ID NO: 9, a reverse primer containing SEQ ID NO: 10, and a probe containing SEQ ID NO: 11; And

iii) a forward primer containing SEQ ID NO: 15, a reverse primer containing SEQ ID NO: 16, and a probe containing SEQ ID NO: 17.

[0093] In some embodiments of the invention, the PCR assay for amplification of the NDRG4 gene includes a second pair of primers and a second probe, wherein the second pair of primers and the second probe are selected from the group consisting of:

iv) a forward primer containing SEQ ID NO: 6, a reverse primer containing SEQ ID NO: 7, and a probe containing SEQ ID NO: 8;

v) a forward primer containing SEQ ID NO: 12, a reverse primer containing SEQ ID NO: 13, and a probe containing SEQ ID NO: 14; And

vi) a forward primer containing SEQ ID NO: 18, a reverse primer containing SEQ ID NO: 19, and a probe containing SEQ ID NO: 20.

[0094] In some embodiments of the invention, the method includes using:

i) a forward primer containing SEQ ID NO: 3, a reverse primer containing SEQ ID NO: 4, and a probe containing SEQ ID NO: 5 for detecting the methylation state or level of at least one CpG dinucleotide of the BMP3 gene in the sample, and

ii) a forward primer containing SEQ ID NO: 6, a reverse primer containing SEQ ID NO: 7, and a probe containing SEQ ID NO: 8 for detecting the methylation state or level of at least one CpG dinucleotide of the NDRG4 gene in the sample.

[0095] In some embodiments of the invention, the method includes using:

i) a forward primer containing SEQ ID NO: 9, a reverse primer containing SEQ ID NO: 10, and a probe containing SEQ ID NO: 11 for detecting the methylation state or level of at least one CpG dinucleotide of the BMP3 gene in the sample; And

ii) a forward primer containing SEQ ID NO: 12, a reverse primer containing SEQ ID NO: 13, and a probe containing SEQ ID NO: 14 for detecting the methylation state or level of at least one CpG dinucleotide of the NDRG4 gene in the sample.

[0096] In some embodiments of the invention, the method includes using:

i) a forward primer containing SEQ ID NO: 15, a reverse primer containing SEQ ID NO: 16, and a probe containing SEQ ID NO: 17 for detecting the methylation state or level of at least one CpG dinucleotide of the BMP3 gene in the sample; And

ii) a forward primer containing SEQ ID NO: 18, a reverse primer containing SEQ ID NO: 19, and a probe containing SEQ ID NO: 20 for detecting the methylation state or level of at least one CpG dinucleotide of the NDRG4 gene in the sample.

[0097] In some embodiments of the invention, the first probe and the second probe comprise a fluorescent donor and an acceptor fluorophore. In some embodiments, the first probe and the second probe are TAQMAN® probes.

[0098] In some embodiments, the mutant KRAS gene contains at least one KRAS mutation selected from the group consisting of G12D, G12V, G12C, G13D, G12A, G12R, G12S, and G13C.

[0099] In some embodiments, the stool immunoassay includes an antibody conjugated to colloidal gold.

[00100] In some embodiments, method step c) and step d) include using the B2M gene as a control gene.

[00101] In some embodiments of the invention, the method includes using:

(1) a positive standard and a negative standard for detecting BMP3 methylation in the sample, and

(2) a positive standard and a negative standard to detect NDRG4 methylation in the sample.

[00102] In some embodiments, the positive standard for detecting BMP3 methylation comprises a polynucleotide sequence

GTTAGTTTGGTCGGGTGTTTTTAAAAATAAAGCGAGGAGGGAAGGTATAGATAGATTTTGAAAATATTCGGGTTATATACGTCGCGATTTATAGTTTTTTTTTAGCGTTGGAGTGGAGACGGCGTTCGTAGCGTTTTGCGCGGGTGAGGTTCGCGTAGTTGTTGGGGAAGAGTTTATTTGTTAGGTTGCGTTGGGTTAGCGTAGTAAGTGGGGTTGGTCGTTATTTCGTTGTA TTCGGTCGCGTTTCGGGTTTCGTGCGTTTTCGTTTTAG (SEQ ID NO: 67).

[00103] In some embodiments, the negative standard for detecting BMP3 methylation comprises a polynucleotide sequence

GTTAGTTTGGTTGGGTGTTTTTAAAAATAAAGTGAGGAGGGAAGGTATAGATAGATTTTGAAAATATTTGGGTTATATATGTTGTGATTTATAGTTTTTTTTTAGTGTTGGAGTGGAGATGGTGTTTGTAGTGTTTTGTGTGGGTGAGGTTTGTGTAGTTGTTGGGGAAGAGTTTATTTGTTAGGTTGTGTTGGGTTAGTGTAGTAAGTGGGGTTGGTTGTTATTTTGTTG TATTTGGTTGTGTTTTGGGGTTTTGTGTGTTTTTGTTTTAG (SEQ ID NO: 68).

[00104] In some embodiments, the positive standard for detecting NDRG4 methylation comprises a polynucleotide sequence

TGAGAAGTCGGCGGGGGCGCGGATCGATCGGGGTGTTTTTTAGGTTTCGCGTCGCGGTTTTCGTTCGTTTTTTCGTTCGTTTATCGGGTATTTTAGTCGCGTAGAAGGCGGAAGTTACGCGCGAGGGATCGCGGTTCGTTCGGGATTAGTTTTAGGTTCGGTATCGTTTCGCGGGTCGAGCGTTTATATTCGTTAAATTTACGCGGGTACGTTTTTCGCGGCGTATCGTTTTTAG TT (SEQ ID NO: 69).

[00105] In some embodiments, the negative standard for detecting NDRG4 methylation includes a polynucleotide sequence

TGAGAAGTTGGTGGGGGTGTGGATTGATTGGGGTGTTTTTTAGGTTTTGTGTTGTGGTTTTTGTTTGTTTTTTTGTTTGTTTATTGGGTATTTTAGTTGTGTAGAAGGTGGAAGTTATGTGTGAGGGATTGTGGTTTGTTTGGGATTAGTTTTAGGTTTGGTATTGTTTTGTGGGTTGAGTGTTTATATTTGTTAAATTTATGTGGGTATGTTTTTGTGGTGTATTGTTTT TAGTT (SEQ ID NO: 70).

In some embodiments, the method includes amplifying a quality control standard in step c) and step d).

[00106] The present invention also relates to a method for diagnosing and treating colorectal cancer (RCC) and/or advanced adenoma (AA) in a patient in need thereof, wherein the method includes determining the presence or absence of RCCA and/or PA in the patient using the kit according to the invention, and treating the patient depending on the presence or absence of POCR and/or PA.

[00107] The present invention also relates to a method for diagnosing and treating colorectal cancer (RCC) and/or advanced adenoma (AA) in a patient in need thereof, wherein the method includes determining the presence or absence of RCCA and/or PA in the patient using the method described here, and treating the patient depending on the presence or absence of POC and/or PA.

Brief description of drawings

[00108] Figures 1A-1D show the results of the prediction of the presence of the CpG region and the relative positions of the amplicons of the two genes BMP3 and NDRG4. "Y", "R" represent degenerate bases. Figure 1A shows the result of the CpG region prediction of the promoter of BMP3 genes; Figure 1B shows the relative positions of the BMP3 gene amplicons; Figure 1C shows the result of the CpG region prediction of the promoter of NDRG4 genes; Figure 1D shows the relative positions of the NDRG4 gene amplicons.

[00109] Figure 2A shows the difference in CpG sites of BMP methylation between white and Asian populations. Figure 2B shows the difference in CpG methylation sites of the NDRG4 gene between white and Asian populations.

[00110] Figure 3A shows the sensitivity amplification assay curve for BMP3 along with primers and probes in Preferred Group 1. Figure 3B shows the sensitivity amplification assay curve for NDRG4 along with primers and probes in Preferred Group 1. Figure 3C shows the assay amplification curve sensitivity amplification curve for BMP3 along with primers and probes in preferred group 2. Figure 3D shows the sensitivity amplification curve for NDRG4 along with primers and probes in preference group 2. Figure 3E shows the sensitivity amplification curve for BMP3 along with primers and probes in preference group 3. Figure 3F shows the sensitivity amplification assay curve for NDRG4 along with primers and probes in preference group 3. Figure 3G shows the sensitivity amplification assay curve for BMP3, along with primers and probes in comparison group 1. Figure 3H shows the amplification curve sensitivity amplification curve for NDRG4 along with primers and probes in comparative group 1. Figure 3I shows the sensitivity amplification curve for BMP3, along with primers and probes in comparison group 2. Figure 3J shows the sensitivity amplification curve for NDRG4 along with primers and probes in comparison group 2. Figure 3K shows the sensitivity amplification curve for BMP3, along with primers and probes in comparison group 3. Figure 3L shows the sensitivity amplification curve for NDRG4, along with primers and probes in comparison group 3.

[00111] Figure 4A shows the specificity amplification curve for BMP3 along with primers and probes in preference group 1. Figure 4B shows the specificity amplification curve for NDRG4 along with primers and probes in preference group 1. Figure 4C shows the specificity amplification curve for NDRG4 along with primers and probes in preference group 1. Figure 4C shows the specificity amplification curve for NDRG4. specificity for BMP3 along with primers and probes in preference group 2. Figure 4D shows the specificity amplification curve for NDRG4 along with primers and probes in preference group 2. Figure 4E shows the specificity amplification curve for BMP3 along with primers and probes in preference group 3. Figure 4F shows the specificity amplification curve for NDRG4, along with primers and probes in preference group 3. Figure 4G shows the specificity amplification curve for BMP3, along with primers and probes in comparison group 1. Figure 4H shows the amplification curve analytical specificity amplification curve for NDRG4 along with primers and probes in comparative group 1. Figure 4I shows the analytical specificity amplification curve for BMP3, along with primers and probes in comparative group 2. Figure 4J shows the analytical specificity amplification curve for NDRG4 along with primers and probes in Comparative Group 2. Figure 4K shows the analytical specificity amplification curve for BMP3, along with primers and probes in Comparative Group 3. Figure 4L shows the analytical specificity amplification curve for NDRG4, along with primers and probes in Comparative Group 3.

[00112] Figures 5A-5C show amplification curves using primers and probes in Preferred Group 1, Preferred Group 2, and Preferred Group 3, respectively, to detect BMP3 methylation in a clinical sample. Figures 5D-5F show the amplification curves using primers and probes in Comparative Group 1, Comparative Group 2, and Comparative Group 3, respectively, to detect BMP3 methylation in the same assay.

[00113] Figures 6A-6C show amplification curves using primers and probes in Preferred Group 1, Preferred Group 2, and Preferred Group 3, respectively, to detect NDRG4 methylation in a clinical sample. Figures 6D-6F show the amplification curves using primers and probes in Comparative Group 1, Comparative Group 2, and Comparative Group 3, respectively, to detect NDRG4 methylation in the same assay.

Detailed Description of the Invention

Definitions

[00114] References to “one embodiment,” “one embodiment,” “one example,” and “example” indicate that the embodiment(s) or example(s) so described may include a particular feature, structure, characteristic, property, element or limitation, however, not every embodiment or example necessarily includes such particular feature, structure, characteristic, property, element or limitation. Additionally, repeated use of the phrase “in one embodiment” does not necessarily refer to the same embodiment, although it may.

[00115] As used herein, the terms “nucleic acid” or “oligonucleotide” or “polynucleotide” mean at least two nucleotides covalently linked to each other. The description of one strand also determines the sequence of the complementary strand. Thus, the nucleic acid also includes a complementary strand to the described single strand. Many nucleic acid variants can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also includes essentially identical nucleic acids and their complements. One strand is a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, the nucleic acid also includes a probe that hybridizes under stringent hybridization conditions. Nucleic acids may be single-stranded or double-stranded, or they may contain portions of both double-stranded and single-stranded sequences. The nucleic acid may be DNA, either genomic, cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribonucleotides and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine. Nucleic acids can be obtained by chemical synthesis or recombinant methods.

[00116] As used herein, the term "individual in need" refers to an animal or person who has been diagnosed with cancer and is at risk of developing cancer (e.g., an individual with a genetic predisposition to cancer, an individual with a medical and/or family history history, an individual who has been exposed to carcinogens, occupational hazards, and environmental hazards), and/or who has suspicious clinical signs of cancer (eg, blood in the stool or melena, unexplained pain, sweating, unexplained fever, unexplained weight loss to the point of anorexia, changes in bowel behavior (constipation and/or diarrhea), tenesmus (feeling of incomplete bowel movements, especially for rectal cancer), anemia and/or general weakness). Additionally or alternatively, the individual in need thereof may be a healthy individual undergoing a routine health screening.

[00117] As used herein, the term “about” means ±10%.

[00118] The phrase "consisting essentially of" means that the composition or method may include additional ingredients and/or steps, but only if these additional ingredients and/or steps do not significantly change the essential and novel properties of the claimed composition or method.

[00119] The articles “a”, “an” and “the” used in this application, used with singular nouns, can also refer to plural nouns, unless the context of the description indicates otherwise. Thus, for example, the term “compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.

[00120] As used herein, the term “stringent hybridization conditions” means conditions under which a first nucleic acid sequence (eg, a probe) will hybridize to a second nucleic acid sequence (eg, a target), for example, in a complex mixture of nucleic acids. The stringency of hybridization depends on the sequence and will differ in different cases. Stringent conditions may be selected where the temperature is approximately 5-10°C lower than the melting temperature (Tm) for a particular sequence at a particular ionic strength and pH. Tm may be the temperature (at a particular ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (since the target sequences are present in excess at that Tm, and 50% probes are in a state of equilibrium). Severe conditions may be those where the salt concentration is less than about 1.0 M sodium ion, such as about 0.01 to 1.0 M sodium ion (or other salts) at a pH of 7.0 to 8 ,3, and the temperature is at least about 30°C for short probes (eg, about 10-50 nucleotides) and at least about 60°C for long probes (eg, more than about 50 nucleotides). Harsh conditions can also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, the positive signal may be at least 2-10 times the background hybridization signal. Examples of stringent hybridization conditions include the following: 50% formamide, 5 x SSC and 1% SDS, incubated at 42°C, or 5 x SSC, 1% SDS, incubated at 65°C, washed in 0.2 x SSC and 0.1% SDS at 65°C.

[00121] As used herein, the term "substantially complementary" means that the first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of the second sequence in the region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40 , 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides, or that the two sequences hybridize under stringent hybridization conditions.

[00122] As used herein, the term "substantially identical" means that the first and second sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98 % or 99% identical in the area of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or, in the case of nucleic acids, the first sequence is substantially complementary to complement second sequence.

[00123] As used herein, the term “diagnosis” refers to the classification of pathology or symptoms, determination of the severity of the pathology (eg, grade or stage), monitoring the progression of the pathology, predicting the outcome of the pathology, and/or prospects for recovery.

[00124] The expression “consisting essentially of” means that the composition or method may include additional ingredients and/or steps, but only if these additional ingredients and/or steps do not significantly change the essential and novel properties of the claimed composition or the claimed method.

1. DNA sequences containing hypermethylated CpG sites in the promoter region of the BMP3 and NDRG4 genes in the Chinese population with PCOS and PA.

[00125] The present invention relates to DNA sequences containing well-described hypermethylated CpG sites in the promoter region of the BMP3 and NDRG4 genes in Asian populations (eg, Chinese populations), which can be used as markers for the diagnosis of POC and PA.

[00126] In some embodiments, the present invention provides the natural BMP3 gene sequence shown below (5' to 3'), where potentially methylated sites are indicated by a superscript "m":

GCCAGTTTGGC ^m CGGGTGTTCCCAAAAATAAAG ^m CGAGGAGGGAAGGTACAGACAGATCTTGAAAACACC ^m CGGGCCACACA ^m CGC ^m CG ^m CGACCTACAGCTCTTTCTCAG ^{m CGTTGGAGTGGAGA} m CGG ^{m CGCCCGCAG m} CGCCCTG ^m CG ^m CGGGTGAGGTC ^m CG ^m CGCAGCTGCTGGGGAAGAGCCCACCTGTCAGGCTG ^m CGCTGGGTC AG m CGCAGCAAGTGGGGCTGGC ^m CGCTATCT ^m CGCTGCACCCGGC ^m CG ^m CGTCC ^m CGGGCTC ^m CGTG ^m CGCCCT ^m CGCCCCAG( SEQ ID NO: 65).

[00127] In some embodiments, the present invention provides the natural NDRG4 gene sequence shown below (5' to 3'), where potentially methylated sites are indicated by a superscript "m"::

TGAGAAGT ^M CGG ^{M CGGGG M} CGGAT ^M CGGGGGGGTCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC ^{M CGT M} CGGTCCC ^M CGCCC M ^{CGCCCC M} CGGGGCACCCCCAGC M ^{CGCAGGG M} CGGGG M CGGGG M CGCAGGG ^{M CGCAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGM AAGCCA M Cg M Cg M} Cgaggag ^{M Cg M} Cggtc ^M Cgtc ^M cgggactagccccaggcc ^m cggcac ^m cgccc ^m cg ^m cgggc ^m cgag ^m cgcccacacc ^m cgccaaaccca ^m cg ^m cgggca ^m cgcccc ^m cg ^m cgg ^m cgcac ^m cgcccccagcc(SEQ ID NO.: 66).

{00128] After treating natural genomic DNA or a fragment thereof with one or more reagents to convert cytosine bases that are not methylated to uracil (e.g., bisulfite) or another base that is markedly different from cytosine in terms of hybridization properties, a the converted BMP3 gene sequence (5' to 3') shown below, with potentially methylated sites indicated by a superscript "m":

GTTAGTTTGGT ^m CGGGTGTTTTTAAAAATAAAG ^m CGAGGAGGGAAGGTATAGATAGATTTTGAAAATATT ^m CGGGTTATATA ^m CGT ^m CG ^m CGATTTATAGTTTTTTTTTAG ^m CGTTGGAGTGGAGA ^m CGG ^m CGTT ^m CGTAG ^m CGTTTTG ^m CG ^m CGGGTGAGGTT ^m CG ^m CGTAGTTGTTGGGGAAGAGTTTATTTGTT AGGTTG ^m CGTTGGGTTAG ^m CGTAGTAAGTGGGGTTGGT ^m CGTTATTT ^m CGTTGTATT ^m CGGT ^m CG ^m CGTTT ^m CGGGTTT ^m CGTG ^m CGTTTT ^m CGTTTTAG (SEQ ID NO:1).

[00129] After treating natural genomic DNA or a fragment thereof with one or more reagents to convert cytosine bases that are not methylated to uracil (e.g., bisulfite) or another base that is markedly different from cytosine in terms of hybridization properties, a the converted NDRG4 gene sequence (5' to 3') shown below, with potentially methylated sites indicated by a superscript "m":

tgagaagt ^m cgg ^m cgggggg ^m cg ^m cggat ^m cgaT ^m cggggtgtTTTTTaggTtT ^m cg ^m cgt ^m cg ^m cggtTTT ^m cgTt ^m cgTTTtTT ^m cgTT ^m cgTTTaT ^m cgggTaTTTTagT ^m cg ^m cgTagaagg ^m cggaagTTa ^m cg ^m cg ^m cgagggaT ^m cg ^m cggt T ^m cgt T ^m cgggaTtagTTTTaggTT ^m cggTaT ^m cgTTT ^m cg ^m cgggT ^m cgag ^m cgTTTaTaTT ^m cgTTaaaTTTa ^m cg ^m cgggTa ^m cgTTTT ^m cg ^m cgg ^m cgTaT ^m cgTTTTTagTT(SEQ ID NO:2).

[00130] DNA sequences containing specifically marked hypermethylated CpG sites in the promoter region of the BMP3 and NDRG4 genes in Asian populations according to the invention are particularly suitable for diagnosing POC and/or PA in Asian populations. For example, primers and probes can be designed to target one or more specific methylation sites in the BMP3 and/or NDRG4 genes as a means to determine the status and level of BMP3 and/or NDRG4 methylation, and therefore to determine the tumor status of a patient needing it.

2. Three pairs of preferred primers and probes for detecting methylation of the BMP3 and NDRG4 genes, respectively, and the corresponding reagents.

[00131] The present invention provides three pairs of preferred primers and probes for determining the methylation levels of the BMP3 and NDRG4 genes, respectively. These primers and probes were designed to target methylated CpG sites commonly found in Asian populations (eg, Chinese populations).

[00132] These specific pairs of preferred primers and probes have surprisingly higher sensitivity and specificity in diagnosing POC and PA, especially in diagnosing PA in Asian populations, compared to the primers and probes found in commercially available products such as Cologuard®.

The primer and probe sequences are shown below.

Gene Group Primer/probe ID sequence Sequences (5 -3'） BMP3 Preferably 1 Direct primer SEQ ID NO.:3 TTTGAAAATATTCGGGTTATATACGTCGC Reverse primer SEQ ID NO.:4 ATAAACTCTTCCCCAACAACTACGCGAA Probe SEQ ID NO.:5 AGCGTTGGAGTGGAGACGGCGTTCG Preferably 2 Direct primer SEQ ID NO.:9 AATATTCGGGTTATATACGTCGCGA Reverse primer SEQ ID NO.:10 GCAACCTAAACAAATAAACTCTTCCCCAA Probe SEQ ID NO.:11 TGGAGTGGAGACGGCGTTCGTAGCGT Preferably 3 Direct primer SEQ ID NO.:15 AATATTCGGGTTATATACGTCGCGATT Reverse primer SEQ ID NO.:16 ACTTACTACGCTAACCCAACG Probe SEQ ID NO.:17 TAGCGTTGGAGTGGAGACGGCGTTCGTA NDRG4 Preferably 1 Direct primer SEQ ID NO.:6 ATCGATCGGGGTGTTTTTTAGGTTTC Reverse primer SEQ ID NO.:7 CCTTCTACGCGACTAAAATACCCGAT Probe SEQ ID NO.:8 CGTCGCGGTTTTCGTTCGTTTTTTCGTTCGT Preferably 2 Direct primer SEQ ID NO.:12 GCGGGTGAGAAGTCGGC Reverse primer SEQ ID NO.:13 GTAACTTCCGCCTTCTACGC Probe SEQ ID NO.:14 TAGGTTTCGCGTCGCGGTTTTCGTT Preferably 3 Direct primer SEQ ID NO.:18 CGGTTTTCGTTCGTTTTTTCG Reverse primer SEQ ID NO.:19 AACCTAAAACTAATCCCGAACGAACC Probe SEQ ID NO.:20 TCGTTTATCGGGTATTTTAGTCGCGTAG

[00133] The oligonucleotides of the invention advantageously allow highly specific amplification of hypermethylated CpG sites in the promoter region of BMP3 or NDRG4 in a biological sample taken from an Asian patient.

[00134] Some embodiments of the invention provide oligonucleotides that are partially or fully complementary to the sequence of SEQ ID NO: 3-20.

[00135] Some embodiments provide oligonucleotides having one or more modifications from the probe sequence, such as SEQ ID NOs: 5, 11, 17, 8, 14, and 20. In some embodiments, the modification may occur at The 5'-end and/or 3'-end of one of the nucleotide sequences presented in SEQ ID NO: 5, 11, 17, 8, 14 and 20.

[00136] Examples of modified base fragments that can be used to modify nucleotides at any position in its structure include, but are not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-ioduracil, hypoxanthine, xanthine, acetylcytosine , 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N-6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2 -methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetic acid, pseudoracyl, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-hydroxyacetic acid methyl ester , uracil-5-hydroxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil and 2,6-diaminopurine, etc.

[00137] Examples of modified sugar moieties that can be used to modify a nucleotide at any position in its structure include, but are not limited to: arabinose, 2-fluoroarabinose, xylose and hexose, or a modified phosphate backbone component such as phosphorothioate, phosphorodithioate, phosphoramidothioate, phosphoramidate, phosphodiamidate, methylphosphonate, alkyl phosphotriester or formacetal or analogs thereof.

[00138] In some embodiments, the oligonucleotide in the sequences of SEQ ID NOs: 5, 11, 17, 8, 14 and 20 is replaced with a non-natural nucleotide, such as an artificial nucleic acid. Artificial nucleic acids include, but are not limited to, peptide nucleic acid (PNA), morpholino nucleic acid, locked nucleic acid (LNA), glycol nucleic acid (GNA), and threose nucleic acid (TNA). Each of them differs from natural DNA or RNA by changes in the backbone of the molecule.

[00139] In some embodiments, the probe of the invention contains a label at its 5' end.

[00140] In some embodiments, the label at the 5' end of the probe contains a fluorescent dye, such as a fluorophore. The fluorophore used here is a fluorescent chemical compound that can re-emit light after being exposed to light. Fluorophores typically contain multiple combined aromatic groups or planar or cyclic molecules with multiple π bonds. Non-proteinaceous organic fluorophores include, but are not limited to, xanthine derivatives (eg, fluorescein, rhodamine, Oregon green, eosin, and Texas red); cyanine derivatives (e.g. cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine and merocyanine), squaraine derivatives and ring-substituted squaraines (e.g. Seta, SeTau and Square dyes), naphthalene derivatives (e.g. dansyl and sold derivatives), coumarin derivatives, derivatives oxadiazole (eg pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole); anthracene derivatives (eg, anthraquinones including DRAQ5, DRAQ7 and orange CyTRAK); pyrene derivatives (cascade blue, etc.), oxazine derivatives (for example, Nile red, Nile blue, cresyl violet, oxazine 170, etc.); acridine derivatives (for example, proflavine, acridine orange, acridine yellow, etc.); arylmethine derivatives (eg, auramine, crystal violet, malachite green); tetrapyrrole derivatives (for example, porphin, phthalocyanine, bilirubin). Specific examples include, but are not limited to, VIC, PET, Texas Red, Cy3, Cy5, FAM (6-carboxyfluorescein), HEX (6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein ), ROX (5(6)-carboxy-X-rhodamine), JOE (6-carboxy-4',5'-dichloro-21,71-dimethoxyfluorescein), TET (5'-tetrachlorofluorescein phosphoramidite), NED (fluorescein -benzoxanthine), TAMRA (6-carboxy-N, N, N, N-tetramethylrhodamine), FITC (fluorescein isothiocyanate). Examples of specific fluorophores that can be used in the probes described herein are known to those skilled in the art and include those described in US Pat. No. 5,866,366 to Nazarenko et al., such as 4-acetamido-4'-isothiocyanatostilbene-2,2'- disulfonic acid; acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate ( Lucifer yellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide; brilliant yellow; coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcouluarin (coumaran 151); cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5',5'-dibromopyrogallol-sulfonephthalein (bromopyrogallol red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and its derivatives such as eosin and eosin isothiocyanate; erythrosine and derivatives such as erythrosine B and erythrosine isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE ), fluorescein, fluorescein isothiocyanate (FITC), QFITC (XRITC), 6-carboxyfluorescein (HEX) and TET (tetramethylfluorescein); fluorescamine; IR144; IR1446; malachite green isothiocyanate; 4-methylumbelliferone; ortho-cresolphthalein; nitrotyrosine; pararosaniline; phenol red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl-1-pyrene butyrate; reactive red 4 (CIBACRON™, brilliant red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyradamine (R6G), lissamine-rhodamine sulfonyl chloride B, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine isothiocyanate X, N, N,N ',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine and tetramethylrhodamine isothiocyanate (TRITC); sulforhodamine B; sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); riboflavin; rosolic acid and terbium chelate derivatives; LightCycler red 640; Cy5.5; and Cy56-carboxyfluorescein; boron dipyrromethene difluoride (BODIPY); acridine; stilbene; 6-carboxy-X-rhodamine (ROX); Cy3; Cy3.5, Cy5, Cy5.5, VIC® (Applied Biosystems); LC red 640; LC red 705; Oregon Green™; CALRedTM; red 640; and Yakima yellow; LighterCycler®Cyan500; LighterCycler®; red 610; Alexa 647; Alexa 555; 5-(2-aminoethyl)amino-1-naphthalenesulfonic acid (EDANS); tetramethylrhodamine (TMR); tetramethylrhodamine isocyanate (TMRITC), fluorescein isocyanate (FITC), γ-rhodamine, derivatives thereof, or any combination thereof, and the like. Other fluorescent dyes are described in US patent numbers: 5866366, 6818431, 6056859, 9140688, 9581587, 6165765, 6485909, 8158358, 7625723, 7560236, 7867701, 915091 2, 7960543, 65551383, 6881570, 8198026, 5625081, 8445291, 9194801, 8835110, 7893227, 9243289, 7427674, 9512493, in US Patent Application Publications: 20170152552, 20030170672, 20160281151, 20130084558, 20060281100, 20140234833, 20150072340, 20050089910, 20090081677, 20140024022201801711393, 200601418886, 20010018185, 20110151446 and WO/2000/017330A1, WO/20 08 /030071A1, WO/2013/049631A1, WO/2016/179090A1, WO/2016/123895A1, WO/2003/079022A1, each of which is incorporated herein in its entirety by reference.

[00141] In some embodiments, the probe of the invention comprises a fluorescent donor and an acceptor fluorophore. As used herein, an acceptor fluorophore (eg, a "fluorescence quencher") is a fluorophore that absorbs energy from a donor fluorophore, for example, in the range of about 400 to 900 nm. Acceptor fluorophores typically absorb light at a wavelength that is typically at least 10 nm higher (eg, at least 20 nm higher) than the maximum absorption wavelength of the donor fluorophore. Acceptor fluorophores have an excitation spectrum that overlaps with the emission of the donor fluorophore, so that the energy emitted by the donor can excite the quencher. Any acceptor fluorophores known to those skilled in the art can be used. In a specific example, the acceptor fluorophore is a dark quencher such as Dabcyl, QSY7 (Molecular Probes), QSY9 (Molecular Probes), QSY21 (Molecular Probes), QSY33 (Molecular Probes), BLACK HOLE QUENCHERS™ (Glen Research, e.g., BHQ-1, BHQ-2, BHQ-3), ECLIPSE™ dark quencher (Epoch Biosciences), DDQ-I, DDQ-II, Dabcyl, Eclipse, or IOWA BLACK™ (Integrated DNA Technologies, e.g. Iowa Black FQ, Iowa Black RQ). Other fluorescence quenchers are described in US Patent NOs: 9957546, US9274008, US Patent Publications NOs: 20140295422, 20090042205, 20160281182, 20180142284, 20140147929, and WO/2009/009615A 1, WO/2016/160572A1, WO/2016/178953A1, WO/ 2018/229663A1, WO/2010/051544A2, WO/2013/152220A2, which are incorporated herein by reference in their entirety. A quencher can reduce or suppress the emission of a donor fluorophore. In such an example, instead of detecting an increase in the emission signal from the acceptor fluorophore when it is in sufficient proximity to the donor fluorophore (or detecting a decrease in the emission signal from the acceptor fluorophore when it is at a significant distance from the donor fluorophore), an increase in the emission signal from the donor fluorophore may be detected. donor fluorophore when the quencher is at a considerable distance from the donor fluorophore (or a decrease in the emission signal from the donor fluorophore when it is in sufficient proximity to the acceptor quencher fluorophore).

[00142] In some embodiments, the primers and probes of the invention are based on fluorescence resonance energy transfer (FRET). Examples of FRET oligonucleotides that can be used to detect amplicons include linear oligoprobes such as HybProbes, 5' nuclease oligoprobes such as TAQMAN® probes, hairpin oligoprobes such as molecular beacons, Scorpion and UniPrimers, probes, minor groove binding and self-fluorescent amplicons such as Sunrise primers.

[00143] In some embodiments, the primers and/or probes of the invention are labeled with other functional molecules, such as biotin, haptens, antigens, chemical groups, radioactive substances, enzyme markers, and the like. Detection of a labeled amplification product can be carried out, for example, using fluorescence methods, chemiluminescence methods, densitometry methods, photometric methods, precipitation reactions, enzymatic reactions, including enzymatic activation reactions, SPR methods (surface plasmon resonance), ellipsometry methods, index measurements refraction, reflectance measurements and similar methods.

[00144] In some embodiments, the primers and probes described herein can be used in quantitative PCR to determine the methylation status and level of the BMP3 and/or NDRG4 gene in a patient. In some embodiments, an additional reaction may be included to amplify one or more control genes. In some embodiments, the control gene is a patient gene whose activity is not affected by the presence or absence of POC and PA, and is not affected by the methylation status and level of BMP3 and NDRG4. In some embodiments, control genes include, but are not limited to, β-globin (HBB), telomerase (TERT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), albumin (ALB), β-actin (ACTB), microglobulin beta 2 (B2M) and T cell receptor γ (TRG).

[00145] In some embodiments, the B2M gene is used as a reference gene in a quantitative PCR assay to detect the methylation status and level of BMP3/NDRG4.

[00146] In some embodiments, one or more other controls may be included, including, but not limited to, a matrix-free control (to detect reagent or equipment contamination and confirm positive results); non-amplification control (to detect background fluorescence generated by degradable labeled probes) and positive control (to detect inhibitors or dysfunction, and to confirm that reagents and equipment are in working order).

[00147] In some embodiments of the invention, qPCR is used to determine the presence of amplification of a methylated BMP3 gene or a methylated NDRG4 gene in a sample. The detected signal from the BMP3 or NDRG4 probe is quantified either from a standard curve or by comparing the Ct values with that of a reference gene. To normalize results, analysis is often performed using housekeeping genes. The cycle threshold (Ct) is defined as the number of cycles required for the fluorescent signal to exceed a specified threshold (eg, above the background level, such as the amplification level in a negative control sample). In some embodiments of the invention, the threshold is automatically determined using a program in the qPCR device or other suitable methods. In some embodiments, the threshold is set slightly higher (eg, approximately 0.01%, 0.1%, 1%, 5%, or 10% higher) than the final fluorescence value in the negative control sample.

[00148] In some embodiments, if the Ct value associated with BMP3 or NDRG4 amplification in a test sample is no more than (≤) about 35, 34, 33, 32, 31, 30 or less, then the sample is determined to be containing methylated BMP3 or NDRG4, and the patient is diagnosed with POCR and/or PA (positive), and otherwise, the sample is defined as not containing methylated BMP3 or NDRG4, and the patient is considered to lack RFOC or PA (negative). For control gene amplification, if the Ct value associated with control gene amplification in the sample is not greater than (≤) approximately 34, 33, 32, 31, 30, 29 or less, then the control gene amplification is determined to be positive, otherwise, amplification of the control gene will be considered negative. If control gene amplification is determined to be negative, the test result is invalid.

[00149] In some embodiments, the difference between the Ct value associated with BMP3 and the Ct value associated with reference gene amplification (ΔCt=Ct _{of the gene of interest} - Ct _{of the reference gene} ) is referred to as ΔCt1. In some embodiments, if ΔCt1 does not exceed a predetermined cutoff value (≤ cutoff value), then the sample is determined to be BMP3 methylated (positive), ie, the patient has POC or PA. In some embodiments, if ΔCt1 is greater than a predetermined critical value (> critical value), then the sample does not have BMP3 methylation (negative) and the patient is considered healthy. In some embodiments, the critical value is the corresponding ΔCt value for a sample containing 5 ng/μl of nucleotide sequence having a 1% degree of methylation, such as about 8, 9, or 10.

[00150] In some embodiments, the difference between the Ct value associated with NDRG4 and the Ct value associated with reference gene amplification (ΔCt=Ct _{of the gene of interest} - Ct _{of the reference gene} ) is referred to as ΔCt2. In some embodiments, if ΔCt2 does not exceed a predetermined critical value (≤ critical value), then the sample is determined to have NDRG4 methylation (positive), ie, the patient has POCR or PA. In some embodiments, if ΔCt2 is greater than a predetermined critical value (> critical value), then the sample does not have NDRG4 methylation (negative result) and the patient is considered healthy. In some embodiments, the critical value is the corresponding ΔCt value for a sample containing 5 ng/μl of nucleotide sequence having a 1% degree of methylation, such as about 8, 9, or 10.

[00151] The preferred primers and probes of the invention have surprisingly high sensitivity and specificity for the detection of POCA and PA in Asian populations, and especially for the detection of PA, compared with those of commercially available products such as Cologuard®.

[00152] As used herein, the term "sensitivity" is defined as the level of sensitivity at which patients who actually have POC and/or PA in a given group are correctly identified. In some embodiments, the sensitivity is at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, according to at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more, or 100%. In some embodiments, the number of individuals in the group is at least about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000 , 9000, 10000 or more.

[00153] As used herein, the term "specificity" is defined as the degree of specificity at which patients who actually do not have POC or PA in a given group are correctly diagnosed as not having the disease. In some embodiments, the specificity is at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more, or 100%. In some embodiments, the number of individuals in the group is at least about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000 , 9000, 10000 or more.

[00154] As shown in the examples, the preferred primers and probes provide remarkably high sensitivity and specificity for detecting BMP3 and NDRG4 methylation, which provides surprisingly high sensitivity and specificity for diagnosing POC and/or PA. For example, the sensitivity for diagnosing POC using three pairs of preferred primers and probes for the BMP3 gene is at least 85%; the sensitivity of diagnosing POC using three pairs of preferred primers and probes for the NDRG4 gene is at least 90%; the sensitivity for diagnosing PA using three pairs of preferred primers and BMP3 gene probes is at least 66%; and the sensitivity for diagnosing PA using three pairs of preferred primers and NDRG4 gene probes is at least 73%. In addition, the specificity for diagnosing POC and PA using three pairs of preferred primers and BMP3 gene probes is approximately 97% - 100% (eg, at least approximately 97.8%); and the specificity for diagnosing POC and PA using three pairs of preferred primers and NDRG4 gene probes is approximately 97% to 100% (eg, at least about 97.8%).

3. Three sets of preferred primer and probe combinations for diagnosing POC and/or PA in patients.

[00155] The present invention also provides three sets of preferred primer and probe combinations for detecting methylation levels of the BMP3 and NDRG4 genes for the purpose of determining the presence or absence of POC or PA in a patient. These specific combinations have remarkably high sensitivity and specificity for the detection of POC and PA in Asian populations, especially in the diagnosis of PA, compared with those of existing commercial products such as Cologuard®.

The sequences of three sets of preferred primers and probes are presented below:

Combination No. Gene Group Primers/Probes ID sequence Subsequence
(5' - 3') 4 BMP3 Preferably 1 Direct primer SEQ ID NO.:3 TTTGAAAATATTCGGGTTATATACGTCGC Reverse primer SEQ ID NO.:4 ATAAACTCTTCCCCAACAACTACGCGAA Probe SEQ ID NO.:5 AGCGTTGGAGTGGAGACGGCGTTCG NDRG4 Preferably 1 Direct primer SEQ ID NO.:6 ATCGATCGGGGTGTTTTTTAGGTTTC Reverse primer SEQ ID NO.:7 CCTTCTACGCGACTAAAATACCCGAT Probe SEQ ID NO.:8 CGTCGCGGTTTTCGTTCGTTTTTTCGTTCGT 5 BMP3 Preferably 1 Direct primer SEQ ID NO.:3 TTTGAAAATATTCGGGTTATATACGTCGC Reverse primer SEQ ID NO.:4 ATAAACTCTTCCCCAACAACTACGCGAA Probe SEQ ID NO.:5 AGCGTTGGAGTGGAGACGGCGTTCG NDRG4 Preferably 2 Direct primer SEQ ID NO.:12 GCGGGTGAGAAGTCGGC Reverse primer SEQ ID NO.:13 GTAACTTCCGCCTTCTACGC Probe SEQ ID NO.:14 TAGGTTTCGCGTCGCGGTTTTCGTT 7 BMP3 Preferably 2 Direct primer SEQ ID NO.:9 AATATTCGGGTTATATACGTCGCGA Reverse primer SEQ ID NO.:10 GCAACCTAAACAAATAAACTCTTCCCCAA Probe SEQ ID NO.:11 TGGAGTGGAGACGGCGTTCGTAGCGT NDRG4 Preferably 1 Direct primer SEQ ID NO.:6 ATCGATCGGGGTGTTTTTTAGGTTTC Reverse primer SEQ ID NO.:7 CCTTCTACGCGACTAAAATACCCGAT Probe SEQ ID NO.:8 CGTCGCGGTTTTCGTTCGTTTTTTCGTTCGT

[00156] As shown in the Examples, preferred combinations of BMP3 and NDRG4 primer probe sets provide remarkably high sensitivity and specificity in detecting BMP3 and NDRG4 methylation, and thereby provide surprisingly high sensitivity and specificity in diagnosing POC and/or PA. In some embodiments, this sensitivity and specificity is achieved when the same test also includes a KRAS gene test and a hemoglobin test, as explained in Example 5. For example, the overall sensitivity for diagnosing POC using three the preferred combinations of primers and probes for the BMP3 and NDRG4 genes when performing a combined analysis for the KRAS gene and hemoglobin is at least 95%; the sensitivity for diagnosing PA using three preferred combinations of primers and probes of the BMP3 and NDRG4 genes when performing a combined analysis for the KRAS gene and hemoglobin is at least 93%; the sensitivity of diagnosing POC+PA using three preferred combinations of primers and probes of the BMP3 and NDRG4 genes when performing a combined analysis for the KRAS gene and hemoglobin is at least 97%; and the specificity for diagnosing POC+PA using three preferred combinations of primers and probes of the BMP3 and NDRG4 genes when performing a combined KRAS gene and hemoglobin test is at least 97%.

4. Kit for diagnosing RPOC and PA

[00157] The present invention relates to kits for detecting BMP3/NDRG4 methylation and/or diagnosing POC/PA in a patient in need thereof. In some embodiments, these kits are particularly suitable for Asian patients, such as Chinese patients.

[00158] In some embodiments, the kit includes: (1) at least one of three sets of preferred primer and probe combinations for the diagnosis of POC and PA (combinations Nos. 4, 5, and 7 in Table 20) and corresponding reagents for .PCR; (2) means for detecting seven mutations in the coding region of the KRAS gene (ie, G12D, G13D, G12V, G12C, G12S, G12A and G13R), such as suitable primers and probes and appropriate qPCR reagents; (3) means for detecting hemoglobin in a stool sample, such as reagents derived from FIT technology (eg, anti-hemoglobin antibody and reagents for detecting the complex formed by antibody and hemoglobin in a stool sample).

[00159] In some embodiments, the kit includes at least one set of the preferred primer-probe combination set forth below:

(1) Three sets of preferred primer and probe combinations are presented below:

NO. Gene Group Primer/Probe ID sequence Sequence （5' - 3'） 4 BMP3 Preferably 1 Direct primer SEQ ID NO.:3 TTTGAAAATATTCGGGTTATATACGTCGC Reverse primer SEQ ID NO.:4 ATAAACTCTTCCCCAACAACTACGCGAA Probe SEQ ID NO.:5 AGCGTTGGAGTGGAGACGGCGTTCG NDRG4 Preferably 1 Direct primer SEQ ID NO.:6 ATCGATCGGGGTGTTTTTTAGGTTTC Reverse primer SEQ ID NO.:7 CCTTCTACGCGACTAAAATACCCGAT Probe SEQ ID NO.:8 CGTCGCGGTTTTCGTTCGTTTTTTCGTTCGT 5 BMP3 Preferably 1 Direct primer SEQ ID NO.:3 TTTGAAAATATTCGGGTTATATACGTCGC Reverse primer SEQ ID NO.:4 ATAAACTCTTCCCCAACAACTACGCGAA Probe SEQ ID NO.:5 AGCGTTGGAGTGGAGACGGCGTTCG NDRG4 Preferably 2 Direct primer SEQ ID NO.:12 GCGGGTGAGAAGTCGGC Reverse primer SEQ ID NO.:13 GTAACTTCCGCCTTCTACGC Probe SEQ ID NO.:14 TAGGTTTCGCGTCGCGGTTTTCGTT 7 BMP3 Preferably 2 Direct primer SEQ ID NO.:9 AATATTCGGGTTATATACGTCGCGA Reverse primer SEQ ID NO.:10 GCAACCTAAACAAATAAACTCTTCCCCAA Probe SEQ ID NO.:11 TGGAGTGGAGACGGCGTTCGTAGCGT NDRG4 Preferably 1 Direct primer SEQ ID NO.:6 ATCGATCGGGGTGTTTTTTAGGTTTC Reverse primer SEQ ID NO.:7 CCTTCTACGCGACTAAAATACCCGAT Probe SEQ ID NO.:8 CGTCGCGGTTTTCGTTCGTTTTTTCGTTCGT

(2) Reagents for triplex quantitative PCR to determine the methylation level of the BMP3 and NDRG4 genes.

[00160] In some embodiments, the kits contain reagents for performing multiplex PCR to simultaneously detect BMP3 and NDRG4 methylation. In some embodiments of the invention, the multiplex PCR is quantitative PCR.

[00161] In some embodiments, the reagents include Taq DNA polymerase. In some embodiments, the final concentration of Taq DNA polymerase is approximately 2 units/reaction. In some embodiments of the invention, the reagents include MgCl ₂ . In some embodiments, the final concentration of MgCl ₂ in the reaction is 2 mM. In some embodiments of the invention, the reagents include dNTPs. In some embodiments, the dNTP has a final concentration of 0.2 mM. In some embodiments, the reagents include from about 0.5 mM to 0.75 mM primers for amplification of BMP3, NDRG4 and control genes. In some embodiments, the reagents include from about 0.1 mM to 0.25 mM probes hybridized to DNA sequences of BMP3, NDRG4, and control genes. In some embodiments, the reagents further include a PCR buffer, such as a concentrated PCR buffer (eg, 5× or 10×), which can be diluted to a final concentration of 1×. In some embodiments, B2M is a control gene that has been amplified for quality control in quantitative PCR.

(3) Primers and probes to detect seven mutations in the coding region of the KRAS gene.

[00162] In some embodiments, the kits contain means for detecting mutations in the KRAS gene. In some embodiments, the kits contain primers and probes that have been designed to amplify and detect seven mutant hotspots in exon 12 and exon 13 in the open reading frame of the KRAS gene, where the mutations are G12D, G13D, G12V, G12C, G12S , G12A and G13R. In some embodiments, the primer and probe sequences are as follows:

Name Primer/Probe ID sequence Sequences (5' - 3') G12D-F Direct primer SEQ ID NO.:35 AACTTGTGGTAGTTGGAGGTGA G13D-F Direct primer SEQ ID NO.:36 AACTTGTGGTAGTTGGAGCTGGGGGA G12V-F Direct primer SEQ ID NO.:37 AACTTGTGGTAGTTGGAGTTGT G12C-F Direct primer SEQ ID NO.:38 AAACTTGTGGTAGTTGGGGCTT G12S-F Direct primer SEQ ID NO.:39 AAACTTGTGGTAGTTGGTGCTA G12A-F Direct primer SEQ ID NO.:40 AACTTGTGGTAGTTGGAGCAGC G12R-F Direct primer SEQ ID NO.:41 AAACTTGTGGTAGTTGGAGCTC Kras-R Reverse primer SEQ ID NO.:42 GAATGGTCCTGCACCAGTAATATG ACTB-F Direct primer SEQ ID NO.:43 AGGGCTTCTTGTCCTTTCCTT ACTB-R Reverse primer SEQ ID NO.:44 CGTGCTCGATGGGGTACTTC KRAS-P Probe SEQ ID NO.:45 AGGCAAGAGTGCCTTGACGATACAGC ACTB-P Probe SEQ ID NO.:46 CGTGATGGTGGGCATGGGTCAGAAGGA

(4) Reagents for multiplex quantitative PCR used to detect codon mutations in the KRAS gene

[00163] The present invention also provides a multiplex quantitative PCR system for detecting all seven KRAS gene mutations. In some embodiments, the multiplex quantitative PCR includes Taq DNA polymerase at a final concentration of 2.5 units/reaction, MgCl ₂ at a final concentration of 1 mM, dNTP at a final concentration of 0.1 mM, 0.3-0.9 μM primers for amplification of the KRAS and ACTB genes, 0.05-0.1 µM probes hybridized with the DNA sequences of the KRAS and ACTB genes, and 1 × PCR buffer. ACTB is a reference gene and amplified for qPCR quality control.

(5) Stool hemoglobin test kit

[00164] In some embodiments of the invention, the kits contain reagents for determining hemoglobin. In some embodiments of the invention, hemoglobin was qualitatively assessed using an enzyme-linked immunosorbent assay (ELISA).

5. Logistic regression model

[00165] In some embodiments, after obtaining the results of the BMP3/NDRG4 methylation assay, KRAS mutation assay, and hemoglobin assay, all results were compiled and subjected to logistic regression to determine the presence or absence of POC and/or PA in patients .

[00166] In some embodiments of the invention, the method includes calculating a P value of the overall cancer index, where P=e ^K /(1+e ^K ), where e denotes a natural constant.

[00167] In some embodiments, K is defined as K=a*ΔCt1+b*ΔCt2+c*ΔCt3+d*FIT+X, where a, b, c, d, X are clinical constants. ΔCt1, ΔCt2, and ΔCt3 are the Ct values of BMP3, NDRG4, and KRAS subtracted from those of the control genes.

[00168] In some embodiments, if the P value is equal to or greater than a predetermined threshold, then the test result will be positive, otherwise the test result will be negative. A positive result indicates that the person may have POC or PA, while a negative result indicates that the person is healthy.

6. Treatment methods

[00169] In some embodiments, the methods of the invention include treating patients in need thereof after the patients have been classified as having colorectal cancer and/or adenoma. In some embodiments, such treatment includes, but is not limited to, surgery, chemotherapy, radiation therapy, immunotherapy, palliative care, and exercise.

[00170] As used herein, the term “treatment regimen” means a treatment protocol that defines the type of treatment, dosage, schedule, and/or duration of treatment for an individual in need thereof (eg, an individual who has been diagnosed with a pathology). The chosen treatment regimen can be radical, which is expected to lead to the best clinical outcome (for example, complete cure of the pathology), or more moderate, which may alleviate the symptoms of the pathology, but at the same time does not provide a complete cure for the patient with the pathology. It should be noted that in some cases, the treatment regimen may be associated with some discomfort for the patient or with adverse side effects (for example, damage to healthy cells or tissues). The type of treatment may include surgery (eg, removal of the lesion, affected cells, tissue, or organ), cell replacement therapy, administration of a therapeutic drug (eg, receptor agonists, antagonists, hormones, chemotherapeutic agents) locally or systemically, external source radiation therapy (eg external radiation) and/or internal source (eg brachytherapy) and/or any combination thereof. The dosage, schedule and duration of treatment may vary depending on the severity of the pathology and the type of treatment chosen, and specialists in the field can independently adjust the type of treatment depending on the dose, schedule and duration of treatment.

[00171] In some embodiments, methods of treatment include, but are not limited to, administration of fluorouracil, capecitabine, oxaliplatin, irinotecan, UFT, FOLFOX, FOLFOXIRI and FOLFIRI, antiangiogenic drugs such as bevacizumab, and epidermal growth factor receptor inhibitors (EGF receptor inhibitors). e.g. cetuximab and panitumumab).

7. Advantages of the present invention

Without being limited to any particular theory, the inventors of the present invention note at least the following advantages:

(1) Detailed methylated CpG sites of the promoter regions of BMP3 and NDRG4 genes in the Chinese population are presented and can be used as a biomarker for the diagnosis of POC and/or PA.

(2) The kit is more suitable for diagnosing POC and PA in the Chinese population than other similar mt-sDNA-based products such as Cologuard®, since the present invention targets CpG methylation sites specific to Chinese, while previously used products were intended for the white population.

(3) The sensitivity and specificity for diagnosing POC are higher compared to those of other similar mt-sDNA products.

(4) The sensitivity and specificity of PA detection are significantly higher compared to those of other similar mt-sDNA products.

(5) This method is noninvasive and easy to sample at home, and therefore has good patient compliance, and it can be widely used as a screening method for POC and PA. This method reduces the morbidity and mortality of PCOS in Asian populations.

EXAMPLES

Example 1

Detection of methylated CpG sites in the promoter regions of the BMP3 and NDRG4 genes, respectively, in Chinese populations with POC and PA.

(1) Sample collection

[00172] A total of 191 colonic FFPE tissue samples were collected from patients with POCC and PA that were confirmed by colonoscopy, including 50 colorectal cancer tissues and 49 pairs of adjacent normal tissues, 46 malignant adenoma tissues, and 46 pairs of additional normal tissues. fabrics.

(2) DNA extraction

[00173] Genomic DNAs were extracted from FFPE samples using the TaKaRa MiniBEST FFPE DNA Extraction Kit (catalog number: 9782). The steps of this procedure are detailed below:

i. Remove a 30 mg paraffin section of tissue with a sterile scalpel and remove excess paraffin.

ii. Place the paraffin section of tissue in a 1.5 ml centrifuge tube and add 500 µl DP buffer, mix and incubate in water at 80°C for 1 minute, and then vortex for 10 seconds. Add 180 µl GL buffer and shake.

iii. The mixture was centrifuged at 12,000 rpm for 1 minute at room temperature, and then the solution was separated into two layers (an upper oil phase and a lower aqueous phase). 20 μL of proteinase K (20 mg/mL) and 10 μL of RNase (10 mg/mL) were added to the lower aqueous phase and mixed thoroughly by pipetting up and down. At the same time, care was taken not to damage the layers. And then the mixture was placed in a water bath at 56°C for 1 hour.

iv. The solution obtained in the previous step was incubated at 90°C for 30 minutes and cooled to room temperature. Then, 200 μL of GB buffer and 200 μL of 100% ethanol were added to the solution and mixed by shaking for 10 seconds. It was then centrifuged at 12,000 rpm for 1 minute at room temperature, and the solution was separated into two layers (upper oil phase and lower aqueous phase).

v. A spin column was placed in a collection tube and the lower aqueous phase obtained in the previous step was added to the column. At the same time, care had to be taken not to damage the layers. Centrifugation was then performed at 12,000 rpm for 2 minutes at room temperature, and the waste was discarded.

vi. 500 μL of WA buffer was added to the spin column and centrifuged at 12,000 rpm for 1 minute at room temperature, and then the waste was discarded.

vii. 500 μL of WB buffer was added to the spin column and centrifuged at 12,000 rpm for 1 minute at room temperature, and then the waste was discarded. This procedure was repeated one more time.

viii. A spin column was placed in the collection tube and centrifuged at 12,000 rpm for 2 minutes at room temperature.

ix. A spin column was added to a new 1.5 mL centrifuge tube, then 50-100 μL of sterilized water or elution buffer was added to the center of the septum of the spin column, and then left for 5 minutes at room temperature.

x. Centrifuge at 12,000 rpm for 2 minutes at room temperature, and DNA was eluted.

xi. Eluted DNA was quantified using a Nanodrop 2000 fluorimeter and stored at -20°C for later use.

(3) Prediction of CpG regions in the promoter regions of BMP3 and NDRG4 genes and design of primers for amplicon sequencing.

i. Prediction of CpG regions in the promoter regions of the BMP3 and NDRG4 genes.

[00174] The promoter sequences of the BMP3 and NDRG4 genes were downloaded, including approximately 1000-1500 bp of DNA sequence. upstream of the transcription start site (TSS) and 5'UTR region. CpG sequence regions were predicted using the MethPrimer program (www.urogene.org/methprimer/). As shown in Figures 1A-1D, the two larger of the three CpG regions of the BMP3 gene were located at approximately 400 bp. upstream of the TSS and the entire 5'UTR region (chr4: 81951752-81952760), and only one CpG region of the NDRG4 gene was located at approximately 500 bp. upstream of the TSS and partial 5'UTR region (chr16: 58497061-58497938). The human reference genome construct is GRCh37/hg19.

ii. Design of a primer to amplify the sequence of CpG regions predicted in the BMP3 and NDRG4 genes.

[00175] Based on the length of the amplified sequences and the length of the sequencing reads, four and five pairs of primers were designed for the BMP3 and NDRG4 genes, respectively. There is as much overlap as possible between adjacent amplicons so that the CpG regions of two genes can be completely sequenced. The primers of the two genes are listed in Table 1, and the relative positions of the amplicons of the two genes are shown in Figures 1A-1D.

Table 1. Primers for sequencing amplicons of the BMP3 and NDRG4 genes

Gene Amplicon Primer ID sequence Sequence (5' -3') BMP3 Amplicon 1 Direct primer 1 SEQ ID NO.:47 AGTTTGGTGTAAGTTAAGAG BMP3 Reverse primer 1 SEQ ID NO.:48 CTAACTCTATTTTTAAAACRCCA BMP3 Amplicon 2 Direct primer2 SEQ ID NO.:49 GTTTTAATTTTTTGGAAAAGGTAA BMP3 Reverse primer 2 SEQ ID NO.:50 ACCTAACAAATAAACTCTTCC BMP3 Amplicon 3 Direct primer 3 SEQ ID NO.:51 GAAGGTATAGATAGATTTTGAA BMP3 Reverse primer 3 SEQ ID NO.:52 CACCTACACAACTTTACRAAACT BMP3 Amplicon 4 Direct primer 4 SEQ ID NO.:53 GTATTTAGTTATGGTTGGGGYGAGTA BMP3 Reverse primer 4 SEQ ID NO.:54 CTCACCTACTACTACCGCCCR NDRG4 Amplicon 1 Direct primer 1 SEQ ID NO.:55 AGGTTTTTGAGTTTTTGGTTTTTTTT NDRG4 Reverse primer 1 SEQ ID NO.:56 CCCTCCAAACCCCCTATAAC NDRG4 Amplicon 2 Direct primer2 SEQ ID NO.:57 GGATGGGGATGTTTTTGTAG NDRG4 Reverse primer 2 SEQ ID NO.:58 RGRGAAAACCTAAAAAACACC NDRG4 Amplicon 3 Direct primer 3 SEQ ID NO.:59 GYGGAGYGGGTGAGAAGT NDRG4 Reverse primer 3 SEQ ID NO.:60 CRAACAACCAAAACCCCTC NDRG4 Amplicon 4 Direct primer 4 SEQ ID NO.:61 GTTYGTTYGGGATTAGTTTTAGG NDRG4 Reverse primer 4 SEQ ID NO.:62 CRCAAACRAAAAACRAAAC NDRG4 Amplicon 5 Direct primer 5 SEQ ID NO.:63 GYGGYGTTTTYGTTTTTG NDRG4 Reverse primer 5 SEQ ID NO.:64 CRACRACTAAAAATCCCCAA

[00176] DNA samples were subjected to bisulfite treatment as described below.

(4) Bisulfite treatment

i. The extracted DNA was left at room temperature to thaw and the DNA concentration was adjusted to 20 ng/μl. Then, 40 μL of diluted DNA was added to a 1.5 ml centrifuge tube, followed by 4 μL of 3 M NaOH solution and incubated at 42°C for 20 minutes.

ii. 400 μl of conversion solution was added, mixed and incubated at 50°C for 16 hours in the dark.

iii. 550 μl of binding solution was added, mixed, and the solution was transferred to a DNA purification column. Centrifuged at 13,000 rpm for 90 seconds and the waste was discarded. Then it was centrifuged again for 3 minutes and the waste was discarded.

iv. 600 μl of 90% ethanol was added to the DNA purification column, centrifuged at 13,000 rpm for 90 seconds, and the waste was discarded. Then centrifuged again at 13,000 rpm for 15 seconds.

v. 300 μl of desulfurization solution (0.3 M NaOH in 90% ethanol) was added to purify the DNA, then left for 30 minutes at room temperature. It was then centrifuged at 13,000 rpm for 90 seconds and the waste was discarded.

vi. 600 μL of 90% ethanol was added, centrifuged at 13,000 rpm for 90 seconds, and the waste was discarded. This step was repeated one more time and centrifuged again at 13,000 rpm for 3 minutes.

vii. A DNA purification column was added to a new 1.5 mL centrifuge tube and 40 μL of eluate was added. The tube was incubated at 50°C for 30 minutes and centrifuged at 13,000 rpm for 90 seconds. The converted DNA solution was stored at -20°C for further use.

[00177]

(5) Library construction and amplicon sequencing

(a) Multiplex PCR amplification

i. The Master PCR mixture was prepared as follows:

table 2

No. Reagents Final concentration 1 Multiprex PCR mixture Master 2 × QIAGNE (QIAGEN, catalog no.: 206143) 1× 2 10 × primer mix (2 µM each) 0.2 µM 3 RNase-free water up to 40 µl

ii. The mixture was shaken gently and 40 μl of the mixture was pipetted into each PCR tube, followed by 10 μl of bisulfate-treated DNA.

iii. The mixture was gently shaken and PCR amplification was performed as follows: one cycle of denaturation at 95°C for 15 minutes, 35 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 90 seconds and extension at 72°C for 90 seconds, and one extension cycle at 72°C for 10 minutes and finally the mixture was kept continuously at 4°C.

iv. 5 μl of PCR products were mixed with 6× loading buffer (TakaRa, cat. no. 9156) and loaded onto a 1% (wt/vol) agarose gel with control DNA marker DL2000 (TakaRa, cat. no. 3427Q) and subjected to electrophoresis at 120V for 40 minutes.

v. If nonspecific amplification was observed, the remaining 45 μl of PCR products after electrophoresis should be recovered according to the kit instructions (QIAGEN, catalog number: 28704).

[00178]

(b) Purification and quantification of PCR products

The PCR product was purified using the MinElute PCR purification kit according to the kit instructions (QIAGEN, part number: 28004). The purified PCR product was quantified using the BR Qubit™ dsDNA Assay Kit (catalog number: Q32850).

[00179]

(c) Adapter ligation

The adapters were ligated to the purified PCR products using the NEBNext Rapid Ligation Module (NEB, part number: E6056L), and the reaction mixture was prepared as follows:

Table 3. Reaction mixture for adapter ligation

No. Reagents Volume (µl)/reaction 1 NEBNext Rapid Ligation Buffer (5×) 10 2 Adapter 5 3 DNA ligase T4 Quick 5 4 RNase-free water 10

30 μL of the mixture was added to each PCR tube, followed by 20 μL of purified PCR products, respectively. The solution was mixed gently, incubated at 20°C for 15 minutes and kept at 4°C for 10 minutes.

[00180]

(d) Purification of ligation products

i. Agencourt AMpure XP spheres (Beckman, part number: A63882) were kept at room temperature for subsequent use.

ii. PCR tubes were centrifuged at 280×g for 1 minute at 20°C, and 50 μl of ligation products were transferred to a new 96-well PCR plate.

iii. AMpure XP beads were vortexed for 30 seconds to ensure even distribution and 56 µl of beads were added to each well of the PCR plate. The mixture was gently stirred 10 times and kept at room temperature for 5 minutes.

iv. The 96-well PCR plate was placed on a 96-well magnetic plate and held for 2 minutes until the supernatant became clear. The 96-well PCR plate was then kept on a 96-well magnetic plate and the supernatant was removed, after which 200 μL of freshly prepared 80% ethanol was added to each well and incubated at room temperature for 30 seconds, and the supernatant was removed.

v. The 96-well PCR plate was kept on a 96-well magnetic plate, after which 200 μL of freshly prepared 80% ethanol was added to each well. Then incubated at room temperature for 30 seconds. The supernatant was discarded and the remaining ethanol was removed using a 10 μL pipette.

vi. The 96-well PCR plate was kept on a magnetic plate and the spheres were allowed to dry naturally for 10 minutes.

vii. The 96-well PCR plate was removed from the magnetic plate, and 27.5 μL of 10 mM Tris (pH 8.5) was added to each well of the 96-well plate. The spheres were gently pipetted up and down and Tris was added 10 times until the spheres were completely distributed. The plate was kept at room temperature for 2 minutes.

viii. The 96-well PCR plate was placed on a magnetic plate and left for 2 minutes until the supernatant became clear. 25 μl of supernatant was pipetted into a new PCR tube and stored at -20°C for later use.

[00181]

(f) PCR amplification and product purification

PCR Master Mix was prepared as described below and mixed gently. 40 μL of PCR mixture was added to each PCR tube, followed by 5 μL of purified ligation products.

Table 4. Reaction mixture for PCR

NO. Reagents Final concentration 1 NEBNext Ultra Q5 Master Mix
(NEB, part number: M0544) 1× 2 Forward primer (10 µM) 1 µM 3 Reverse primer (10 µM) 1 µM 4 Water without nuclease Up to 40 µl

PCR tubes were gently shaken and quickly centrifuged, and then PCR was performed under the following conditions: one cycle of denaturation at 98°C for 30 seconds, 8 to 15 cycles of denaturation at 98°C for 10 seconds, and extension at 65°C for for 75 seconds, one cycle of denaturation at 65°C for 5 minutes and finally holding at 4°C. PCR products were purified as described in step (d) “Purification of Ligation Products.”

[00182]

(f) Library detection and sequencing

The concentration and size distribution of purified PCR products were analyzed using the QubitTM BR dsDNA Assay Kit (catalog number: Q32850) and an Agilent 2100 Bioanalyzer, respectively. Libraries of equal molar concentration were pooled and sequenced on an Illumina HiSeq2500 with read length PE125.

[00183]

(g) Analysis of sequencing data

The raw data were demultiplexed according to the sample indices, and the sequencing read data were mapped to the BMP3 and NDRG4 reference gene sequences using the SHRiMP V2.04 program. Methylated CpG sites were identified based on the mapping results. Finally, 26 and 39 CpG sites of the BMP3 and NDRG4 genes were found to be hypermethylated in tissues with POC and PA compared with adjacent normal tissues (p < 0.05), indicating that these methylated CpG sites may be used as DNA biomarkers for the early diagnosis of POC and PA.

Table 5. Frequency of methylation of CpG sites of the BMP3 gene in POC tissues and in adjacent normal tissues

CpG Sites Chromosome coordinates
(hg19) Localization at the transcription start site
(NM_001201.3） Number of methylated samples divided by the total number of samples with RPOC Percentage of methylation in RPOC Number of methylated samples divided by total number of normal samples Percentage of methylation in normal sample P-value #1 81952078 -41 34/50 0.68 2/49 0.04 <0.01 #2 81952099 -20 38/50 0.76 2/49 0.04 <0.01 #3 81952135 17 33/50 0.66 1/49 0.02 <0.01 #4 81952146 28 34/50 0.68 2/49 0.04 <0.01 #5 81952149 31 34/50 0.68 1/49 0.02 <0.01 #6 81952151 33 35/50 0.7 2/49 0.04 <0.01 #7 81952172 54 33/50 0.66 0/49 0.00 <0.01 #8 81952186 68 33/50 0.66 1/49 0.02 <0.01 #9 81952189 71 36/50 0.72 1/49 0.02 <0.01 #10 81952193 75 37/50 0.74 0/49 0.00 <0.01 #eleven 81952198 80 34/50 0.68 1/49 0.02 <0.01 #12 81952205 87 37/50 0.74 2/49 0.04 <0.01 #13 81952207 89 37/50 0.74 2/49 0.04 <0.01 #14 81952218 100 34/50 0.68 1/49 0.02 <0.01 #15 81952220 102 37/50 0.74 2/49 0.04 <0.01 #16 81952255 137 35/50 0.7 0/49 0.00 <0.01 #17 81952266 148 33/50 0.66 1/49 0.02 <0.01 #18 81952285 167 38/50 0.76 1/49 0.02 <0.01 #19 81952293 175 36/50 0.72 1/49 0.02 <0.01 #20 81952302 184 35/50 0.7 1/49 0.02 <0.01 #21 81952306 188 33/50 0.66 1/49 0.02 <0.01 #22 81952308 190 36/50 0.72 0/49 0.00 <0.01 #23 81952313 195 35/50 0.7 0/49 0.00 <0.01 #24 81952320 202 35/50 0.7 2/49 0.04 <0.01 #25 81952324 206 38/50 0.76 2/49 0.04 <0.01 #26 81952330 212 33/50 0.66 2/49 0.04 <0.01

Table 6. Frequency of methylation of CpG sites of the BMP3 gene in PA tissues and in adjacent normal tissues

CpG Sites Chromosome coordinates
(hg19) Localization at the transcription start site
(NM_001201.3) Number of methylated samples divided by the total number of samples with PA Percentage of methylation in PA Number of methylated samples divided by the total number of samples with PA Percentage of methylation in normal sample P-value #1 81952078 -41 30/50 0.60 2/49 0.04 <0.01 #2 81952099 -20 29/50 0.58 1/49 0.02 <0.01 #3 81952135 17 32/50 0.64 2/49 0.04 <0.01 #4 81952146 28 29/50 0.58 2/49 0.04 <0.01 #5 81952149 31 30/50 0.60 2/49 0.04 <0.01 #6 81952151 33 32/50 0.64 0/49 0.00 <0.01 #7 81952172 54 32/50 0.64 2/49 0.04 <0.01 #8 81952186 68 32/50 0.64 2/49 0.04 <0.01 #9 81952189 71 28/50 0.56 0/49 0.00 <0.01 #10 81952193 75 29/50 0.58 1/49 0.02 <0.01 #eleven 81952198 80 29/50 0.58 0/49 0.00 <0.01 #12 81952205 87 31/50 0.62 1/49 0.02 <0.01 #13 81952207 89 29/50 0.58 0/49 0.00 <0.01 #14 81952218 100 32/50 0.64 1/49 0.02 <0.01 #15 81952220 102 28/50 0.56 1/49 0.02 <0.01 #16 81952255 137 29/50 0.58 0/49 0.00 <0.01 #17 81952266 148 31/50 0.62 0/49 0.00 <0.01 #18 81952285 167 32/50 0.64 1/49 0.02 <0.01 #19 81952293 175 33/50 0.66 0/49 0.00 <0.01 #20 81952302 184 32/50 0.64 1/49 0.02 <0.01 #21 81952306 188 30/50 0.60 0/49 0.00 <0.01 #22 81952308 190 31/50 0.62 1/49 0.02 <0.01 #23 81952313 195 29/50 0.58 1/49 0.02 <0.01 #24 81952320 202 32/50 0.64 1/49 0.02 <0.01 #25 81952324 206 31/50 0.62 2/49 0.04 <0.01 #26 81952330 212 32/50 0.64 1/49 0.02 <0.01

Table 7. Frequency of methylation of CpG sites of the NDRG4 gene in PA tissues and in adjacent normal tissues

CpG Sites Chromosome coordinates
(hg19) Localization at the transcription start site
(NM_0020465.3) Number of methylated samples divided by the total number of samples with RPOC Percentage of methylation in RPOC Number of methylated samples divided by total number of normal samples Percentage of methylation in normal sample P-value #1 chr16:58497349 -200 38/50 0.75 1/49 0.02 <0.01 #2 chr16:58497352 -197 35/50 0.70 2/49 0.04 <0.01 #3 chr16:58497358 -191 35/50 0.70 2/49 0.04 <0.01 #4 chr16:58497360 -189 39/50 0.78 1/49 0.03 <0.01 #5 chr16:58497365 -184 39/50 0.78 1/49 0.03 <0.01 #6 chr16:58497369 -180 39/50 0.77 1/49 0.02 <0.01 #7 chr16:58497388 -161 37/50 0.74 1/49 0.02 <0.01 #8 chr16:58497390 -159 37/50 0.73 1/49 0.03 <0.01 #9 chr16:58497393 -156 40/50 0.79 1/49 0.03 <0.01 #10 chr16:58497395 -154 37/50 0.74 1/49 0.02 <0.01 #eleven chr16:58497402 -147 38/50 0.76 1/49 0.03 <0.01 #12 chr16:58497406 -143 39/50 0.78 1/49 0.02 <0.01 #13 chr16:58497414 -135 37/50 0.74 1/49 0.03 <0.01 #14 chr16:58497418 -131 36/50 0.72 0/49 0.00 <0.01 #15 chr16:58497425 -124 36/50 0.72 1/49 0.02 <0.01 #16 chr16:58497438 -111 36/50 0.72 2/49 0.04 <0.01 #17 chr16:58497440 -109 36/50 0.71 1/49 0.02 <0.01 #18 chr16:58497449 -100 37/50 0.73 2/49 0.04 <0.01 #19 chr16:58497458 -91 39/50 0.77 2/49 0.04 <0.01 #20 chr16:58497460 -89 37/50 0.74 1/49 0.03 <0.01 #21 chr16:58497462 -87 38/50 0.75 1/49 0.03 <0.01 #22 chr16:58497470 -79 35/50 0.70 1/49 0.02 <0.01 #23 chr16:58497472 -77 36/50 0.71 2/49 0.04 <0.01 #24 chr16:58497477 -72 36/50 0.72 1/49 0.03 <0.01 #25 chr16:58497481 -68 37/50 0.74 1/49 0.02 <0.01 #26 chr16:58497499 -50 37/50 0.73 1/49 0.02 <0.01 #27 chr16:58497505 -44 40/50 0.79 2/49 0.04 <0.01 #28 chr16:58497510 -39 40/50 0.79 1/49 0.02 <0.01 #29 chr16:58497512 -37 40/50 0.79 2/49 0.04 <0.01 #thirty chr16:58497517 -32 35/50 0.70 1/49 0.02 <0.01 #31 chr16:58497521 -28 39/50 0.77 1/49 0.02 <0.01 #32 chr16:58497531 -18 38/50 0.76 1/49 0.03 <0.01 #33 chr16:58497542 -7 36/50 0.71 1/49 0.02 <0.01 #34 chr16:58497544 -5 37/50 0.74 1/49 0.03 <0.01 #35 chr16:58497550 2 37/50 0.74 0/49 0.00 <0.01 #36 chr16:58497556 8 38/50 0.75 1/49 0.03 <0.01 #37 chr16:58497558 10 39/50 0.78 2/49 0.04 <0.01 #38 chr16:58497561 13 36/50 0.71 2/49 0.04 <0.01 #39 chr16:58497566 18 35/50 0.70 1/49 0.03 <0.01

Table 8. Frequency of methylation of CpG sites of the NDRG4 gene in PA tissues and in adjacent normal tissues

CpG Sites Chromosome coordinates
(hg19) Localization at the transcription start site
(NM_020465.3） Number of methylated samples divided by the total number of samples with PA Percentage of methylation in PA Number of methylated samples divided by total number of normal samples Percentage of methylation in normal sample P-value #1 chr16:58497349 -200 28/46 0.60 1/46 0.02 <0.01 #2 chr16:58497352 -197 28/46 0.60 1/46 0.02 <0.01 #3 chr16:58497358 -191 27/46 0.59 1/46 0.03 <0.01 #4 chr16:58497360 -189 29/46 0.62 1/46 0.03 <0.01 #5 chr16:58497365 -184 30/46 0.65 2/46 0.04 <0.01 #6 chr16:58497369 -180 28/46 0.61 2/46 0.04 <0.01 #7 chr16:58497388 -161 29/46 0.64 1/46 0.03 <0.01 #8 chr16:58497390 -159 29/46 0.64 1/46 0.03 <0.01 #9 chr16:58497393 -156 30/46 0.65 1/46 0.02 <0.01 #10 chr16:58497395 -154 28/46 0.60 2/46 0.04 <0.01 #eleven chr16:58497402 -147 29/46 0.62 1/46 0.03 <0.01 #12 chr16:58497406 -143 29/46 0.62 1/46 0.03 <0.01 #13 chr16:58497414 -135 28/46 0.61 1/46 0.02 <0.01 #14 chr16:58497418 -131 30/46 0.65 1/46 0.02 <0.01 #15 chr16:58497425 -124 29/46 0.64 2/46 0.04 <0.01 #16 chr16:58497438 -111 29/46 0.64 1/46 0.02 <0.01 #17 chr16:58497440 -109 29/46 0.62 2/46 0.04 <0.01 #18 chr16:58497449 -100 29/46 0.64 1/46 0.03 <0.01 #19 chr16:58497458 -91 29/46 0.62 1/46 0.02 <0.01 #20 chr16:58497460 -89 27/46 0.59 1/46 0.03 <0.01 #21 chr16:58497462 -87 29/46 0.62 1/46 0.02 <0.01 #22 chr16:58497470 -79 29/46 0.64 1/46 0.02 <0.01 #23 chr16:58497472 -77 30/46 0.65 2/46 0.04 <0.01 #24 chr16:58497477 -72 30/46 0.65 2/46 0.04 <0.01 #25 chr16:58497481 -68 28/46 0.60 2/46 0.04 <0.01 #26 chr16:58497499 -50 29/46 0.63 1/46 0.02 <0.01 #27 chr16:58497505 -44 28/46 0.61 1/46 0.02 <0.01 #28 chr16:58497510 -39 27/46 0.59 1/46 0.02 <0.01 #29 chr16:58497512 -37 30/46 0.65 2/46 0.04 <0.01 #thirty chr16:58497517 -32 29/46 0.62 2/46 0.04 <0.01 #31 chr16:58497521 -28 29/46 0.64 1/46 0.02 <0.01 #32 chr16:58497531 -18 29/46 0.62 2/46 0.04 <0.01 #33 chr16:58497542 -7 29/46 0.64 1/46 0.02 <0.01 #34 chr16:58497544 -5 29/46 0.63 1/46 0.02 <0.01 #35 chr16:58497550 2 29/46 0.62 1/46 0.02 <0.01 #36 chr16:58497556 8 28/46 0.61 1/46 0.03 <0.01 #37 chr16:58497558 10 30/46 0.65 1/46 0.03 <0.01 #38 chr16:58497561 13 29/46 0.63 1/46 0.02 <0.01 #39 chr16:58497566 18 30/46 0.65 1/46 0.02 <0.01

EXAMPLE 2

Comparison of CpG sites of differential methylation of the BMP3 and NDRG4 genes associated with POC in different races.

[00184] We analyzed microarray methylation data for the BMP3 and NDRG4 genes in the TCGA database (Illumina 450 human methylation data) and found that there were five significantly different CpG methylation sites in the promoter region of the NDRG4 genes between white and nonwhite populations. Asia (Figure 2 and Table 9). To further confirm the sites of differential CpG methylation, tissue samples and blood samples were collected from 106 patients with POC and PsA in China. DNA was extracted and treated with bisulfate. The promoter regions of the BMP3 and NDRG4 genes were amplified and sequenced. The authors analyzed the sequencing data and found that the CpG hypermethylation sites in the Asian population differed from the CpG hypermethylation sites in the white population taken from the TCGA database, and the CpG hypermethylation sites differed in tissue samples from POC and PA. Regarding these different CpG methylation sites, we have produced a detection kit, and in particular, for the detection of POC and PA in the Asian population (for example, in the Chinese population).

Table 9. Differences in CpG methylation sites of the BMP3 and NDRG4 genes in white and Asian populations

NO. Gene Methylation site One-way ANOVA Coordinates (hg19) Gene region Methylation percentage (White>Asian) 1 BMP3 cg19675063 p=0.001735, (f=4.408) 81975265 Exon 3 Y 2 NDRG4 cg27147718 p=0.00004290, (f=6.558) 58496542 promoter Y 3 NDRG4 cg04190807 p=0.01761, (f=3.036) 58497230 promoter N 4 NDRG4 cg00687686 p=0.01657, (f=3.072) 58497236 promoter N 5 NDRG4 cg04942472 p=0.01955, (f=2.973) 58497239 promoter N 6 NDRG4 cg01466678 p=0.02327, (f=2.867) 58497395 promoter N 7 NDRG4 cg05469759 p=0.03612, (f=2.599) 58498456 Intron 1 N 8 NDRG4 cg00262031 p=0.02585, (f=2.803) 58498574 Intron 1 N 9 NDRG4 cg06650115 p=0.01221, (f=3.256) 58498585 Intron 1 N 10 NDRG4 cg04005075 p=0.01938, (f=2.978) 58498636 Intron 1 N eleven NDRG4 cg09324514 p=0.0004509, (f=5.194) 58498710 Intron 1 N 12 NDRG4 cg16812519 p=0.01862, (f=3.002) 58498754 Intron 1 N 13 NDRG4 cg26824423 p=0.007544, (f=3.543) 58498818 Intron 1 N 14 NDRG4 cg05333442 p=0.003665, (f=3.970) 58533743 Intron 3 N 15 NDRG4 cg01343363 p=0.01099, (f=3.319) 58533808 Intron 3 Y 16 NDRG4 cg27113419 p=0.006302, (f=3.649) 58533979 Intron 3 Y 17 NDRG4 cg05725404 p=0.01145, (f=3.294) 58534157 Intron 3 N

EXAMPLE 3

Screening of primers and probes for the BMP3 and NDRG4 genes

(1) Design and selection of primers and probes for the BMP3 and NDRG4 genes.

[00185] Primers and probes for qPCR were designed based on the methylated CpG sites of the BMP3 and NDRG4 genes. Three pairs of preferred primers and probes were identified. The preferred primers and probes were compared with several other primer and probe candidates for the BMP3 and NDRG4 genes with positive controls and negative controls. Information about primers and probes is presented in Table 10.

Table 10. Information on preferred and other primers and probes

Gene Group Primer/Probe Primer/Probe ID Sequence （5' - 3'） BMP3 Preferably 1 Direct primer SEQ ID NO.:3 TTTGAAAATATTCGGGTTATATACGTCGC BMP3 Preferably 1 Reverse primer SEQ ID NO.:4 ATAAACTCTTCCCCAACAACTACGCGAA BMP3 Preferably 1 Probe SEQ ID NO.:5 AGCGTTGGAGTGGAGACGGCGTTCG NDRG4 Preferably 1 Direct primer SEQ ID NO.:6 ATCGATCGGGGTGTTTTTTAGGTTTC NDRG4 Preferably 1 Reverse primer SEQ ID NO.:7 CCTTCTACGCGACTAAAATACCCGAT NDRG4 Preferably 1 Probe SEQ ID NO.:8 CGTCGCGGTTTTCGTTCGTTTTTTCGTTCGT BMP3 Preferably 2 Direct primer SEQ ID NO.:9 AATATTCGGGTTATATACGTCGCGA BMP3 Preferably 2 Reverse primer SEQ ID NO.:10 GCAACCTAAACAAATAAACTCTTCCCCAA BMP3 Preferably 2 Probe SEQ ID NO.:11 TGGAGTGGAGACGGCGTTCGTAGCGT NDRG4 Preferably 2 Direct primer SEQ ID NO.:12 GCGGGTGAGAAGTCGGC NDRG4 Preferably 2 Reverse primer SEQ ID NO.:13 GTAACTTCCGCCTTCTACGC NDRG4 Preferably 2 Probe SEQ ID NO.:14 TAGGTTTCGCGTCGCGGTTTTCGTT BMP3 Preferably 3 Direct primer SEQ ID NO.:15 AATATTCGGGTTATATACGTCGCGATT BMP3 Preferably 3 Reverse primer SEQ ID NO.:16 ACTTACTACGCTAACCCAACG BMP3 Preferably 3 Probe SEQ ID NO.:17 TAGCGTTGGAGTGGAGACGGCGTTCGTA NDRG4 Preferably 3 Direct primer SEQ ID NO.:18 CGGTTTTCGTTCGTTTTTTCG NDRG4 Preferably 3 Reverse primer SEQ ID NO.:19 AACCTAAAACTAATCCCGAACGAACC NDRG4 Preferably 3 Probe SEQ ID NO.:20 TCGTTTATCGGGTATTTTAGTCGCGTAG BMP3 Control 1 Direct primer SEQ ID NO.:21 GAGTGGAGACGGCGTTCGTA BMP3 Control 1 Reverse primer SEQ ID NO.:22 CCACTTACTACGCTAACCCAACG BMP3 Control 1 Probe SEQ ID NO.:23 CGGGTGAGGTTCGCGTAGTTGTTGGG NDRG4 Control 1
Control 2，
Control 3 Direct primer SEQ ID NO.:24 GGGTGTTTTTTAGGTTTCGCGTC NDRG4 Control 1 Reverse primer SEQ ID NO.:25 CGTAACTTCCGCCTTCTACGC NDRG4 Control 1，
Control 2，
Control 3 Probe SEQ ID NO.:26 ACGCGACTAAAATACCCGATAAACGAACGAAAAAACGAAC BMP3 Control 2 Direct primer SEQ ID NO.:27 TTAGGTTGCGTTGGGTTAGCG BMP3 Control 2 Reverse primer SEQ ID NO.:28 ACTCCGAAAACGCAAAAAACCG BMP3 Control 2 Probe SEQ ID NO.:29 ATTCGGTCGCGTTTCGGGTTTCGTGC NDRG4 Control 2 Reverse primer SEQ ID NO.:30 GACCCGCGAAACGATACCG BMP3 Control 3 Direct primer SEQ ID NO.:31 TATTCGGGTTATATACGTCGC BMP3 Control 3 Reverse primer SEQ ID NO.:32 CTTACTACGCTAACCCAACG BMP3 Control 3 Probe SEQ ID NO.:33 CCCAACAACTACGCGAACCTCACCCG NDRG4 Control 3 Reverse primer SEQ ID NO.:34 TCGCGCGTAACTTCCGCCTT

(2) Comparison of preferred primers and probes with positive and negative controls of the BMP3 and NDRG4 genes

[00186] Standard samples with varying levels of methylation were prepared by adding positive control DNA to negative control DNA of the BMP3 and NDRG4 genes, respectively, in various ratios (Table 11). Analytical sensitivity was compared by amplifying a standard DNA sample with preferred and control primers and probes for the BMP3 and NDRG4 genes, respectively. Analytical specificity was compared by amplifying the negative control DNA of the BMP3 and NDRG4 genes with the preferred and control primers and probes of the BMP3 and NDRG4 genes, respectively, while the amount of negative control DNA was 10 ⁴ copies, 10 ⁵ copies, 10 ⁶ copies, 10 ⁷ copies and 10 ⁸ copies per reaction.

Table 11. Standard sample of BMP3 and NDRG4 genes

Methylation ratio Methylated DNA copy number Total DNA copy number 1 10 ⁵ 10 ⁵ 1/10 10 ⁴ 10 ⁵ 1/10 ² 10 ³ 10 ⁵ 1/10 ³ 10 ² 10 ⁵ 1/10 ⁴ 10 10 ⁵

[00187] The master mixture was prepared as described in Table 5, and qPCR conditions included first denaturation at 95°C for 1 minute, followed by 50 cycles of denaturation at 95°C for 20 seconds and extension at 60°C for 1 minute.

Table 12. Final concentrations of reagents for quantitative PCR with methylation

No. Reagents Final concentration 1 10× PCR buffer 1× 2 _MgCl2 2 mm 3 dNTP 0.2 mM 4 DNA polymerase Taq 2 units 5 Each primer 0.75 mM 6 Each probe 0.25 mM 7 DNA matrix 2 µl 8 Ultrapure water up to 50 µl

[00188] As shown in Table 13 and Figures 3A-3L, 1/10 ⁴ methylated DNA can be detected using three pairs of preferred primers and probes, but three pairs of controls cannot be detected.

[00189] As shown in Table 13 and Figures 3A-3L, the preferred three pairs of primers and probes BMP3 and NDRG4 according to the invention are able to consistently detect methylation levels down to one in ten thousand, while the three pairs of comparative primers and probes are not able to detect even one in ten thousand methylation levels.

Table 13: Comparison of analytical sensitivities of preferred and control primers and probes for the BMP3 and NDRG4 genes

Gene Methylation ratio Methylated DNA copy number Total DNA copy number Preferred Primers and Probes Control primers and probes Preferably 1 Preferably 2 Preferably 3 Control 1 Control 2 Control 3 BMP3 1 10 ⁵ 10 ⁵ Y Y Y Y Y Y 1/10 10 ⁴ 10 ⁵ Y Y Y Y Y Y 1/10 ² 10 ³ 10 ⁵ Y Y Y Y Y Y 1/10 ³ 10 ² 10 ⁵ Y Y Y Y N N 1/10 ⁴ 10 10 ⁵ Y Y Y N N N NDRG4 1 10 ⁵ 10 ⁵ Y Y Y Y Y Y 1/10 10 ⁴ 10 ⁵ Y Y Y Y Y Y 1/10 ² 10 ³ 10 ⁵ Y Y Y Y Y Y 1/10 ³ 10 ² 10 ⁵ Y Y Y N N Y 1/10 ⁴ 10 10 ⁵ Y Y Y N N N

Y: detected, N: undetermined.

[00190] Analytical sensitivity amplification curves of BMP3 and NDRG4 are shown in Figures 3A-3L for each of the preferred combinations compared to control groups.

Primers and probes BMP3 NDRG4 Preferably 1 Figure 3A Figure 3B Preferably 2 Figure 3C 3D figure Preferably 3 Figure 3E Figure 3F Control 1 Figure 3G Figure 3H Control 2 Figure 3I Figure 3J Control 3 Figure 3K Figure 3L

[00191] As shown in Table 14 and Figures 4A-4L, the three pairs of preferred primers and probes for the BMP3 and NDRG4 genes had no amplification signals at various concentrations of unmethylated DNA, while the comparison primers and probes exhibited varying levels of nonspecific amplification.

Table 14. Comparison of analytical specificity of preferred and control primers and probes

BMP3
(Copies for reaction) NDRG4
(Copies for reaction) Preferred Primers and Probes Control primers and probes Preferably 1 Preferably 2 Preferably 3 Control
1 Control 2 Control 3 10 ⁸ 10 ⁸ N N N Y Y Y 10 ⁷ 10 ⁷ N N N N Y Y 10 ⁶ 10 ⁶ N N N N N N 10 ⁵ 10 ⁵ N N N N N N 10 ⁴ 10 ⁴ N N N N N N

Y: no amplification, N: non-specific amplification.

Analytical specificity amplification curves of BMP3 and NDRG4

Primers and probes BMP3 NDRG4 Preferably 1 Figure 4A Figure 4B Preferably 2 Figure 4C 4D figure Preferably 3 Figure 4E Figure 4F Control 1 Figure 4G Figure 4H Control 2 Figure 4I Figure 4I Control 3 Figure 4K Figure 4L

Example 4

Confirmation of preferred and comparative methylation primers and probes is carried out on stool samples with the BMP3 and NDRG4 genes.

[00192] The methylation levels of the BMP3 and NDRG4 genes in 81 stool samples were determined using three pairs of preferred and comparative primers and probes. The three comparison primers and probes are:

Comparative primers and probes 1: forward primer BMP3 SEQ ID NO: 21, reverse primer BMP3 SEQ ID NO: 22 and probe BMP3 SEQ ID NO: 23; forward primer NDRG4 SEQ ID NO: 24, reverse primer NDRG4 SEQ ID NO: 25 and probe NDRG4 SEQ ID NO: 26;

Comparative primers and probes 2: forward primer BMP3 SEQ ID NO: 27, reverse primer BMP3 SEQ ID NO: 28 and probe BMP3 SEQ ID NO: 29; forward primer NDRG4 SEQ ID NO: 24, reverse primer NDRG4 SEQ ID NO: 30 and probe NDRG4 SEQ ID NO: 26;

Comparative primers and probes 3: forward primer BMP3 SEQ ID NO: 31, reverse primer BMP3 SEQ ID NO: 32 and probe BMP3 SEQ ID NO: 33; forward primer NDRG4 SEQ ID NO: 24, reverse primer NDRG4 SEQ ID NO: 34 and probe NDRG4 SEQ ID NO: 26.

[00193] Information about the stool samples is presented in Table 15.

Table 15. Statistics for 81 stool samples

Colonoscopy Number Norm 46 Adenomas 15 Colorectal cancer 20 Total 81

[00194] Stool DNA was extracted from the samples according to the method described below.

(1) Stool DNA extraction:

i. Add 40 ml of lysate to 4-6 g of stool samples, shake thoroughly, and then incubate at 50°C for 16 hours.

ii. Then centrifuge at 5000 rpm for 10 minutes. In this case, you should pay attention to the scales for weighing before centrifugation. After centrifugation is completed, the centrifuge tubes are carefully removed and shaken, but not vigorously.

iii. 9 ml of the supernatant is pipetted into a new 50 ml centrifuge tube, and then 1 ml of extraction adjuvant, 60 μl of magnetic beads and 10 ml of isopropanol are added. Then mix for 10 seconds and incubate at 65°C for 20 minutes. During incubation, stir up and down once every 5 minutes.

iv. After incubation, the 50 ml centrifuge tube is placed on a magnetic stand and left for 3 minutes until the supernatant becomes clear, after which the supernatant is discarded.

v. The 50 mL centrifuge tube is then removed from the magnetic stand and 12 mL of wash solution is added. The solution is stirred until the magnetic spheres fall from the tube contained in the wells and kept for 3 minutes, and then the 50 ml centrifuge tube is again placed on the magnetic stand for 3 minutes until the supernatant becomes clear, after which the supernatant discarded.

vi. Add 15 ml of 80% ethanol solution, shake until the spheres fall out of the tube contained in the wells, and incubate for 3 minutes. The tube is then placed back on the magnetic stand for 3 minutes until the supernatant becomes clear, after which the supernatant is discarded.

vii. The previous stage is repeated one more time.

viii. The liquid remaining at the bottom is pipetted, while the tube is kept open and incubated at 65°C for 5 minutes. The tube is then removed from the magnetic stand and the spheres are allowed to dry, and 1.5 ml of pre-warmed eluent I is added. The spheres are transferred from the well to eluent I using a 1000 µl pipette, after which the mixture is transferred to a 2 ml centrifuge tube and the cap is closed. , and then incubated at 65°C for 5 minutes.

ix. Then centrifuge at 13,000 rpm for 3 minutes, 600 µl of the supernatant is pipetted into a new 1.5 ml tube, and then 600 µl of binding solution is added and mixed thoroughly.

x. 600 µl of the above mixture is transferred to a DNA purification column, centrifuged at 13000 rpm for 1 minute and the waste is discarded.

xi. The remaining mixture is processed as in the previous step and centrifuged at 13,000 rpm for 2 minutes.

xii. 600 μl of 90% ethanol solution is added to the DNA purification column, centrifuged at 13,000 rpm for 1 minute, and the waste is discarded.

xii. The previous stage is repeated 2 times.

xiv. Then centrifuge at 13,000 rpm for 3 minutes and the DNA purification column is placed in a new 1.5 ml centrifuge tube. The DNA purification column is opened and incubated at 65°C for 5 minutes until dry.

xv. 100 µl of pre-warmed eluate II is added dropwise to the middle of the DNA purification column, the cap is closed and incubated at 65°C for 5 minutes. Then centrifuge at 13,000 rpm for 2 minutes. The resulting solution of eluted DNA is stored at 2-8°C until further use. The temperature for long-term storage should be from -25°C to -15°C.

[00195]

(2) Bisulfite treatment

A detailed description of the stages of the procedures is given in Example 3.

[00196]

(3) qPCR

The Master mixture for quantitative PCR was prepared as follows:

Table 16

No. Reagents Final concentration 1 10× PCR buffer 1× 2 _MgCl2 2 mm 3 dNTP 0.2 mM 4 DNA polymerase Taq 2 units 5 Each primer 0.75 mM 6 Each probe 0.25 mM 7 DNA matrix 2 µl 8 Ultrapure water up to 50 µl

QPCR reaction conditions: One cycle of denaturation at 95°C for 2 minutes, 50 cycles of denaturation at 95°C for 20 seconds and extension at 95°C for 1 minute. The B2M gene is a control gene for quality control of the QPCR reaction.

(4) Results

[00197] As shown in Table 17, the sensitivity for diagnosing POC and PA in 81 stool samples using three pairs of preferred primers and probes for the BMP3 and NDRG4 genes is up to 85.0-95.0% (POC by BMP3 methylation), 66.7 % -73.3% (PA for BMP3 methylation) and 90.0% - 95.0% (PA for NDRG4 methylation) and 73.3% -86.7% (PA for NDRG4 methylation), respectively. Additionally, the specificity for diagnosing POC and PA using BMP3 methylation data or NDRG4 methylation data is approximately 97.8% - 100.0%.

[00198] However, the sensitivity of diagnosing POC and PA in 81 stool samples using three pairs of comparative primers and probes of the BMP3 and NDRG4 genes is 85.0-90.0% (POC by BMP3 methylation), 46.7-60.0% ( PA for BMP3 methylation) and 90.0% -95.0% (RPOC for NDRG4 methylation) and 66.7% -73.3%, respectively (PA for NDRG4 methylation). In addition, the overall specificity for diagnosing POC and PA using BMP3 methylation data or NDRG4 methylation data increased to approximately 91.3–93.5% and 93.5–95.7%, respectively.

[00199] It is clear that the preferred primers and probes are superior to the comparative primers and probes in the analysis of clinical samples, especially in the diagnosis of PsA.

Table 17.

Results of statistical analysis of the studied clinical samples

BMP3 Data Preferably 1 Preferably 2 Preferably 3 Sample groups RPOC PA Norm RPOC PA Norm RPOC PA Norm Number of positive samples detected 19/20 11/15 0/46 18/20 11/15 1/46 17/20 10/15 1/46 Sensitivity 95.0% 73.3% / 90.0% 73.3% / 85.0% 66.7% / Specificity 100.0% 97.8% 97.8% General compliance 93.8% 91.4% 88.9% Amplification curve Figure 5A Figure 5B Figure 5C Data Control 1 Control 2 Control 3 Sample groups RPOC PA Norm RPOC PA Norm RPOC PA Norm Number of positive samples examined 18/20 9/15 3/46 17/20 8/15 4/46 17/20 7/15 4/46 Sensitivity 90.0% 60.0% / 85.0% 53.3% / 85.0% 46.7% / Specificity 93.5% 91.3% 91.3% General compliance 86.4% 82.7% 81.5% Amplification curve Figure 5D Figure 5E Figure 5F NDRG4 Data Preferably 1 Preferably 2 Preferably 3 Sample groups RPOC PA Norm RPOC PA Norm RPOC PA Norm Number of positive samples studied 19/20 13/15 0/46 19/20 12/15 0/46 18/20 11/15 1/46 Sensitivity 95.0% 86.7% / 95.0% 80.0% / 90.0% 73.3% / Specificity 100.0% 100.0% 97.8% General compliance 96.3% 95.1% 91.4% Data Figure 6A Figure 6B Figure 6C Sample groups Control 1 Control 2 Control 3 Number of positive samples examined RPOC PA Norm RPOC PA Norm RPOC PA Norm Sensitivity 18/20 10/15 2/46 18/20 10/15 3/46 19/20 11/15 3/46 Specificity 90.0% 66.7% / 90.0% 66.7% / 95.0% 73.3% / General compliance 95.7% 93.5% 93.5% Data 88.9% 87.7% 90.1% Amplification curve Figure 6D Figure 6E Figure 6F

Table 18.

Detection results for 81 stool samples

No, Floor Age Colonoscopy Ct BMP3 NDRG4 Preferred Primers and Probes Comparative primers and probes Preferred Primers and Probes Comparative primers and probes 1 2 3 1 2 3 1 2 3 1 2 3 1 Man 50 RPOC - stage I 33.9 32.1 37.5 32.7 35.8 36.2 U 33.6 35 33.9 38.8 40.1 2 Man 51 RPOC stage II 34.9 32.2 29.1 41.3 37.6 39.9 39.4 38.4 33.8 34 38.1 40.3 3 Woman 56 RPOC stage III 35.1 33.1 31.4 34.9 41.2 35.1 30.7 30.4 33.2 34.2 42.6 35 4 Man 72 RPOC stage I 35.7 U U 33.4 40.1 36 37.2 38.9 U U U 36.7 5 Woman 75 RPOC stage II 31.9 36.6 32.7 37.9 36.3 34.5 35.4 37.4 34.5 36.4 33 40.5 6 Woman 55 RPOC stage II 37.5 35.1 29.5 43 39.8 39.7 34.4 35.9 35.6 35.1 37.4 42 7 Woman 50 RPOC stage II 36.2 32.2 38.8 39.7 37.2 37.3 33.6 37.4 38.8 42.3 36.4 37.5 8 Man 62 RPOC stage Ⅳ U U U U U U 36.1 34.5 39.9 41 41.8 40.4 9 Woman 74 RPOC stage I 35.7 37.7 29.3 38 36 44 29.6 32.3 36.6 42.6 36.8 33 10 Woman 73 RPOC stage II 31.7 29.7 33.9 32.6 42 32.1 36.5 35.7 39.7 39.3 33.5 31.8 eleven Man 52 RPOC stage II 34 40 35.9 39.5 38.1 42.4 36.9 U 36.5 42.6 37.8 36.6 12 Woman 57 RPOC stage II 34.5 29.1 29.3 34.6 42.6 41.3 37.7 31.8 35.6 42.3 39 42.6 13 Man 51 RPOC stage I 37.6 34 33.2 38.8 U U 31.9 35.4 35.9 39.1 35.1 34.6 14 Man 60 RPOC stage II 36.2 31.7 35.6 40.1 42.2 33.1 29.6 31.7 29.1 33.8 36.4 36.5 15 Woman 49 RPOC stage III 31.1 37 29.4 41.9 35.8 34.3 34.4 33.6 35.6 40.3 39.2 39 16 Man 49 RPOC stage IV 34 35.5 U 41.8 35.9 37.5 37.6 32.3 32.3 38.1 U 31.3 17 Man 48 RPOC stage I 38.1 37.1 35.8 39 40.7 38.3 29.4 30.7 U U 37.1 U 18 Man 54 RPOC stage II 36.1 33.7 35.2 U U U 29.4 37 33.5 34.7 34 41.5 19 Woman 60 RPOC stage III 29.2 36.4 33.6 38.4 36.5 33.7 32.9 30.1 35.1 33.5 38.5 41.3 20 Woman 62 RPOC stage IV 36.4 34.7 38.5 39.5 37.9 38.9 36.4 29.4 33.1 35.8 43.1 42.8 21 Woman 50 PA 38.7 U U 35.3 U U 34.6 37 U U U 40.9 22 Woman 56 PA U 35.6 thirty 40.9 38.6 35 34.6 U 29.3 42.7 33.4 U 23 Man 58 PA U U 29.6 39.3 38 41.1 39.3 35.1 39.5 39.4 U 37.4 24 Man 67 PA 36.3 32.8 34.2 U U U 33.9 33 32.7 36 35.5 U 25 Man 51 PA 35.9 33.7 33.9 36.4 38 39 32.7 33.9 U U 35.8 40.4 26 Woman 71 PA 29.3 thirty 35.4 40.3 37.7 34.4 30.7 34.9 38.3 33.8 41.1 33 27 Man 65 PA U 30.3 U U U U 34.8 34 36.8 41.9 42.7 36.1 28 Man 62 PA 37.5 29.2 U U U U U U 36.8 43.1 43.9 32.8 29 Woman 45 PA 31 37.6 37.4 37.2 38.5 37.8 30.8 31.7 30.8 U U U thirty Man 67 PA 29.7 34.3 29.8 31.3 U U 33.9 U U U U U 31 Woman 53 PA 36.3 U U U 35.4 34.6 29.5 34.9 37.7 39.6 40 42.9 32 Woman 68 PA 38.4 37.3 30.4 U U U U 37.2 U U U 35.1 33 Woman 50 PA 36.9 39 32.1 36.6 U U 39.5 29.1 30.7 37.1 37.6 36.6 34 Woman 48 PA U U U U 37.3 35.8 38.3 32.2 32.4 36.2 36 43.9 35 Man 50 PA 35.5 29.1 32.7 39.4 42.7 U 29.8 33.8 32.9 36.4 38.2 42 36 Man 45 Norm U U U U U U U U U U U U 37 Woman 50 Norm U U U U U U U U U U U U 38 Man 59 Norm U U U U U U U U U U U U 39 Man 73 Norm U U U U U U U U U U U U 40 Man 47 Norm U U U U U U U U U U U U 41 Man 71 Norm U U U U U U U U U U U U 42 Woman 45 Norm U U U U U U U U U U U U 43 Woman 62 Norm U U U U U U U U U U U U 44 Man 55 Norm U U U U U U U U U U U U 45 Woman 54 Norm U U U U U U U U U U U U 46 Man 63 Norm U U U 39.4 U U U U U U U U 47 Woman 53 Norm U U U U U U U U U 41.2 U U 48 Man 67 Norm U U U U 37.2 41.2 U U U U U U 49 Man 69 Norm U U U U U U U U U U U U 50 Man 54 Norm U U U U U U U U U U U U 51 Man 50 Norm U U U U U U U U U U U U 52 Woman 65 Norm U U U U U U U U U U 40.3 41 53 Woman 56 Norm U U U U U U U U U U U U 54 Man 67 Norm U 35.6 U U 41.3 U U U U U U U 55 Man 70 Norm U U U U U U U U U U U U 56 Woman 75 Norm U U U U U U U U U U U U 57 Woman 50 Norm U U U U U U U U U U U U 58 Woman 66 Norm U U U U U U U U U U U U 59 Man 49 Norm U U U U U 41.6 U U U U U U 60 Man 71 Norm U U U 40.8 U U U U U U U U 61 Man 70 Norm U U U U U U U U U U 40.5 U 62 Man 73 Norm U U U U U U U U U U U U 63 Man 48 Norm U U U U 38.5 U U U U U U U 64 Man 52 Norm U U U U U U U U U U U 39.6 65 Man 71 Norm U U U U U U U U U U U U 66 Woman 60 Norm U U U U U 41.9 U U 35.9 39.4 U U 67 Woman 65 Norm U U U U U U U U U U U U 68 Woman 74 Norm U U U U U U U U U U U U 69 Man 71 Norm U U U U U U U U U U U U 70 Woman 73 Norm U U U U U U U U U U U U 71 Man 49 Norm U U U U U U U U U U U U 72 Woman 69 Norm U U U U U U U U U U U U 73 Man 59 Norm U U U 42.7 41.9 42.5 U U U U U U 74 Man 71 Norm U U 37.1 U U U U U U U U U 75 Woman 54 Norm U U U U U U U U U U 38.9 35.2 76 Man 71 Norm U U U U U U U U U U U U 77 Woman 63 Norm U U U U U U U U U U U U 78 Man 74 Norm U U U U U U U U U U U U 79 Man 62 Norm U U U U U U U U U U U U 80 Woman 75 Norm U U U U U U U U U U U U 81 Man 48 Norm U U U U U U U U U U U U

Notes: U: not determined.

Example 5

Screening of combinations of primers and probes for methylation of the BMP3 and DNRG4 genes

(1) DNA extraction from feces

[00200] DNA was extracted from stool according to the protocol of Example 3.

(2) Determination of methylation of BMP3 and NDRG4 genes

[00201] Primer and probe combinations for the BMP3 and NDRG4 genes are shown below. qPCR was carried out with nine combinations, respectively, as described in the steps of Example 3.

Table 19. Primer and probe combinations

Combination No. BMP3 NDRG4 1 Preferably 3 Preferably 1 2 Preferably 3 Preferably 2 3 Preferably 3 Preferably 3 4 Preferably 1 Preferably 1 5 Preferably 1 Preferably 2 6 Preferably 1 Preferably 3 7 Preferably 2 Preferably 1 8 Preferably 2 Preferably 2 9 Preferably 2 Preferably 3

The sequences of primers and probes for nine combinations of BMP3 and NDRG4 genes are presented below:

Table 20. Sequences of primers and probes in combinations

Combination No. Gene Group Primer/probe ID sequence Sequence （5' - 3'） 1 BMP3 Preferably 3 Direct primer SEQ ID NO.:15 AATATTCGGGTTATATACGTCGCGATT Reverse primer SEQ ID NO.:16 ACTTACTACGCTAACCCAACG Probe SEQ ID NO.:17 TAGCGTTGGAGTGGAGACGGCGTTCGTA NDRG4 Preferably 1 Direct primer SEQ ID NO.:6 ATCGATCGGGGTGTTTTTTAGGTTTC Reverse primer SEQ ID NO.:7 CCTTCTACGCGACTAAAATACCCGAT Probe SEQ ID NO.:8 CGTCGCGGTTTTCGTTCGTTTTTTCGTTCGT 2 BMP3 Preferably 3 Direct primer SEQ ID NO.:15 AATATTCGGGTTATATACGTCGCGATT Reverse primer SEQ ID NO.:16 ACTTACTACGCTAACCCAACG Probe SEQ ID NO.:17 TAGCGTTGGAGTGGAGACGGCGTTCGTA NDRG4 Preferably 2 Direct primer SEQ ID NO.:12 GCGGGTGAGAAGTCGGC Reverse primer SEQ ID NO.:13 GTAACTTCCGCCTTCTACGC Probe SEQ ID NO.:14 TAGGTTTCGCGTCGCGGTTTTCGTT 3 BMP3 Preferably 3 Direct primer SEQ ID NO.:15 AATATTCGGGTTATATACGTCGCGATT Reverse primer SEQ ID NO.:16 ACTTACTACGCTAACCCAACG Probe SEQ ID NO.:17 TAGCGTTGGAGTGGAGACGGCGTTCGTA NDRG4 Preferably 3 Direct primer SEQ ID NO.:18 CGGTTTTCGTTCGTTTTTTCG Reverse primer SEQ ID NO.:19 AACCTAAAACTAATCCCGAACGAACC Probe SEQ ID NO.:20 TCGTTTATCGGGTATTTTAGTCGCGTAG 4 BMP3 Preferably 1 Direct primer SEQ ID NO.:3 TTTGAAAATATTCGGGTTATATACGTCGC Reverse primer SEQ ID NO.:4 ATAAACTCTTCCCCAACAACTACGCGAA Probe SEQ ID NO.:5 AGCGTTGGAGTGGAGACGGCGTTCG NDRG4 Preferably 1 Direct primer SEQ ID NO.:6 ATCGATCGGGGTGTTTTTTAGGTTTC Reverse primer SEQ ID NO.:7 CCTTCTACGCGACTAAAATACCCGAT Probe SEQ ID NO.:8 CGTCGCGGTTTTCGTTCGTTTTTTCGTTCGT 5 BMP3 Preferably 1 Direct primer SEQ ID NO.:3 TTTGAAAATATTCGGGTTATATACGTCGC Reverse primer SEQ ID NO.:4 ATAAACTCTTCCCCAACAACTACGCGAA Probe SEQ ID NO.:5 AGCGTTGGAGTGGAGACGGCGTTCG NDRG4 Preferably 2 Direct primer SEQ ID NO.:12 GCGGGTGAGAAGTCGGC Reverse primer SEQ ID NO.:13 GTAACTTCCGCCTTCTACGC Probe SEQ ID NO.:14 TAGGTTTCGCGTCGCGGTTTTCGTT 6 BMP3 Preferably 1 Direct primer SEQ ID NO.:3 TTTGAAAATATTCGGGTTATATACGTCGC Reverse primer SEQ ID NO.:4 ATAAACTCTTCCCCAACAACTACGCGAA Probe SEQ ID NO.:5 AGCGTTGGAGTGGAGACGGCGTTCG NDRG4 Preferably 3 Direct primer SEQ ID NO.:18 CGGTTTTCGTTCGTTTTTTCG Reverse primer SEQ ID NO.:19 AACCTAAAACTAATCCCGAACGAACC Probe SEQ ID NO.:20 TCGTTTATCGGGTATTTTAGTCGCGTAG 7 BMP3 Preferably 2 Direct primer SEQ ID NO.:9 AATATTCGGGTTATATACGTCGCGA Reverse primer SEQ ID NO.:10 GCAACCTAAACAAATAAACTCTTCCCCAA Probe SEQ ID NO.:11 TGGAGTGGAGACGGCGTTCGTAGCGT NDRG4 Preferably 1 Direct primer SEQ ID NO.:6 ATCGATCGGGGTGTTTTTTAGGTTTC Reverse primer SEQ ID NO.:7 CCTTCTACGCGACTAAAATACCCGAT Probe SEQ ID NO.:8 CGTCGCGGTTTTCGTTCGTTTTTTCGTTCGT 8 BMP3 Preferably 2 Direct primer SEQ ID NO.:9 AATATTCGGGTTATATACGTCGCGA Reverse primer SEQ ID NO.:10 GCAACCTAAACAAATAAACTCTTCCCCAA Probe SEQ ID NO.:11 TGGAGTGGAGACGGCGTTCGTAGCGT NDRG4 Preferably 2 Direct primer SEQ ID NO.:12 GCGGGTGAGAAGTCGGC Reverse primer SEQ ID NO.:13 GTAACTTCCGCCTTCTACGC Probe SEQ ID NO.:14 TAGGTTTCGCGTCGCGGTTTTCGTT 9 BMP3 Preferably 2 Direct primer SEQ ID NO.:9 AATATTCGGGTTATATACGTCGCGA Reverse primer SEQ ID NO.:10 GCAACCTAAACAAATAAACTCTTCCCCAA Probe SEQ ID NO.:11 TGGAGTGGAGACGGCGTTCGTAGCGT NDRG4 Preferably 3 Direct primer SEQ ID NO.:18 CGGTTTTCGTTCGTTTTTTCG Reverse primer SEQ ID NO.:19 AACCTAAAACTAATCCCGAACGAACC Probe SEQ ID NO.:20 TCGTTTATCGGGTATTTTAGTCGCGTAG

[00202] The QPCR Master Mix used in the reaction is shown below:

Table 21. Master mix for quantitative PCR for BMP3/NDRG4

No. Reagents Final concentration 1 10× PCR buffer 1× 2 _MgCl2 2 mm 3 dNTP 0.2 mM 4 DNA polymerase Taq 2 units 5 Each primer 0.75 mM 6 Each probe 0.25 mM 7 DNA matrix 2 µl 8 Ultrapure water up to 50 µl

[00203] The qPCR reaction conditions are one cycle of denaturation at 95°C for 2 minutes and 50 cycles of denaturation at 95°C for 20 seconds and extension at 60°C for 1 minute.

(3) Detection of KRAS gene variants

[00204] Seven mutation hotspots were detected at codons 12 and 13 of the KRAS gene. The seven mutants are G12D, G13D, G12V, G12C, G12S, G12A, and G13R, and the primer and probe sequences are shown in Table 22.

Table 22. Primers and probes for detecting seven mutations of the KRAS gene

Primer name Primer/Probe ID sequence Sequence (5' - 3') G12D-F Direct primer SEQ ID NO.:35 AACTTGTGGTAGTTGGAGGTGA G13D-F Direct primer SEQ ID NO.:36 AACTTGTGGTAGTTGGAGCTGGGGGA G12V-F Direct primer SEQ ID NO.:37 AACTTGTGGTAGTTGGAGTTGT G12C-F Direct primer SEQ ID NO.:38 AAACTTGTGGTAGTTGGGGCTT G12S-F Direct primer SEQ ID NO.:39 AAACTTGTGGTAGTTGGTGCTA G12A-F Direct primer SEQ ID NO.:40 AACTTGTGGTAGTTGGAGCAGC G12R-F Direct primer SEQ ID NO.:41 AAACTTGTGGTAGTTGGAGCTC Kras-R Reverse primer SEQ ID NO.:42 GAATGGTCCTGCACCAGTAATATG ACTB-F Direct primer SEQ ID NO.:43 AGGGCTTCTTGTCCTTTCCTT ACTB-R Reverse primer SEQ ID NO.:44 CGTGCTCGATGGGGTACTTC KRAS-P Probe SEQ ID NO.:45 AGGCAAGAGTGCCTTGACGATACAGC ACTB-P Probe SEQ ID NO.:46 CGTGATGGTGGGCATGGGTCAGAAGGA

The QPCR Master Mix used in the reaction is presented below:

Table 23. Master mix for KRAS quantitative PCR

No. Reagents Final concentration 1 G12A-F 0.72 µM 2 G12C-F 0.60 µM 3 G12D-F 0.72 µM 4 G12R-F 0.48 µM 5 G12S-F 0.90 µM 6 G12V-F 0.72 µM 7 G13D-F 0.48 µM 8 Kras-R 0.90 µM 9 Kras-P 0.10 µM 10 ACTB-F 0.30 µM eleven ACTB-R 0.30 µM 12 ACTB-P 0.05 µM 13 5× PCR buffer, -Mg ²⁺ 1× 14 _MgCl2 1.0 mM 15 dNTP 0.1 mM 16 DNA polymerase Taq 2.5 units 17 DNA matrix 2 µl 18 Ultrapure water Up to 50 µl

[00205] The qPCR reaction conditions are one cycle of denaturation at 95°C for 5 minutes and 45 cycles of denaturation at 95°C for 15 seconds, annealing at 71°C for 60 seconds, and then extension at 55° C for 50 seconds.

[00206] Quantitative PCR for quality control was performed using ACTB as the control gene.

(4) Test for hemoglobin in stool

[00207] Fecal hemoglobin was determined using a fecal immunochemical test (FIT), and the result was either positive or negative.

(5) Calculation of quantities using the formula

[00208] The Ct value for detection of the BMP3, NDRG4 and KRAS genes by qPCR, as well as the positive or negative result of the fecal hemoglobin test, was entered into the logistic regression formula as shown below:

P=e ^K /(1+e ^K )

[00209] where: P means general index,

K=a*ΔCt1+b*ΔCt2+c*ΔCt3+d*FIT+X, where e means natural constant, a, b, c, d, X mean clinical constants. ΔCt1, ΔCt2 and ΔCt3 represent the Ct value of target genes subtracted from the values of control genes.

[00210] The result of the analysis is positive if the P value is equal to or greater than a predetermined threshold, and otherwise the result is negative. A positive result indicates that the individual may be diagnosed with POC or PA.

(6) Analysis result

[00211] Eighty-one stool samples from Example 3 were analyzed, and the results of various primer and probe combinations for the BMP3 and NDRG4 genes are presented in Table 24.

Table 24. Results of analysis of 81 stool samples

Combination No. BMP3 NDRG4 Sensitivity
(RPOC+PA) Specificity
(RPOC+PA) Number of samples positive for RPOC Number of samples positive for PA Number of samples assessed as negative in a healthy person Sensitivity
(RPOC) Sensitivity
(PA) 1 Preferably 3 Preferably 1 94.3% 97.8% 19/20 14/15 45/46 95.0% 93.3% 2 Preferably 3 Preferably 2 91.4% 97.8% 18/20 14/15 45/46 90.0% 93.3% 3 Preferably 3 Preferably 3 80.0% 95.6% 16/20 12/15 44/46 80.0% 80.0% 4 Preferably 1 Preferably 1 97.1% 100.0% 20/20 14/15 46/46 100.0% 93.3% 5 Preferably 1 Preferably 2 97.1% 97.8% 20/20 14/15 45/46 100.0% 93.3% 6 Preferably 1 Preferably 3 88.6% 95.6% 19/20 12/15 44/46 95.0% 80.0% 7 Preferably 2 Preferably 1 97.1% 97.8% 19/20 15/15 45/46 95.0% 100.0% 8 Preferably 2 Preferably 2 94.3% 97.8% 19/20 14/15 45/46 95.0% 93.3% 9 Preferably 2 Preferably 3 85.7% 95.6% 19/20 11/15 44/46 95.0% 73.3%

[00212] It is obvious that: (1) Combinations Nos. 4, 5 and 7 are superior to the other six combinations. Considering that all primers and probes tested are preferred and superior to other primers and probes (see Example 3), these three specific combinations are superior to any known BMP3 and NDRG4 primer/probe combinations. (2) The sensitivity and specificity of the BMP3, NDRG4, KRAS and fecal hemoglobin detection kits for POC and PA are significantly better than the sensitivity and specificity of methylation detection of a single BMP3 or NDRG4 gene. (3) The sensitivity and specificity of the kit for diagnosing POC in Asian populations (eg, Chinese populations) is clearly superior to that of existing similar products such as Cologuard®. (4) The sensitivity and specificity of the kit for diagnosing PsA in Asian populations (eg, Chinese populations) is significantly better than the sensitivity and specificity of existing similar products such as Cologuard®.

[00213] The description of the invention, including the claims, figures and/or drawings, of each of the patents, patent applications and publications cited herein is incorporated herein in its entirety by reference.

[00214] Unless otherwise specified, all technical and scientific terms used in this application have the generally accepted meaning known to those skilled in the field to which the invention relates. Although any methods and materials similar or equivalent to those described herein may be used to practice the present invention or to perform tests according to the invention, the methods and materials described herein are preferred. All publications, patents, and patent applications cited herein are incorporated herein for all purposes and in their entirety by reference.

[00215] The publications discussed herein are presented solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that the inventors of the present invention are not entitled to predate such publication by reason of a prior invention.

[00216] Although the present invention has been described in combination with its specific embodiments, it should be noted that further modifications may be made thereto, and this application covers any variations, applications or adaptations of the invention substantially in accordance with the principles of the invention, and includes such deviations from the disclosure of the invention as are within known or common practice in the field of technology to which the invention relates and which can be applied to the essential features set forth above and below within the scope of the appended claims.

--->

LIST OF SEQUENCES

<110> Hangzhou New Horizon Health Technology Co. Ltd.

Li, Hui

Li, Cunyao

Zheng, Weixian

Yang, Jiao

Liu, Gang

Lv, Ning

Chen, Yiyou

<120> COLON CANCER SCREENING KIT

PROGRESSIVE ADENOMA AND ITS APPLICATION

<130> NEWH-017/01WO 333709-2028

<150>CN 201810502387.9

<151> 2018-05-23

<150>CN 201810502359.7

<151> 2018-05-23

<160> 70

<170> Patent In version 3.5

<210> 1

<211> 271

<212> DNA

<213> Homo sapiens

<220>

<221> modified_base

<222> (12)..(12)

<223> methylated

<220>

<221> modified_base

<222> (33)..(33)

<223> methylated

<220>

<221> modified_base

<222> (69)..(69)

<223> methylated

<220>

<221> modified_base

<222> (80)..(80)

<223> methylated

<220>

<221> modified_base

<222> (83)..(83)

<223> methylated

<220>

<221> modified_base

<222> (85)..(85)

<223> methylated

<220>

<221> modified_base

<222> (106)..(106)

<223> methylated

<220>

<221> modified_base

<222> (120)..(120)

<223> methylated

<220>

<221> modified_base

<222> (123)..(123)

<223> methylated

<220>

<221> modified_base

<222> (127)..(127)

<223> methylated

<220>

<221> modified_base

<222> (132)..(132)

<223> methylated

<220>

<221> modified_base

<222> (139)..(139)

<223> methylated

<220>

<221> modified_base

<222> (141)..(141)

<223> methylated

<220>

<221> modified_base

<222> (152)..(152)

<223> methylated

<220>

<221> modified_base

<222> (154)..(154)

<223> methylated

<220>

<221> modified_base

<222> (189)..(189)

<223> methylated

<220>

<221> modified_base

<222> (200)..(200)

<223> methylated

<220>

<221> modified_base

<222> (219)..(219)

<223> methylated

<220>

<221> modified_base

<222> (227)..(227)

<223> methylated

<220>

<221> modified_base

<222> (236)..(236)

<223> methylated

<220>

<221> modified_base

<222> (240)..(240)

<223> methylated

<220>

<221> modified_base

<222> (242)..(242)

<223> methylated

<220>

<221> modified_base

<222> (247)..(247)

<223> methylated

<220>

<221> modified_base

<222> (254)..(254)

<223> methylated

<220>

<221> modified_base

<222> (258)..(258)

<223> methylated

<220>

<221> modified_base

<222> (264)..(264)

<223> methylated

<400> 1

gttagtttgg tcgggtgttt ttaaaaataa agcgaggagg gaaggtatag atagattttg 60

aaaatattcg ggttatatac gtcgcgattt atagtttttt tttagcgttg gagtggagac 120

ggcgttcgta gcgttttgcg cgggtgaggt tcgcgtagtt gttggggaag agtttatttg 180

ttaggttgcg ttgggttagc gtagtaagtg gggttggtcg ttatttcgtt gtattcggtc 240

gcgtttcggg tttcgtgcgt tttcgtttta g 271

<210> 2

<211> 236

<212> DNA

<213> Homo sapiens

<220>

<221> modified_base

<222> (9)..(9)

<223> methylated

<220>

<221> modified_base

<222> (12)..(12)

<223> methylated

<220>

<221> modified_base

<222> (18)..(18)

<223> methylated

<220>

<221> modified_base

<222> (20)..(20)

<223> methylated

<220>

<221> modified_base

<222> (25)..(25)

<223> methylated

<220>

<221> modified_base

<222> (29)..(29)

<223> methylated

<220>

<221> modified_base

<222> (48)..(48)

<223> methylated

<220>

<221> modified_base

<222> (50)..(50)

<223> methylated

<220>

<221> modified_base

<222> (53)..(53)

<223> methylated

<220>

<221> modified_base

<222> (55)..(55)

<223> methylated

<220>

<221> modified_base

<222> (62)..(62)

<223> methylated

<220>

<221> modified_base

<222> (66)..(66)

<223> methylated

<220>

<221> modified_base

<222> (74)..(74)

<223> methylated

<220>

<221> modified_base

<222> (78)..(78)

<223> methylated

<220>

<221> modified_base

<222> (85)..(85)

<223> methylated

<220>

<221> modified_base

<222> (98)..(98)

<223> methylated

<220>

<221> modified_base

<222> (100)..(100)

<223> methylated

<220>

<221> modified_base

<222> (109)..(109)

<223> methylated

<220>

<221> modified_base

<222> (118)..(118)

<223> methylated

<220>

<221> modified_base

<222> (120)..(120)

<223> methylated

<220>

<221> modified_base

<222> (122)..(122)

<223> methylated

<220>

<221> modified_base

<222> (130)..(130)

<223> methylated

<220>

<221> modified_base

<222> (132)..(132)

<223> methylated

<220>

<221> modified_base

<222> (137)..(137)

<223> methylated

<220>

<221> modified_base

<222> (141)..(141)

<223> methylated

<220>

<221> modified_base

<222> (159)..(159)

<223> methylated

<220>

<221> modified_base

<222> (165)..(165)

<223> methylated

<220>

<221> modified_base

<222> (170)..(170)

<223> methylated

<220>

<221> modified_base

<222> (172)..(172)

<223> methylated

<220>

<221> modified_base

<222> (177)..(177)

<223> methylated

<220>

<221> modified_base

<222> (181)..(181)

<223> methylated

<220>

<221> modified_base

<222> (191)..(191)

<223> methylated

<220>

<221> modified_base

<222> (202)..(202)

<223> methylated

<220>

<221> modified_base

<222> (204)..(204)

<223> methylated

<220>

<221> modified_base

<222> (210)..(210)

<223> methylated

<220>

<221> modified_base

<222> (216)..(216)

<223> methylated

<220>

<221> modified_base

<222> (218)..(218)

<223> methylated

<220>

<221> modified_base

<222> (221)..(221)

<223> methylated

<220>

<221> modified_base

<222> (226)..(226)

<223> methylated

<400> 2

tgagaagtcg gcggggggcgc ggatcgatcg gggtgttttt taggtttcgc gtcgcggttt 60

tcgttcgttt tttcgttcgt ttatcgggta ttttagtcgc gtagaaggcg gaagttacgc 120

gcgagggatc gcggttcgtt cgggattagt tttaggttcg gtatcgtttc gcgggtcgag 180

cgtttatatt cgttaaattt acgcgggtac gttttcgcgg cgtatcgttt ttagtt 236

<210> 3

<211> 29

<212> DNA

<213> Homo sapiens

<400> 3

tttgaaaata ttcggggttat atacgtcgc 29

<210> 4

<211> 28

<212> DNA

<213> Homo sapiens

<400> 4

ataaactctt ccccaacaac tacgcgaa 28

<210> 5

<211> 25

<212> DNA

<213> Homo sapiens

<400> 5

agcgttggag tggagacggc gttcg 25

<210> 6

<211> 26

<212> DNA

<213> Homo sapiens

<400> 6

atcgatcggg gtgtttttta ggtttc 26

<210> 7

<211> 26

<212> DNA

<213> Homo sapiens

<400> 7

ccttctacgc gactaaaata cccgat 26

<210> 8

<211> 31

<212> DNA

<213> Homo sapiens

<400> 8

cgtcgcggtt ttcgttcgtt ttttcgttcg t 31

<210> 9

<211> 25

<212> DNA

<213> Homo sapiens

<400> 9

aatattcggg ttatatacgt cgcga 25

<210> 10

<211> 28

<212> DNA

<213> Homo sapiens

<400> 10

gcaacctaac aaataaactc ttccccaa 28

<210> 11

<211> 26

<212> DNA

<213> Homo sapiens

<400> 11

tggagtggag acggcgttcg tagcgt 26

<210> 12

<211> 17

<212> DNA

<213> Homo sapiens

<400> 12

gcgggtgaga agtcggc 17

<210> 13

<211> 20

<212> DNA

<213> Homo sapiens

<400> 13

gtaacttccg ccttctacgc 20

<210> 14

<211> 25

<212> DNA

<213> Homo sapiens

<400> 14

taggtttcgc gtcgcggttt tcgtt 25

<210> 15

<211> 27

<212> DNA

<213> Homo sapiens

<400> 15

aatattcggg ttatatacgt cgcgatt 27

<210> 16

<211> 21

<212> DNA

<213> Homo sapiens

<400> 16

acttactacg ctaacccaac g 21

<210> 17

<211> 28

<212> DNA

<213> Homo sapiens

<400> 17

tagcgttgga gtggagacgg cgttcgta 28

<210> 18

<211> 21

<212> DNA

<213> Homo sapiens

<400> 18

cggttttcgt tcgttttttc g 21

<210> 19

<211> 26

<212> DNA

<213> Homo sapiens

<400> 19

aacctaaaac taatcccgaa cgaacc 26

<210> 20

<211> 28

<212> DNA

<213> Homo sapiens

<400> 20

tcgtttatcg ggtattttag tcgcgtag 28

<210> 21

<211> 20

<212> DNA

<213> Homo sapiens

<400> 21

gagtggagac ggcgttcgta 20

<210> 22

<211> 23

<212> DNA

<213> Homo sapiens

<400> 22

ccacttacta cgctaaccca acg 23

<210> 23

<211> 26

<212> DNA

<213> Homo sapiens

<400> 23

cgggtgaggt tcgcgtagtt gttggg 26

<210> 24

<211> 23

<212> DNA

<213> Homo sapiens

<400> 24

gggtgttttt taggtttcgc gtc 23

<210> 25

<211> 21

<212> DNA

<213> Homo sapiens

<400> 25

cgtaacttcc gccttctacg c 21

<210> 26

<211> 40

<212> DNA

<213> Homo sapiens

<400> 26

acgcgactaa aatacccgat aaacgaacga aaaaacgaac 40

<210> 27

<211> 21

<212> DNA

<213> Homo sapiens

<400> 27

ttaggttgcg ttggggttagc g 21

<210> 28

<211> 22

<212> DNA

<213> Homo sapiens

<400> 28

actccgaaaa cgcaaaaaac cg 22

<210> 29

<211> 26

<212> DNA

<213> Homo sapiens

<400> 29

attcggtcgc gtttcgggtt tcgtgc 26

<210> 30

<211> 19

<212> DNA

<213> Homo sapiens

<400> 30

gacccgcgaa acgataccg 19

<210> 31

<211> 21

<212> DNA

<213> Homo sapiens

<400> 31

tattcgggtt atatacgtcg from 21

<210> 32

<211> 20

<212> DNA

<213> Homo sapiens

<400> 32

cttactacgc taacccaacg 20

<210> 33

<211> 26

<212> DNA

<213> Homo sapiens

<400> 33

cccaacaact acgcgaacct cacccg 26

<210> 34

<211> 20

<212> DNA

<213> Homo sapiens

<400> 34

tcgcgcgtaa cttccgcctt 20

<210> 35

<211> 22

<212> DNA

<213> Homo sapiens

<400> 35

aacttgtggt agttggaggt ga 22

<210> 36

<211> 25

<212> DNA

<213> Homo sapiens

<400> 36

aacttgtggt agttggagct gggga 25

<210> 37

<211> 22

<212> DNA

<213> Homo sapiens

<400> 37

aacttgtggt agttggagtt gt 22

<210> 38

<211> 22

<212> DNA

<213> Homo sapiens

<400> 38

aaacttgtgg tagttggggc tt 22

<210> 39

<211> 22

<212> DNA

<213> Homo sapiens

<400> 39

aaacttgtgg tagttggtgc ta 22

<210> 40

<211> 22

<212> DNA

<213> Homo sapiens

<400> 40

aacttgtggt agttggagca gc 22

<210> 41

<211> 22

<212> DNA

<213> Homo sapiens

<400> 41

aaacttgtgg tagttggagc tc 22

<210> 42

<211> 24

<212> DNA

<213> Homo sapiens

<400> 42

gaatggtcct gcaccagtaa tatg 24

<210> 43

<211> 21

<212> DNA

<213> Homo sapiens

<400> 43

agggcttctt gtcctttcct t 21

<210> 44

<211> 20

<212> DNA

<213> Homo sapiens

<400> 44

cgtgctcgat ggggtacttc 20

<210> 45

<211> 26

<212> DNA

<213> Homo sapiens

<400> 45

aggcaagagt gccttgacga tacagc 26

<210> 46

<211> 27

<212> DNA

<213> Homo sapiens

<400> 46

cgtgatggtg ggcatgggtc agaagga 27

<210> 47

<211> 20

<212> DNA

<213> Homo sapiens

<400> 47

agtttggtgt aagttaagag 20

<210> 48

<211> 21

<212> DNA

<213> Homo sapiens

<400> 48

ctaactctat tttaaacrcc a 21

<210> 49

<211> 23

<212> DNA

<213> Homo sapiens

<400> 49

gttttaattt ttggaaaagg taa 23

<210> 50

<211> 21

<212> DNA

<213> Homo sapiens

<400> 50

acctaacaaa taaactcttc from 21

<210> 51

<211> 22

<212> DNA

<213> Homo sapiens

<400> 51

gaaggtatag atagattttg aa 22

<210> 52

<211> 24

<212> DNA

<213> Homo sapiens

<400> 52

cacctaacac aactttacra aact 24

<210> 53

<211> 26

<212> DNA

<213> Homo sapiens

<400> 53

gtatttagtt atggttgggg ygagta 26

<210> 54

<211> 21

<212> DNA

<213> Homo sapiens

<400> 54

ctcacctact actaccgccc r 21

<210> 55

<211> 25

<212> DNA

<213> Homo sapiens

<400> 55

aggtttttga gtttttggtt ttttt 25

<210> 56

<211> 20

<212> DNA

<213> Homo sapiens

<400> 56

ccctccaaac cccctataac 20

<210> 57

<211> 20

<212> DNA

<213> Homo sapiens

<400> 57

ggatggggat gtttttgtag 20

<210> 58

<211> 20

<212> DNA

<213> Homo sapiens

<400> 58

rgrgaaacct aaaaaacacc 20

<210> 59

<211> 18

<212> DNA

<213> Homo sapiens

<400> 59

gyggagyggg tgagaagt 18

<210> 60

<211> 20

<212> DNA

<213> Homo sapiens

<400> 60

craacaacca aaaacccctc 20

<210> 61

<211> 23

<212> DNA

<213> Homo sapiens

<400> 61

gttygttygg gattagtttt agg 23

<210> 62

<211> 19

<212> DNA

<213> Homo sapiens

<400> 62

crcaaacraa aaacraaac 19

<210> 63

<211> 18

<212> DNA

<213> Homo sapiens

<400> 63

gyggygtttt ygtttttg 18

<210> 64

<211> 20

<212> DNA

<213> Homo sapiens

<400> 64

cracractaa aaatccccaa 20

<210> 65

<211> 271

<212> DNA

<213> Homo sapiens

<220>

<221> modified_base

<222> (12)..(12)

<223> methylated

<220>

<221> modified_base

<222> (33)..(33)

<223> methylated

<220>

<221> modified_base

<222> (69)..(69)

<223> methylated

<220>

<221> modified_base

<222> (80)..(80)

<223> methylated

<220>

<221> modified_base

<222> (83)..(83)

<223> methylated

<220>

<221> modified_base

<222> (85)..(85)

<223> methylated

<220>

<221> modified_base

<222> (106)..(106)

<223> methylated

<220>

<221> modified_base

<222> (120)..(120)

<223> methylated

<220>

<221> modified_base

<222> (123)..(123)

<223> methylated

<220>

<221> modified_base

<222> (132)..(132)

<223> methylated

<220>

<221> modified_base

<222> (139)..(139)

<223> methylated

<220>

<221> modified_base

<222> (141)..(141)

<223> methylated

<220>

<221> modified_base

<222> (152)..(152)

<223> methylated

<220>

<221> modified_base

<222> (154)..(154)

<223> methylated

<220>

<221> modified_base

<222> (189)..(189)

<223> methylated

<220>

<221> modified_base

<222> (200)..(200)

<223> methylated

<220>

<221> modified_base

<222> (219)..(219)

<223> methylated

<220>

<221> modified_base

<222> (227)..(227)

<223> methylated

<220>

<221> modified_base

<222> (240)..(240)

<223> methylated

<220>

<221> modified_base

<222> (242)..(242)

<223> methylated

<220>

<221> modified_base

<222> (247)..(247)

<223> methylated

<220>

<221> modified_base

<222> (254)..(254)

<223> methylated

<220>

<221> modified_base

<222> (258)..(258)

<223> methylated

<220>

<221> modified_base

<222> (264)..(264)

<223> methylated

<400> 65

gccagtttgg ccgggtgttc ccaaaaataa agcgaggagg gaaggtacag acagatcttg 60

aaaacacccg ggccacacac gccgcgacct acagctcttt ctcagcgttg gagtggagac 120

ggcgcccgca gcgccctgcg cgggtgaggt ccgcgcagct gctggggaag agcccacctg 180

tcaggctgcg ctgggtcagc gcagcaagtg gggctggccg ctatctcgct gcacccggcc 240

gcgtcccggg ctccgtgcgc cctcgcccca g 271

<210> 66

<211> 236

<212> DNA

<213> Homo sapiens

<220>

<221> modified_base

<222> (9)..(9)

<223> methylated

<220>

<221> modified_base

<222> (12)..(12)

<223> methylated

<220>

<221> modified_base

<222> (18)..(18)

<223> methylated

<220>

<221> modified_base

<222> (20)..(20)

<223> methylated

<220>

<221> modified_base

<222> (25)..(25)

<223> methylated

<220>

<221> modified_base

<222> (29)..(29)

<223> methylated

<220>

<221> modified_base

<222> (48)..(48)

<223> methylated

<220>

<221> modified_base

<222> (50)..(50)

<223> methylated

<220>

<221> modified_base

<222> (53)..(53)

<223> methylated

<220>

<221> modified_base

<222> (55)..(55)

<223> methylated

<220>

<221> modified_base

<222> (62)..(62)

<223> methylated

<220>

<221> modified_base

<222> (66)..(66)

<223> methylated

<220>

<221> modified_base

<222> (74)..(74)

<223> methylated

<220>

<221> modified_base

<222> (78)..(78)

<223> methylated

<220>

<221> modified_base

<222> (85)..(85)

<223> methylated

<220>

<221> modified_base

<222> (98)..(98)

<223> methylated

<220>

<221> modified_base

<222> (100)..(100)

<223> methylated

<220>

<221> modified_base

<222> (109)..(109)

<223> methylated

<220>

<221> modified_base

<222> (118)..(118)

<223> methylated

<220>

<221> modified_base

<222> (120)..(120)

<223> methylated

<220>

<221> modified_base

<222> (122)..(122)

<223> methylated

<220>

<221> modified_base

<222> (130)..(130)

<223> methylated

<220>

<221> modified_base

<222> (132)..(132)

<223> methylated

<220>

<221> modified_base

<222> (137)..(137)

<223> methylated

<220>

<221> modified_base

<222> (141)..(141)

<223> methylated

<220>

<221> modified_base

<222> (159)..(159)

<223> methylated

<220>

<221> modified_base

<222> (165)..(165)

<223> methylated

<220>

<221> modified_base

<222> (170)..(170)

<223> methylated

<220>

<221> modified_base

<222> (172)..(172)

<223> methylated

<220>

<221> modified_base

<222> (177)..(177)

<223> methylated

<220>

<221> modified_base

<222> (181)..(181)

<223> methylated

<220>

<221> modified_base

<222> (191)..(191)

<223> methylated

<220>

<221> modified_base

<222> (202)..(202)

<223> methylated

<220>

<221> modified_base

<222> (204)..(204)

<223> methylated

<220>

<221> modified_base

<222> (210)..(210)

<223> methylated

<220>

<221> modified_base

<222> (216)..(216)

<223> methylated

<220>

<221> modified_base

<222> (218)..(218)

<223> methylated

<220>

<221> modified_base

<222> (221)..(221)

<223> methylated

<220>

<221> modified_base

<222> (226)..(226)

<223> methylated

<400> 66

tgagaagtcg gcggggggcgc ggatcgaccg gggtgtcccc caggctccgc gtcgcggtcc 60

ccgctcgccc tcccgcccgc ccaccgggca ccccagccgc gcagaaggcg gaagccacgc 120

gcgagggacc gcggtccgtc cgggactagc cccaggcccg gcaccgcccc gcgggccgag 180

cgcccacacc cgccaaaccc acgcgggcac gcccccgcgg cgcaccgccc ccagcc 236

<210> 67

<211> 271

<212> DNA

<213> Homo sapiens

<400> 67

gttagtttgg tcgggtgttt ttaaaaataa agcgaggagg gaaggtatag atagattttg 60

aaaatattcg ggttatatac gtcgcgattt atagtttttt tttagcgttg gagtggagac 120

ggcgttcgta gcgttttgcg cgggtgaggt tcgcgtagtt gttggggaag agtttatttg 180

ttaggttgcg ttgggttagc gtagtaagtg gggttggtcg ttatttcgtt gtattcggtc 240

gcgtttcggg tttcgtgcgt tttcgtttta g 271

<210> 68

<211> 271

<212> DNA

<213> Homo sapiens

<400> 68

gttagtttgg ttgggtgttt ttaaaaataa agtgaggagg gaaggtatag atagattttg 60

aaaatatttg ggttatatat gttgtgattt atagtttttt tttagtgttg gagtggagat 120

ggtgtttgta gtgttttgtg tgggtgaggt ttgtgtagtt gttggggaag agtttatttg 180

ttaggttgtg ttgggttagt gtagtaagtg gggttggttg ttattttgtt gtatttggtt 240

gtgttttggg ttttgtgtgt ttttgtttta g 271

<210> 69

<211> 236

<212> DNA

<213> Homo sapiens

<400> 69

tgagaagtcg gcggggggcgc ggatcgatcg gggtgttttt taggtttcgc gtcgcggttt 60

tcgttcgttt tttcgttcgt ttatcgggta ttttagtcgc gtagaaggcg gaagttacgc 120

gcgagggatc gcggttcgtt cgggattagt tttaggttcg gtatcgtttc gcgggtcgag 180

cgtttatatt cgttaaattt acgcgggtac gttttcgcgg cgtatcgttt ttagtt 236

<210> 70

<211> 236

<212> DNA

<213> Homo sapiens

<400> 70

tgagaagttg gtggggggtgt ggattgattg gggtgttttt taggttttgt gttgtggttt 60

ttgtttgttt ttttgtttgt ttattgggta ttttagttgt gtagaaggtg gaagttatgt 120

gtgagggatt gtggtttgtt tgggattagt tttaggtttg gtattgtttt gtgggttgag 180

tgtttatatt tgttaaattt atgtgggtat gtttttgtgg tgtattgttt ttagtt 236

<---

Claims (168)

1. A kit for detecting the presence or absence of colorectal cancer (RCC) in a patient in need thereof, wherein said kit contains: (a) a first pair of primers and a first probe for detecting the methylation state or level of at least one CpG dinucleotide of the BMP3 gene in a biological sample taken from the patient, wherein each of the first pair of primers and the first probe includes a contiguous sequence of at least 16 nucleotides, which is identical to, complementary to, a fragment of the sequence SEQ ID NO. 1 or hybridizes with this fragment under stringent hybridization conditions; (b) a second pair of primers and a second probe for detecting the methylation status or level of at least one CpG dinucleotide of the NDRG4 gene in a biological sample taken from the patient, wherein each of the second pair of primers and the second probe includes a contiguous sequence of at least 16 nucleotides, which is identical to, complementary to, a fragment of the sequence SEQ ID NO. 2 or hybridizes with this fragment under stringent hybridization conditions, where the first pair of primers and the first probe are selected from the group consisting of: i) a forward primer containing SEQ ID NO: 3, a reverse primer containing SEQ ID NO: 4, and a probe containing SEQ ID NO: 5; ii) a forward primer containing SEQ ID NO: 9, a reverse primer containing SEQ ID NO: 10, and a probe containing SEQ ID NO: 11; And iii) a forward primer containing SEQ ID NO: 15, a reverse primer containing SEQ ID NO: 16, and a probe containing SEQ ID NO: 17; or where the second pair of primers and the second probe are selected from the group consisting of: iv) a forward primer containing SEQ ID NO: 6, a reverse primer containing SEQ ID NO: 7, and a probe containing SEQ ID NO: 8; v) a forward primer containing SEQ ID NO: 12, a reverse primer containing SEQ ID NO: 13, and a probe containing SEQ ID NO: 14; And vi) a forward primer containing SEQ ID NO: 18, a reverse primer containing SEQ ID NO: 19, and a probe containing SEQ ID NO: 20. 2. The set according to claim 1, containing (a) a first pair of primers and a first probe; and (b) a second pair of primers and a second probe. 3. The set according to claim 1, where the specified set includes: i) a forward primer containing SEQ ID NO: 3, a reverse primer containing SEQ ID NO: 4, and a probe containing SEQ ID NO: 5, for detecting the methylation state or level of at least one CpG dinucleotide of the BMP3 gene in a biological sample, taken from the patient, and ii) a forward primer containing SEQ ID NO: 6, a reverse primer containing SEQ ID NO: 7, and a probe containing SEQ ID NO: 8, for detecting the methylation state or level of at least one CpG dinucleotide of the NDRG4 gene in a biological sample, taken from the patient. 4. The set according to claim 1, where the specified set includes: i) a forward primer containing SEQ ID NO: 9, a reverse primer containing SEQ ID NO: 10, and a probe containing SEQ ID NO: 11, for detecting the methylation state or level of at least one CpG dinucleotide of the BMP3 gene in a biological sample, taken from the patient, and ii) a forward primer containing SEQ ID NO: 12, a reverse primer containing SEQ ID NO: 13, and a probe containing SEQ ID NO: 14, for detecting the methylation state or level of at least one CpG dinucleotide of the NDRG4 gene in a biological sample, taken from the patient. 5. The set according to claim 1, where the specified set includes: i) a forward primer containing SEQ ID NO: 15, a reverse primer containing SEQ ID NO: 16, and a probe containing SEQ ID NO: 17, for detecting the methylation state or level of at least one CpG dinucleotide of the BMP3 gene in a biological sample, taken from the patient, and ii) a forward primer containing SEQ ID NO: 18, a reverse primer containing SEQ ID NO: 19, and a probe containing SEQ ID NO: 20, for detecting the methylation state or level of at least one CpG dinucleotide of the NDRG4 gene in a biological sample, taken from the patient. 6. Set according to any one of paragraphs. 1-5, where the first probe and the second probe contain a fluorescent donor and an acceptor fluorophore. 7. The kit of claim 6, wherein the first probe and the second probe are TAQMAN® probes. 8. Set according to any one of paragraphs. 1-7, where the set additionally includes: (1) means for detecting the presence or absence of at least one mutation in the KRAS gene in a patient; And (2) means for detecting the presence or absence of hemoglobin in a biological sample taken from a patient. 9. The kit of claim 8, wherein the means for detecting the presence or absence of at least one mutation in the KRAS gene in a patient includes at least one pair of primers capable of amplifying a region of exon 12 and/or exon 13 of the KRAS gene in a polymerase chain reaction ( PCR). 10. The kit of claim 8, wherein the means for detecting the presence or absence of hemoglobin in a biological sample comprises an anti-hemoglobin antibody. 11. The kit of claim 9, wherein the primers are capable of amplifying a region of the KRAS gene containing at least one KRAS mutation selected from the group consisting of G12D, G12V, G12C, G13D, G12A, G12R, G12S and G13C. 12. The kit of claim 10, wherein the antibody is a colloidal gold conjugated antibody. 13. Set according to any one of paragraphs. 1-12, wherein the kit further includes means for amplifying a reference gene for quantification. 14. Set according to any one of paragraphs. 1-12, wherein the kit further contains instructions for use and/or interpretation of the test result obtained using the kit. 15. The kit according to claim 10, where the kit further includes a means for detecting a complex formed by the antibody and hemoglobin in a biological sample. 16. Set according to any one of paragraphs. 1-15, in which the biological sample taken from the patient is a stool sample. 17. Set according to any one of paragraphs. 1-16, wherein the kit further includes a bisulfite reagent and a container suitable for mixing the bisulfite reagent and a biological sample from a patient or polynucleotides derived from a biological sample. 18. Set according to any one of paragraphs. 1-17, where the kit further includes a methylation-sensitive restriction enzyme reagent. 19. Set according to any one of paragraphs. 1-18, where the set additionally includes: (1) a positive standard and a negative standard for detecting BMP3 methylation in a biological sample, and (2) a positive standard and a negative standard for detecting NDRG4 methylation in a biological sample. 20. The set according to claim 19, in which: positive standard for detection of BMP3 methylation includes polynucleotide sequence GTTAGTTTGGTCGGGTGTTTTTAAAAATAAAGCGAGGAGGGAAGGTATAGATAGATTTTGAAAATATTCGGGTTATATACGTCGCGATTTATAGTTTTTTTTTAGCGTTGGAGTGGAGACGGCGTTCGTAGCGTTTTGCGCGGGTGAGGTTCGCGTAGTTGTTGGGGAAGAGTTTATTTGTTAGGTTGCGTTGGGTTAGCGTAGTAAGTGGGGTTGGTCGTTATTTCGTTGTA TTCGGTCGCGTTTCGGGTTTCGTGCGTTTTCGTTTTAG (SEQ ID NO: 67); negative standard for detecting BMP3 methylation includes a polynucleotide sequence GTTAGTTTGGTTGGGTGTTTTTAAAAATAAAGTGAGGAGGGAAGGTATAGATAGATTTTGAAAATATTTGGGTTATATATGTTGTGATTTATAGTTTTTTTTTAGTGTTGGAGTGGAGATGGTGTTTGTAGTGTTTTGTGTGGGTGAGGTTTGTGTAGTTGTTGGGGAAGAGTTTATTTGTTAGGTTGTGTTGGGTTAGTGTAGTAAGTGGGGTTGGTTGTTATTTTGTTG TATTTGGTTGTGTTTTGGGGTTTTGTGTGTTTTTGTTTTAG (SEQ ID NO: 68); positive standard for detecting NDRG4 methylation includes a polynucleotide sequence TGAGAAGTCGGCGGGGGCGCGGATCGATCGGGGTGTTTTTTAGGTTTCGCGTCGCGGTTTTCGTTCGTTTTTTCGTTCGTTTATCGGGTATTTTAGTCGCGTAGAAGGCGGAAGTTACGCGCGAGGGATCGCGGTTCGTTCGGGATTAGTTTTAGGTTCGGTATCGTTTCGCGGGTCGAGCGTTTATATTCGTTAAATTTACGCGGGTACGTTTTTCGCGGCGTATCGTTTTTAG TT (SEQ ID NO.: 69); And negative standard for detecting NDRG4 methylation includes a polynucleotide sequence TGAGAAGTTGGTGGGGGTGTGGATTGATTGGGGTGTTTTTTAGGTTTTGTGTTGTGGTTTTTGTTTGTTTTTTTGTTTGTTTATTGGGTATTTTAGTTGTGTAGAAGGTGGAAGTTATGTGTGAGGGATTGTGGTTTGTTTGGGATTAGTTTTAGGTTTGGTATTGTTTTGTGGGTTGAGTGTTTATATTTGTTAAATTTATGTGGGTATGTTTTTGTGGTGTATTGTTTT TAGTT (SEQ ID NO.: 70). 21. The kit according to claim 13, where the means for amplifying the internal control gene contains primers for amplifying the positive control gene and/or the negative control gene. 22. A method for determining the presence or absence of colorectal cancer (RCC) in a patient in need, where the method includes: a) obtaining genomic DNA from a biological sample of the patient; b) treating genomic DNA(a) or a fragment thereof with one or more reagents to convert cytosine bases that are not methylated to uracil or another base that is markedly different from cytosine in terms of hybridization properties; c) contacting the processed genomic DNA or a processed fragment thereof with a first pair of primers for detecting the presence or absence of methylation sites of the gene encoding bone morphogenesis protein 3 (BMP3) in the patient, and with a second pair of primers for detecting the presence or absence of methylation sites of the gene in the patient, encoding protein NDRG family member 4 (NDRG4), wherein the first pair of primers includes a contiguous sequence of at least 9 nucleotides that is identical to, complementary to, or hybridizes to the fragment of SEQ ID NO: 1 under stringent hybridization conditions, and where the second pair of primers contains a contiguous a sequence of at least 9 nucleotides that is complementary to or hybridizes to the fragment of SEQ ID NO: 2 under stringent hybridization conditions wherein the processed genomic DNA or fragment thereof has either been amplified to produce at least one amplification using a first pair of primers or a second pair primers or were not amplified; And d) determining the presence or absence of POC in the patient based on the presence or absence of the specified amplification, state or methylation level of at least one CpG dinucleotide of the BMP3 gene and the NDRG4 gene in the patient, using a first probe to detect the presence or absence of methylation sites of the BMP3 gene and a second probe to detect the presence or absence of methylation sites of the NDRG4 gene, where the first pair of primers and the first probe are selected from the group consisting of: i) a forward primer containing SEQ ID NO: 3, a reverse primer containing SEQ ID NO: 4, and a probe containing SEQ ID NO: 5; ii) a forward primer containing SEQ ID NO: 9, a reverse primer containing SEQ ID NO: 10, and a probe containing SEQ ID NO: 11; And iii) a forward primer containing SEQ ID NO: 15, a reverse primer containing SEQ ID NO: 16, and a probe containing SEQ ID NO: 17; or where the second pair of primers and the second probe are selected from the group consisting of: iv) a forward primer containing SEQ ID NO: 6, a reverse primer containing SEQ ID NO: 7, and a probe containing SEQ ID NO: 8; v) a forward primer containing SEQ ID NO: 12, a reverse primer containing SEQ ID NO: 13, and a probe containing SEQ ID NO: 14; And vi) a forward primer containing SEQ ID NO: 18, a reverse primer containing SEQ ID NO: 19, and a probe containing SEQ ID NO: 20. 23. The method of claim 22, comprising using (a) a first pair of primers and a first probe, and (b) a second pair of primers and a second probe. 24. The method according to claim 22, which includes the use of: i) a forward primer containing SEQ ID NO: 3, a reverse primer containing SEQ ID NO: 4, and a probe containing SEQ ID NO: 5, for detecting the methylation state or level of at least one CpG dinucleotide of the BMP3 gene in a biological sample, taken from the patient, and ii) a forward primer containing SEQ ID NO: 6, a reverse primer containing SEQ ID NO: 7, and a probe containing SEQ ID NO: 8, for detecting the methylation state or level of at least one CpG dinucleotide of the NDRG4 gene in a biological sample, taken from the patient. 25. The method according to claim 22, which includes the use of: i) a forward primer containing SEQ ID NO: 9, a reverse primer containing SEQ ID NO: 10, and a probe containing SEQ ID NO: 11, for detecting the methylation state or level of at least one CpG dinucleotide of the BMP3 gene in a biological sample, taken from the patient, and ii) a forward primer containing SEQ ID NO: 12, a reverse primer containing SEQ ID NO: 13, and a probe containing SEQ ID NO: 14, for detecting the methylation state or level of at least one CpG dinucleotide of the NDRG4 gene in a biological sample, taken from the patient. 26. The method according to claim 22, which includes the use of: i) a forward primer containing SEQ ID NO: 15, a reverse primer containing SEQ ID NO: 16, and a probe containing SEQ ID NO: 17, for detecting the methylation state or level of at least one CpG dinucleotide of the BMP3 gene in a biological sample, taken from the patient, and ii) a forward primer containing SEQ ID NO: 18, a reverse primer containing SEQ ID NO: 19, and a probe containing SEQ ID NO: 20, for detecting the methylation state or level of at least one CpG dinucleotide of the NDRG4 gene in a biological sample, taken from the patient. 27. Method according to any one of paragraphs. 22-26, wherein the first probe and the second probe comprise a fluorescent donor and an acceptor fluorophore. 28. Method according to any one of paragraphs. 22-26, wherein the first probe and the second probe are TAQMAN® probes. 29. Method according to any one of paragraphs. 22-28, wherein the method further includes the step of detecting the presence or absence of at least one mutation in the KRAS gene in a biological sample taken from the patient, and the step of detecting the presence or absence of hemoglobin in the biological sample taken from the patient. 30. The method of claim 29, wherein the step of detecting the presence or absence of at least one mutation in the KRAS gene in a patient includes using at least one pair of primers capable of amplifying a region of exon 12 and/or exon 13 of the KRAS gene in a polymerase chain reaction ( PCR). 31. The method of claim 29, wherein the step of detecting the presence or absence of hemoglobin in the biological sample includes using an anti-hemoglobin antibody. 32. The method of claim 30, wherein the primers are capable of amplifying a region of the KRAS gene containing at least one KRAS mutation selected from the group consisting of G12D, G12V, G12C, G13D, G12A, G12R, G12S and G13C. 33. The method of claim 31, wherein the antibody is an antibody conjugated to colloidal gold. 34. Method according to any one of paragraphs. 22-33, wherein the BMP3 gene is amplified by quantitative PCR (QPCR), and the method further includes amplifying a first control gene to determine the Ct value of BMP3 amplification as ∆Ct1. 35. Method according to any one of paragraphs. 22-33, wherein the NDRG4 gene is amplified by quantitative PCR (QPCR), and the method further includes amplifying a second control gene to determine the Ct value of the NDRG4 amplification as ΔCt2. 36. Method according to any one of paragraphs. 30-33, wherein amplification of the mutant KRAS gene is carried out using quantitative PCR (QPCR), and the method further includes amplification of a third control gene to determine the Ct value of mutant KRAS amplification as ∆Ct3. 37. The method according to claim 34 or 35, where the first and second control genes are the same. 38. The method of claim 37, wherein the same control gene is a B2M gene. 39. The method of claim 36, wherein the mutant KRAS gene contains a mutation selected from the group consisting of G12D, G13D, G12V, G12C, G12S, G12A and G13R. 40. The method of claim 39, wherein the mutant KRAS gene is amplified using one or more primer pairs selected from the group consisting of: (1) forward primer G12D-F containing SEQ ID NO: 35 and reverse primer Kras-R containing SEQ ID NO: 42; (2) forward primer G13D-F containing SEQ ID NO: 36 and reverse primer Kras-R containing SEQ ID NO: 42; (3) forward primer G12V-F containing SEQ ID NO: 37 and reverse primer Kras-R containing SEQ ID NO: 42; (4) forward primer G12C-F containing SEQ ID NO: 38 and reverse primer Kras-R containing SEQ ID NO: 42; (5) forward primer G12S-F containing SEQ ID NO: 39 and reverse primer Kras-R containing SEQ ID NO: 42; (6) forward primer G12A-F containing SEQ ID NO: 40 and reverse primer Kras-R containing SEQ ID NO: 42; And (7) forward primer G12R-F containing SEQ ID NO: 41, and reverse primer Kras-R containing SEQ ID NO: 42, and where the KRAS probe for qPCR contains SEQ ID NO: 45. 41. The method of claim 36, wherein the third control gene is an ACTB gene. 42. The method according to claim 41, where the primers for qPCR for amplification of the ACTB gene contain SEQ ID NO: 43 and 44, and the probe contains SEQ ID NO: 46. 43. Method according to any one of paragraphs. 22-42, where the specified method includes the use of: (1) a positive standard and a negative standard for detecting BMP3 methylation in the sample, and (2) a positive standard and a negative standard to detect NDRG4 methylation in the sample. 44. Method according to claim 43, where positive standard for detecting BMP3 methylation includes a polynucleotide sequence GTTAGTTTGGTCGGGTGTTTTTAAAAATAAAGCGAGGAGGGAAGGTATAGATAGATTTTGAAAATATTCGGGTTATATACGTCGCGATTTATAGTTTTTTTTTAGCGTTGGAGTGGAGACGGCGTTCGTAGCGTTTTGCGCGGGTGAGGTTCGCGTAGTTGTTGGGGAAGAGTTTATTTGTTAGGTTGCGTTGGGTTAGCGTAGTAAGTGGGGTTGGTCGTTATTTCGTTGTA TTCGGTCGCGTTTCGGGTTTCGTGCGTTTTCGTTTTAG (SEQ ID NO: 67); negative standard for detecting BMP3 methylation includes a polynucleotide sequence GTTAGTTTGGTTGGGTGTTTTTAAAAATAAAGTGAGGAGGGAAGGTATAGATAGATTTTGAAAATATTTGGGTTATATATGTTGTGATTTATAGTTTTTTTTTAGTGTTGGAGTGGAGATGGTGTTTGTAGTGTTTTGTGTGGGTGAGGTTTGTGTAGTTGTTGGGGAAGAGTTTATTTGTTAGGTTGTGTTGGGTTAGTGTAGTAAGTGGGGTTGGTTGTTATTTTGTTG TATTTGGTTGTGTTTTGGGGTTTTGTGTGTTTTTGTTTTAG (SEQ ID NO: 68); positive standard for detecting NDRG4 methylation includes a polynucleotide sequence TGAGAAGTCGGCGGGGGCGCGGATCGATCGGGGTGTTTTTTAGGTTTCGCGTCGCGGTTTTCGTTCGTTTTTTCGTTCGTTTATCGGGTATTTTAGTCGCGTAGAAGGCGGAAGTTACGCGCGAGGGATCGCGGTTCGTTCGGGATTAGTTTTAGGTTCGGTATCGTTTCGCGGGTCGAGCGTTTATATTCGTTAAATTTACGCGGGTACGTTTTTCGCGGCGTATCGTTTTTAG TT (SEQ ID NO.: 69); And negative standard for detecting NDRG4 methylation includes a polynucleotide sequence TGAGAAGTTGGTGGGGGTGTGGATTGATTGGGGTGTTTTTTAGGTTTTGTGTTGTGGTTTTTGTTTGTTTTTTTGTTTGTTTATTGGGTATTTTAGTTGTGTAGAAGGTGGAAGTTATGTGTGAGGGATTGTGGTTTGTTTGGGATTAGTTTTAGGTTTGGTATTGTTTTGTGGGTTGAGTGTTTATATTTGTTAAATTTATGTGGGTATGTTTTTGTGGTGTATTGTTTT TAGTT (SEQ ID NO.: 70). 45. Method according to any one of paragraphs. 22-44, where the method includes amplification of a quality control standard. 46. A method for detecting the presence or absence of colorectal cancer (RCC) in a patient in need thereof, where the method includes using a kit according to any one of claims. 1-21. 47. A method for detecting the presence or absence of colorectal cancer (RCC) in a patient in need, where the method includes: a) obtaining raw genomic DNA from a patient's stool sample; b) treating the genomic DNA(s) or a fragment thereof with one or more reagents to convert cytosine bases that are unmethylated to uracil or another base that is markedly different from cytosine in terms of hybridization properties; c) performing quantitative PCR (qPCR) using the processed genomic DNA (b) as a template and determining the Ct value of the BMP3 gene, subtracted from the value of the first control gene in the patient as ΔCt1; d) performing qPCR using the processed genomic DNA (b) as a template and determining the Ct value of the NDRG4 gene, subtracted from the value of the first control gene in the patient as ΔCt2; e) performing qPCR using raw genomic DNA as a template and determining the Ct value of the mutant KRAS gene, subtracted from the value of the second control gene in the patient as ∆Ct3; f) performing a stool immunochemical test for hemoglobin protein in the stool sample and assessing the result as FIT; g) determining the value of K, where K=a*ΔCt1+b*ΔCt2+c*ΔCt3+d*FIT+X, where a, b, c, d, X are clinical constants; And h) determining the value of the general index P, where P=eK/(1+eK), where e is a natural constant, in this case, if P is equal to or greater than a given threshold, then the patient is diagnosed with RPOC, and if P is less than this threshold, then it is considered that the patient does not have RPOC, where the qPCR for amplification of the BMP3 gene includes the first pair of primers and the first probe selected from the group consisting of: i) a forward primer containing SEQ ID NO: 3, a reverse primer containing SEQ ID NO: 4, and a probe containing SEQ ID NO: 5; ii) a forward primer containing SEQ ID NO: 9, a reverse primer containing SEQ ID NO: 10, and a probe containing SEQ ID NO: 11; And iii) a forward primer containing SEQ ID NO: 15, a reverse primer containing SEQ ID NO: 16, and a probe containing SEQ ID NO: 17; or where the qPCR for amplification of the NDRG4 gene includes a second pair of primers and a second probe selected from the group consisting of: iv) a forward primer containing SEQ ID NO: 6, a reverse primer containing SEQ ID NO: 7, and a probe containing SEQ ID NO: 8; v) a forward primer containing SEQ ID NO: 12, a reverse primer containing SEQ ID NO: 13, and a probe containing SEQ ID NO: 14; And vi) a forward primer containing SEQ ID NO: 18, a reverse primer containing SEQ ID NO: 19, and a probe containing SEQ ID NO: 20. 48. The method of claim 47, wherein the qPCR for amplification of the BMP3 gene includes a first pair of primers and a first probe, and wherein the qPCR for amplification of the NDRG4 gene includes a second pair of primers and a second probe. 49. The method according to claim 47, where the method includes the use of: i) a forward primer containing SEQ ID NO: 3, a reverse primer containing SEQ ID NO: 4, and a probe containing SEQ ID NO: 5 for detecting the methylation state or level of at least one CpG dinucleotide of the BMP3 gene in the sample, and ii) a forward primer containing SEQ ID NO: 6, a reverse primer containing SEQ ID NO: 7, and a probe containing SEQ ID NO: 8 for detecting the methylation state or level of at least one CpG dinucleotide of the NDRG4 gene in the sample. 50. The method according to claim 47, where the method includes the use of: i) a forward primer containing SEQ ID NO: 9, a reverse primer containing SEQ ID NO: 10, and a probe containing SEQ ID NO: 11 for detecting the methylation state or level of at least one CpG dinucleotide of the BMP3 gene in the sample, and ii) a forward primer containing SEQ ID NO: 12, a reverse primer containing SEQ ID NO: 13, and a probe containing SEQ ID NO: 14 for detecting the methylation state or level of at least one CpG dinucleotide of the NDRG4 gene in the sample. 51. The method according to claim 47, where said method includes the use of: i) a forward primer containing SEQ ID NO: 15, a reverse primer containing SEQ ID NO: 16, and a probe containing SEQ ID NO: 17 for detecting the methylation state or level of at least one CpG dinucleotide of the BMP3 gene in the sample, and ii) a forward primer containing SEQ ID NO: 18, a reverse primer containing SEQ ID NO: 19, and a probe containing SEQ ID NO: 20 to determine the methylation status or level of at least one CpG dinucleotide of the NDRG4 gene in the sample. 52. Method according to any one of paragraphs. 47-51, where the first probe and the second probe contain a fluorescent donor and an acceptor fluorophore. 53. Method according to any one of paragraphs. 47-52, where the first probe and the second probe are TAQMAN® probes. 54. Method according to any one of paragraphs. 47-53, wherein the mutant KRAS gene contains at least one KRAS mutation selected from the group consisting of G12D, G12V, G12C, G13D, G12A, G12R, G12S and G13C. 55. Method according to any one of paragraphs. 47-54, in which the stool immunoassay includes an antibody conjugated to colloidal gold. 56. Method according to any one of paragraphs. 47-55, in which step c) and step d) include using the B2M gene as a first control gene. 57. The method of claim 54, wherein the mutant KRAS gene is amplified using one or more primer pairs selected from the group consisting of: (1) forward primer G12D-F containing SEQ ID NO: 35 and reverse primer Kras-R containing SEQ ID NO: 42; (2) forward primer G13D-F containing SEQ ID NO: 36 and reverse primer Kras-R containing SEQ ID NO: 42; (3) forward primer G12V-F containing SEQ ID NO: 37 and reverse primer Kras-R containing SEQ ID NO: 42; (4) forward primer G12C-F containing SEQ ID NO: 38 and reverse primer Kras-R containing SEQ ID NO: 42; (5) forward primer G12S-F containing SEQ ID NO: 39 and reverse primer Kras-R containing SEQ ID NO: 42; (6) forward primer G12A-F containing SEQ ID NO: 40 and reverse primer Kras-R containing SEQ ID NO: 42; And (7) forward primer G12R-F containing SEQ ID NO: 41, and reverse primer Kras-R containing SEQ ID NO: 42, and wherein the amplified qPCR product of the mutant KRAS gene is detected with a KRAS probe containing SEQ ID NO: 45. 58. The method according to claim 57, where the ACTB gene is used as a second control gene in the qPCR to amplify the mutant KRAS gene. 59. The method according to claim 58, where the primers for qPCR for amplification of the ACTB gene contain SEQ ID NO: 43 and 44, and the probe for qPCR of the ACTB gene contains SEQ ID NO: 46. 60. Method according to paragraphs. 47-59, where the method includes using: (1) a positive standard and a negative standard for detecting BMP3 methylation in the sample, and (2) a positive standard and a negative standard to detect NDRG4 methylation in the sample. 61. Method according to claim 60, where positive standard for detecting BMP3 methylation includes a polynucleotide sequence GTTAGTTTGGTCGGGTGTTTTTAAAAATAAAGCGAGGAGGGAAGGTATAGATAGATTTTGAAAATATTCGGGTTATATACGTCGCGATTTATAGTTTTTTTTTAGCGTTGGAGTGGAGACGGCGTTCGTAGCGTTTTGCGCGGGTGAGGTTCGCGTAGTTGTTGGGGAAGAGTTTATTTGTTAGGTTGCGTTGGGTTAGCGTAGTAAGTGGGGTTGGTCGTTATTTCGTTGTA TTCGGTCGCGTTTCGGGTTTCGTGCGTTTTCGTTTTAG (SEQ ID NO: 67); negative standard for detecting BMP3 methylation includes a polynucleotide sequence GTTAGTTTGGTTGGGTGTTTTTAAAAATAAAGTGAGGAGGGAAGGTATAGATAGATTTTGAAAATATTTGGGTTATATATGTTGTGATTTATAGTTTTTTTTTAGTGTTGGAGTGGAGATGGTGTTTGTAGTGTTTTGTGTGGGTGAGGTTTGTGTAGTTGTTGGGGAAGAGTTTATTTGTTAGGTTGTGTTGGGTTAGTGTAGTAAGTGGGGTTGGTTGTTATTTTGTTG TATTTGGTTGTGTTTTGGGGTTTTGTGTGTTTTTGTTTTAG (SEQ ID NO: 68); positive standard for detecting NDRG4 methylation includes a polynucleotide sequence TGAGAAGTCGGCGGGGGCGCGGATCGATCGGGGTGTTTTTTAGGTTTCGCGTCGCGGTTTTCGTTCGTTTTTTCGTTCGTTTATCGGGTATTTTAGTCGCGTAGAAGGCGGAAGTTACGCGCGAGGGATCGCGGTTCGTTCGGGATTAGTTTTAGGTTCGGTATCGTTTCGCGGGTCGAGCGTTTATATTCGTTAAATTTACGCGGGTACGTTTTTCGCGGCGTATCGTTTTTAG TT (SEQ ID NO.: 69); And negative standard for detecting NDRG4 methylation includes a polynucleotide sequence TGAGAAGTTGGTGGGGGTGTGGATTGATTGGGGTGTTTTTTAGGTTTTGTGTTGTGGTTTTTGTTTGTTTTTTTGTTTGTTTATTGGGTATTTTAGTTGTGTAGAAGGTGGAAGTTATGTGTGAGGGATTGTGGTTTGTTTGGGATTAGTTTTAGGTTTGGTATTGTTTTGTGGGTTGAGTGTTTATATTTGTTAAATTTATGTGGGTATGTTTTTGTGGTGTATTGTTTT TAGTT (SEQ ID NO: 70). 62. Method according to any one of paragraphs. 47-61, where the method includes amplification of the quality control standard in step c) and in step d). 63. A method of treating colorectal cancer (RCC) in a patient in need thereof, wherein the method comprises determining the presence or absence of RCCA in the patient using a kit according to any one of claims. 1-21, and treatment of the patient depending on the presence or absence of POC. 64. A method of treating colorectal cancer (RCC) in a patient in need thereof, wherein said method includes determining the presence or absence of RCCC in the patient using the method according to any one of claims. 22-62, and treatment of the patient depending on the presence or absence of POC.