RU2586775C1 - Method for non-invasive diagnosis of colorectal cancer - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and is intended for non-invasive diagnosis of colorectal cancer. Method comprises sampling and transportation of samples of patient's stool, extraction of patient's stool total DNA and determining integrity of DNA by PCR-analysis. To assess quality and quantity of recovered DNA, PCR-analysis of two DNA fragments are carried out in duplex mode: first fragment length 141 CL., located in coding gene TR53, second fragment 153 CL., located in coding BLM gene region. To assess integrity of genomic DNA by PCR-analysis of first fragment is used with length of 800 CL., containing 7, 8 and 9 exons gene TP53, and second fragment length 2340 CL., containing 14 and 15 MLH1 gene exons. Detection of at least one of these two fragments in PCR products indicates presence of colorectal cancer in patient.

EFFECT: invention provides effective diagnosis of colorectal cancer at early stages.

5 cl, 1 ex

Description

The invention relates to the field of biotechnology.

Colorectal cancer (CRC) is one of the most common oncological diseases and leads to high mortality among patients. However, when detecting CRC at the first stage, we will cure in 90% of cases [Duffy M.J. Use of molecular markers for predicting therapy response in cancer patients // J. Cancer Treatment Reviews. - 2011. - P. 151-159], from which it follows that early diagnosis of CRC is extremely important [1].

Today, there is no longer any doubt about the need to screen the population, especially people at risk, for the detection of CRC.

Fibrocolonoscopy (FCC) today is the main test that is used to establish or exclude colorectal pathology [http://www.colonoscopy.ru/patient/procedure1.htm] [2]. However, as a screening test, this method is not always applicable because of its high cost, inconvenience associated with the need for very careful preparation of the patient’s intestines to obtain reliable results, because of the possibility of complications.

As one of the analogues, the commercial test Epi proColon, developed by Epigenomics in Germany, is considered.

The cost of the test in Germany is 99-161 euros, the duration of the analysis is 1 week.

The sensitivity of the test ranges from 68% to 80%, and the specificity is in the range of 70-90% according to various studies.

The colorectal cancer test is based on the detection of the Septin9 methylated gene in plasma.

DNA fragments enter the bloodstream from colorectal carcinomas of the proximal and distal colon and from the rectum (freely circulating DNA - cDNA). These fragments are characterized by the presence of oncogen markers and specific mutations of cancer suppressors.

The epigenetic effects of carcinogenesis include DNA methylation in its early stages. The Septin9 gene encodes the septin-9 protein, which is an oncosuppressor and regulates cell division in the cytoplasm. DNA methylation of this gene stops its active work and “turns off” the synthesis of cancer suppressor protein [Church TR, Wandell M, Lofton-Day C et al. Prospective evaluation of methylated SEPT9 in plasma for detection of asymptomatic colorectal cancer. Gut. 2014 Feb; 63 (2): 317-25] [3].

Technique of execution.

The Epi proColon test consists of three parts: a kit for DNA extraction from blood plasma, a kit containing positive and negative controls, and a kit for Real time PCR.

10 ml of whole blood is taken from a patient into a Vacutainer K2EDTA tube (exclusively these tubes are recommended for use). Within 4 hours after sampling, about 3.5 ml of plasma is removed from the blood by centrifugation (1800 g). Plasma is stored for 72 hours at a temperature of 2 ° C to 8 ° C or 14 days at a temperature of 15 ° C to 25 ° C.

DNA isolation is based on the method of sorption on magnetic balls. After elution, the final volume is 55 μl.

By the bisulfite conversion method, all demethylated cytosine residues found in the cDNA blood sample are converted to thymine. The analysis of throwing is carried out by the duplex method Real time PCR. The sequence in the promoter region of the Septin9 gene (SEPT9_v2) is amplified. At the same time as Septin9, the β-actin gene fragment is amplified for internal control. For one patient, three PCR analyzes are performed to increase accuracy, then the PCR curves are evaluated. If the curve exceeds a predefined threshold for 50 PCR cycles, the reaction is considered positive. If at least one PCR from the subject was positive, the analysis is considered positive.

If yes, colonoscopy recommended

[http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/Medical Devices / MedicalDevicesAdvisoryCommittee / MolecularandClinicalGeneticsPanel / UCM390234.pdf] [4].

The disadvantages of the method:

1) incomplete conversion, which may lead to erroneous analysis results;

2) significant loss of DNA at the conversion stage;

3) the high resource intensity of the methods, which negatively affects the quality and cost of the diagnosis;

4) the analysis requires highly qualified personnel.

As another analog, Cologuard from Exact Sciences Corporation is considered. The cost of Cologuard is $ 600, the declared sensitivity is 92% for colorectal cancer and 69% for benign tumors of the intestinal tract

[http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/MedicalDevices/MedicalDevicesAdvisoryCommittee/MolecularandClinicalGeneticsPanel/UCM391101.pdf] [5].

This is a multi-purpose test, where the sample is analyzed in three categories of biomarkers:

1) analysis of occult blood in feces by the FIT method, that is, the detection of hemoglobin molecules by the immunochemical method;

2) analysis of DNA hypermethylation by two markers NDRG4 and BMP3;

3) detection of mutations at 7 points in the second exon of the KRAS gene (which covers 98% of mutations characteristic of this gene) and analysis of the β-actin gene to monitor the state of genomic DNA.

Technique of execution.

Samples are taken at home and placed in two containers: for DNA analysis and for the detection of globulin.

Within 72 hours of sampling, the sample is delivered for analysis.

Stages of analysis:

1) isolation of DNA from the stool;

2) automated DNA purification and conversion for epigenetic assessment;

3) DNA analysis by QuARTS method - (Quantitative allele-specific real-time target and signal amplification - quantitative allele - specific real-time PCR);

4) the number of hemoglobin molecules in the sample is determined by the method of quantitative enzyme-linked immunosorbent assay (quantitative ELISA).

The conclusion is ready 2 weeks after receiving the sample.

If yes, a colonoscopy is recommended [Thomas F. Imperiale, M.D., David F et al. Multitarget Stool DNA Testing for Colorectal-Cancer Screening N Engl J Med 2014; 370: 1287-1297] [6].

The disadvantages of the method.

This test is characterized by increased complexity, high technological requirements, very high cost and relative duration of the analysis.

In human feces, a small number of intestinal epithelial cells are detected as a result of physiological desquamation. The cells of the intestinal epithelium are rapidly updated. Exfoliated epithelial cells when passing through the intestinal tract can be completely or partially destroyed by enzymes, the DNA of these cells is degraded, as a rule, to sizes of 180-210 n.p. [Kerr JF, Wyllie AN, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972; 26: 239-57] [7].

A somewhat different picture is observed in patients with colorectal tumors. Colorectal cancer cells, along with normal cells, enter the stool of a person, and in patients with neoplasms of the intestinal tract, the amount of DNA in the feces can even be increased in comparison with healthy individuals. A significant amount of tumor cell DNA can remain stable due to impaired apoptosis mechanism or due to the resistance of such cells to various degrading enzymes.

Shown [Boynton K.A., Summerhayes I.C., Ahlquist D.Α., Shuber A.P. DNA integrity as a potential marker for stool_based detection of colorectal cancer // Clin. Chem. - 2003. - Vol. 49. - P. 1058-1065] [8] that DNA fragments isolated from the stool of patients with colorectal tumors have a greater molecular weight than fragments obtained from the stool of healthy individuals.

Therefore, the analysis of the integrity of DNA fragments isolated from stool samples provides great opportunities for the diagnosis of CRC.

Therefore, as a prototype, a method for non-invasive diagnosis of colorectal cancer based on a PCR analysis of the integrity of fecal DNA [Yehya A.N., Yusoff N.M., Khalid I.A. et al. Pilot study of the sensitivity and specificity of the DNA integrity assay for stool-based detection of colorectal cancer in Malaysian patients // Asian Pac J Cancer Prev. - 2012. - Vol. 13 (5). - P. 1869-1872] [9].

The method includes operations: taking and transporting samples of the patient’s stool, extracting total DNA from the patient’s stool, monitoring the quality and quantity of DNA extracted (i.e., is its concentration sufficient for PCR and is it well purified from impurities that may be polymerase inhibitors - an enzyme by which PCR fragments are synthesized) and PCR analysis of extended DNA fragments (i.e., establishing the integrity of DNA), by the presence of which they judge the presence of colorectal cancer in the patient.

PCR analysis of DNA fragments is carried out by sequentially carrying out a polymerase chain reaction and visualizing its products by electrophoresis.

Stool samples were taken from 64 individuals, including 32 patients with colorectal cancer and 32 healthy volunteers.

For patients with colorectal cancer, the diagnosis was histologically confirmed. Feces were collected 4-5 days after the colonoscopy or before preparing the patient for surgery to avoid traumatic artifacts. Healthy volunteers had a negative occult blood count.

Stool samples (~ 5 g) were transported from the sampling point to the laboratory in ice and genomic DNA was isolated on the same day.

To isolate total DNA from the stool, the QIAamp DNA Stool Mini Kit (Qiagen 51504) was used in accordance with the manufacturer's protocol.

To control the quality of the extracted DNA, UV spectroscopy at 260/280 nm was used, as well as agarose gel electrophoresis.

The amount of DNA was estimated using an Invitrogen Qubit fluorimeter and a BR Assay Kit (Invitrogen Q32853) for double-stranded DNA. The isolated DNA was stored in small aliquots at -20 ° C.

For analysis of extended DNA fragments, a fragment of 1476 bp was used, containing from 6 to 9 exons of the TP53 gene. For PCR, a set of primers was used previously published [Beroud C & Soussi Τ (1998). p53 gene mutation: software and database. Nucleic Acids Res, 1, 200-4] [10]. (Forward = 5 ′ GCCTCTGATTCCTCACTGAT 3 ′ and reverse = 5 ′ AAGACTTAGTACCTGAAGGGT 3 ′).

Invitrogen PCR reagents (10342-020) were used, including Taq DNA polymerase.

PCR temperature conditions:

initial denaturation at 95 ° C / 3 min; 35 cycles 95 ° C / 30 s; 58 ° C / 2 min; 72 ° C / 2 min; final polymerization 72 ° C / 5 min in an Eppendorf Mastercycler® pro apparatus.

5 μl of the PCR product was electrophoresed on a 0.7% agarose gel and photographed using the FluorChem M Fluorescent Imaging System.

Out of 32 patients with colorectal cancer, 18 revealed 1476 bp DNA fragments, while none of the healthy individuals had similar fragments in the stool. Thus, the sensitivity and specificity of the method for the detection of colorectal cancer were 56.3% and 100%, respectively.

The disadvantage of this method is the low sensitivity of detection of colorectal cancer. In this work, a reliable correlation was found between tumor size and test sensitivity. So, for tumors less than 3 cm in size, the sensitivity of the method is 38%, and more than 3 cm - 78.5% (p = 0.0356), that is, the detection of a tumor in the early stages is difficult.

Low sensitivity is primarily determined by the use of only one target gene (TP53), since a deletion in this gene can lead to a false negative conclusion.

In addition, the method involves the use of expensive, highly sensitive imported equipment. UV spectroscopy at 260/280 nm was used to determine the quality of the extracted DNA, and the amount of DNA was evaluated using an Invitrogen Qubit fluorimeter and a BR Assay Kit (Invitrogen Q32853). The use of such equipment limits the possibility of mass screening of the population.

The objective of the present invention is to develop a method for early non-invasive diagnosis of colorectal cancer, providing large-scale screening of the population with economic affordability.

The technical effect consists in increasing the sensitivity of the method, simplifying the method while maintaining 100% specificity without the use of expensive, complex equipment.

The technical effect is achieved by the fact that in the known method of non-invasive diagnosis of colorectal cancer, based on the analysis of DNA integrity from the patient’s stool, including the collection and transportation of patient’s stool samples, isolation of total DNA from the patient’s stool, quality control of the extracted DNA and PCR analysis of extended DNA fragments , i.e. establishing the integrity of DNA, by the presence of which they judge the presence of colorectal cancer in a patient, it is new that stool samples are transported in a medium containing 0.5 M EDTA, and PCR analysis of two short DNA fragments is carried out to assess the quality and quantity of extracted DNA less than 200 n.p. in duplex mode: the first fragment is 141 bp in length located in the coding region of the TP53 gene, the second fragment is 153 bp located in the coding region of the BLM gene, and two extended DNA fragments are used to assess the integrity of genomic DNA by PCR analysis : the first fragment with a length of 800 bp containing 7, 8 and 9 exons of the TP53 gene, the second fragment with a length of 2340 bp containing 14 and 15 exons of the MLH1 gene, and for both of these fragments the polymerase chain reaction is carried out using a whale " Evrogen Encyclo PCR ", which includes Encyclo polymerase, and found ie these fragments or at least one of these two fragments in the PCR products indicates the presence of colorectal cancer in a patient.

In this case, PCR analysis of the first short DNA fragments with a length of 141 n.p. carried out using the direct primer 5′-ATTCACTTTTATCACCTTTCCTTGCC-3 ′, the reverse primer: 5′-CCACTTGATAAGAGGTCCCAAGACTT-3, PCR analysis of the second short DNA fragments 153 np in length carried out using a direct primer 5′-CTCTGCCACCAGGAAGAATC-3 ′, the reverse primer: 5′-ACAGCAGTGCTTGTGAGAAC-3 ′; and the presence of the first extended fragment with a length of 800 n.p. when analyzing the integrity of the DNA, it is determined using the selected forward primer: 5′-GCCTCATCTTGGGCCTGTGTTATCTC-3 ′, reverse primer: 5′-CCACTTGATAAGAGGTCCCAAGACTT-3 ′, and the presence of the second extended fragment is 2340 bp in length. when analyzing the integrity of the DNA is determined using the selected forward primer: 5′-AAGTGGGGTTGGTAGGATTCT-3 ′, reverse primer: 5′-ACTATACAATACAGCAACTATCCT-3 ′.

In the inventive method, the quality and quantity of the isolated DNA is evaluated by PCR analysis of short fragments of 141 n.p. and 153 n.p. with the selection of a specific test system developed by the authors that detects genomic DNA in duplex mode.

The quality of the extracted DNA means whether it is well cleared of impurities that can be inhibitors for polymerase, an enzyme that synthesizes PCR fragments, and by quantity, is its concentration sufficient for subsequent PCR of extended fragments. Since genomic DNA normally breaks down into 190-210 bp fragments when passing through the gastrointestinal tract, under optimal conditions for PCR fragments of 141 and / or 153 bp length found in the stool of both healthy and colorectal cancer patients.

In the prototype, UV spectroscopy at 260/280 nm was used to determine the quality of the extracted DNA, and the amount of DNA was estimated using an Invitrogen Qubit fluorimeter and a BR Assay Kit (Invitrogen Q32853). These operations require the use of expensive equipment and highly qualified specialists.

In addition, in the proposed method (unlike the prototype), to determine the integrity of the genomic DNA, two extended DNA fragments (fragments of TP53 and MLH1 genes) are used instead of one extended TP53 gene fragment, which provide higher sensitivity of the test.

The fact is that to date, for colorectal cancer from the literature, two main developmental paths are known in which two different groups of genes are damaged [Fearon, E.R. Molecular genetics of colorectal cancer. Annu. Rev. Pathol. Mech Dis. 2011, 6, 479-507] [11].

Therefore, the authors of the proposed method have chosen both for the analysis of the quality and quantity of DNA, and for the analysis of DNA integrity for one gene fragment from each group, the simultaneous damage of which occurs with a small probability:

the short and long fragments of the TP53 gene belong to the first group of genes,

the short BLM gene fragment and the long MLH1 gene fragment belong to the second group of genes.

Indeed, if the TP53 gene is damaged in the patient with CRC, a fragment of this gene (as in the prototype) may not be detected by PCR analysis, but in the inventive method a fragment of the MLH1 gene will be detected, which increases the sensitivity of the test, because eliminates the possibility of a false negative conclusion that takes place in the prototype.

Thus, the choice of the length of these fragments, their localization in the human genome, the structure of the primers and the conditions for the PCR analysis are those moments that were devoted to experiments in the development of the proposed method.

These fragments 800 np long TP53 gene and 2340 bp fragment MLH1 gene has not been used previously for DNA integrity analysis, which indicates a certain inventive step.

PCR analysis of DNA fragments is carried out by sequentially carrying out a polymerase chain reaction and visualizing its products by electrophoresis.

The combination of all the features (operations) in the present method provides an increase in sensitivity.

An example of a specific implementation.

For three days before sampling, colonoscopy and other invasive manipulations with the rectum of the patient were not performed. The study involved 60 people, including 42 patients with colorectal cancer and 18 people who did not have tumors of the gastrointestinal tract. The presence or absence of a tumor was determined by fibrocolonoscopy. Demographic data were obtained (including for the control group) and clinical and pathological data of patients. After surgery, all tumors were histologically characterized.

Immediately after defecation, about 1 ml of stool is taken and placed in a sealed plastic container containing 1 ml of 0.5 M EDTA. This solution allows you to keep fecal DNA intact for 24 hours, protecting against the action of various enzymes. Selection can be made at home. Isolation of total DNA is performed in the next 24 hours after sampling. Isolation is carried out using a DNA-sorb-B whale (AmpliSens). The extraction method is based on specific reversible sorption of DNA in the presence of chaotropic salts on silica gel particles [http://www.interlabservice.ru/catalog/reagents/index.php?sid=l402&id=8693)] [12].

Assessment of the quality and quantity of DNA extracted from the patient’s stool.

To assess the quality and quantity of the extracted DNA, a preliminary PCR analysis of two short fragments with a length of less than 200 n.p. A test system was developed that detects these DNA fragments in duplex mode, which allowed to reduce the labor costs for PCR by half, and also reduce the consumption of reagents by 1.5 times. The first fragment is 141 n.p. located in the coding region of the TP53 gene (exon 9). Direct primer: 5′-ATTCACTTTTATCACCTTTCCTTGCC-3 ′; reverse primer: 5′-CCACTTGATAAGAGGTCCCAAGACTT-3 ′. The second fragment is 153 n.p. located in the coding region of the BLM gene. Direct primer: 5′-CTCTGCCACCAGGAAGAATC-3 ′; reverse primer: 5′-ACAGCAGTGCTTGTGAGAAC-3 ′.

6-10 ng of DNA were taken into the reaction mixture; 1 unit Taq polymerases (NPO SibEnzyme).

Temperature reaction: initial denaturation 4 min / 95 ° C; 30 cycles, 40 s / 94 ° C, 40 s / 55 ° C, 40 s / 72 ° C; final polymerization of 7 min / 72 ° C in the Tertsik thermocycler (DNA-Technology).

After that, electrophoresis of PCR products was carried out in a polyacrylamide gel and the gel was stained with silver nitrate.

The presence in PCR products of both fragments with a length of less than 200 n.p. or at least one of them indicates that the extracted DNA is suitable in its quality and quantity for further analysis on its integrity.

Two extended DNA fragments were also selected for analysis of DNA integrity during PCR analysis.

The first fragment is 800 bp long, containing 7, 8, and 9 exons of the TP53 gene. We selected primers using the Primer Premier 6 program. Direct primer: 5′-GCCTCATCTTGGGCCTGTGTTATCTC-3 ′; reverse primer: 5′-CCACTTGATAAGAGGTCCCAAGACTT-3 ′.

Temperature reaction: initial denaturation 4 min / 95 ° C; 10 cycles 50 s / 95 ° C, 40 s / 63 ° C, 90 s / 72 ° C; 30 cycles, 50 s / 95 ° C, 40 s / 56 ° C, 90 s / 72 ° C; final polymerization of 7 min / 72 ° C in the Tertsik thermocycler (DNA-Technology).

(Unlike the prototype, when annealing the primers on the matrix, the temperature “step” is used, first 10 cycles at 63 ° C, and then 30 cycles at 56 ° C). The use of temperature “steps” increases the sensitivity and specificity of PCR analysis.

The second fragment is 2340 bp long, containing 14 and 15 exons of the MLH1 gene. Forward primer: 5′-AAGTGGGGTTGGTAGGATTCT-3 ′, reverse primer: 5′-ACTATACAATACAGCAACTATCCT-3 ′.

Temperature reaction: initial denaturation 3 min / 95 ° C; 40 cycles, 30 s / 95 ° C, 40 s / 60 ° C, 2 min / 72 ° C; final polymerization of 7 min / 72 ° C in the Tertsik thermocycler (DNA-Technology).

For both long fragments, the polymerase chain reaction was carried out using an Evrogen Encyclo PCR kit. 1-9 ng DNA was taken into the reaction mixture; 1 unit Encyclo polymerase (The prototype uses Taq DNA polymerase; Encyclo polymerase is preferred for amplification of long DNA fragments).

To visualize PCR products, polyacrylamide gel electrophoresis was performed followed by staining with silver nitrate.

DNA fragments 800 np long and / or 2340 n.a. 32 of 42 patients with colorectal cancer were found in stool. In the control group, such DNA fragments were absent in the stool.

To analyze the data, we used the Graphpad Instat program [http://www.keygenguru.com/serial/graphpad_instat_3_0.html] [13]. Differences between groups were considered significant at the 95% probability level at p <0.05.

The sensitivity and specificity of the method were 76% and 100%, respectively. With a statistical confidence level of more than 95%, there were no statistically significant differences (p = 1) between the sensitivity of the method for patients with I-II (73%) and III-IV (70%) stages, which demonstrates the capabilities of the method for early diagnosis of CRC.

Analysis results can be obtained within 3 days after delivery of the sample to the laboratory.

The method is advisable to apply in a mass population survey, because It is not associated with the use of expensive, complex equipment and highly qualified personnel, and allows the identification of CRC in the early stages.

Bibliography

1. Duffy M.J. Use of molecular markers for predicting therapy response in cancer patients // J. Cancer Treatment Reviews. - 2011 .-- P. 151-159.

2.http: //www.colonoscopy.ru/patient/procedure1.htm.

3. Church TR, Wandell M, Lofton-Day C et al. Prospective evaluation of methylated SEPT9 in plasma for detection of asymptomatic colorectal cancer. Gut. 2014 Feb; 63 (2): 317-25.

4. http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/MedicalDevices/MedicalDevicesAdvisoryCommittee/MolecularandClinicalGeneticsPanel/UCM390234.pdf.

5. http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/MedicalDevices/MedicalDevicesAdvisoryCommittee/MolecularandClinicalGeneticsPanel/UCM391101.pdf.

6. Thomas F. Imperiale, M. D., David F et al. Multitarget Stool DNA Testing for Colorectal-Cancer Screening N Engl J Med 2014; 370: 1287-1297.

7. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972; 26: 239-57.

8. Boynton K.A., Summerhayes I.C., Ahlquist D.Α., Shuber A.P. DNA integrity as a potential marker for stool_based detection of colorectal cancer // Clin. Chem. - 2003. - Vol. 49. - P. 1058-1065.

9. Yehya A.H., Yusoff N.M., Khalid I.A. et al. Pilot study of the sensitivity and specificity of the DNA integrity assay for stool-based detection of colorectal cancer in Malaysian patients // Asian Pac J Cancer Prev. - 2012. - Vol. 13 (5). - P. 1869-1872. - prototype.

10. Beroud C & Soussi Τ (1998). p53 gene mutation: software and database. Nucleic Acids Res, 1, 200-4.

11. Fearon E.R. Molecular genetics of colorectal cancer. Annu. Rev. Pathol. Mech Dis. 2011, 6, 479-507.

12. http://www.interlabservice.ru/catalog/reagents/index.php?sid=1402&id=8693).

13. http://www.keygenguru.com/serial/graphpad_instat_3_0.html.

Claims (5)

1. A method for non-invasive diagnosis of colorectal cancer, based on the analysis of DNA integrity from the patient’s stool, including sampling and transportation of patient’s stool samples, extraction of total DNA from the patient’s stool, quality control and quantity of extracted DNA, and identification of extended fragments for determining DNA integrity by PCR analysis , by the presence of which it is judged that the patient has colorectal cancer, characterized in that the stool samples are transported in a medium containing 0.5 M EDTA, and to assess the quality and quantity of excretion This DNA is carried out by PCR analysis of two short DNA fragments with a length of less than 200 n.p. in duplex mode: the first fragment is 141 bp in length, located in the coding region of the TP53 gene, the second fragment is 153 bp, located in the coding region of the BLM gene, and two extended fragments are used to evaluate the integrity of genomic DNA by PCR analysis: the first fragment is 800 bp long, containing 7, 8 and 9 exons of the TP53 gene, the second fragment is 2340 bp long, containing 14 and 15 exons of the MLH1 gene, and for both extended fragments the polymerase chain reaction is carried out using the Evrogen whale Encyclo PCR ", which includes Encyclo polymerase, and found The presence of these fragments or at least one of these two fragments in PCR products indicates the presence of colorectal cancer in the patient. 2. A non-invasive method for the diagnosis of colorectal cancer according to claim 1, characterized in that the PCR analysis of the first short DNA fragment with a length of 141 n.p. carried out using a direct primer 5′-ATTCACTTTTATCACCTTTCCTTGCC-3 ′, the reverse primer: 5′-CCACTTGATAAGAGGTCCCAAGACTT-3. 3. The method for non-invasive diagnosis of colorectal cancer according to claim 1, characterized in that the PCR analysis of the second short DNA fragment of 153 n.p. carried out using a direct primer 5′-CTCTGCCACCAGGAAGAATC-3 ′, the reverse primer: 5′-ACAGCAGTGCTTGTGAGAAC-3 ′. 4. The method for non-invasive diagnosis of colorectal cancer according to claim 1, characterized in that the presence of the first fragment 800 np in length when analyzing the integrity of the DNA is determined using the selected forward primer: 5′-GCCTCATCTTGGGCCTGTGTTATCTC-3 ′, reverse primer: 5′-CCACTTGATAAGAGGTCCCAAGACTT-3 ′. 5. The non-invasive diagnostic method for colorectal cancer according to claim 1, characterized in that the presence of a second fragment of 2340 n.p. when analyzing the integrity of the DNA is determined using the selected forward primer: 5′-AAGTGGGGTTGGTAGGATTCT-3 ′, reverse primer: 5′-ACTATACAATACAGCAACTATCCT-3 ′.