WO2017101756A1 - Kit de détection du cancer colorectal - Google Patents

Kit de détection du cancer colorectal Download PDF

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WO2017101756A1
WO2017101756A1 PCT/CN2016/109627 CN2016109627W WO2017101756A1 WO 2017101756 A1 WO2017101756 A1 WO 2017101756A1 CN 2016109627 W CN2016109627 W CN 2016109627W WO 2017101756 A1 WO2017101756 A1 WO 2017101756A1
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sample
colorectal cancer
pvpp
alu
amplification
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PCT/CN2016/109627
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Chinese (zh)
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邹鸿志
牛智通
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广州市康立明生物科技有限责任公司
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Priority to MYPI2018702149A priority Critical patent/MY189407A/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • the invention belongs to the field of biomedicine and relates to a kit for detecting colorectal cancer, in particular to a simple colorectal cancer detecting kit using feces as a test sample.
  • the fecal occult blood test is to detect the blood components in the stool (the test object is hemoglobin). If multiple, continuous positive reactions suggest gastrointestinal bleeding, further examination should be done to guard against the occurrence of intestinal tumors.
  • the advantage of this method is that the results are fast and clear, and the results can be semi-quantitative.
  • it has a series of shortcomings: First, its specificity is poor, and it is greatly affected by diet.
  • the patient Before the occult blood test, the patient should be advised to avoid taking iron, animal blood, liver, lean meat and a large number of green leafy vegetables for 3 days. If there is bleeding from the gums, swallow bloody saliva, in order to prevent the fecal occult blood test from being false positive; secondly, The sensitivity is low, and the amount of bleeding >90 ⁇ g/ml can be detected; again, the method has more restrictive conditions: the patient has a long preparation time in advance, and the reaction time is different due to different materials, and the judgment of color development is different, so in the same method In the test, errors can also be generated. Therefore, the patient is required to continuously measure the stool samples of different days in the general test.
  • colonoscopy is the most effective and reliable diagnostic method for the diagnosis of intestinal lesions.
  • the vast majority of patients with early stage bowel cancer can be found and confirmed by endoscopy. It can be inserted through the anus to examine the rectum, sigmoid colon, descending colon, transverse colon, ascending colon and cecum and a small intestine (the ileocecal end) connected to the large intestine.
  • a small intestine the ileocecal end
  • Colonoscopy is the main method that cannot be replaced by other methods of diagnosis and treatment. At present, colonoscopy is the most effective and reliable diagnostic method for the diagnosis of intestinal lesions. However, it also has a series of shortcomings: First, it needs to be prepared before examination. For colonoscopy, it is necessary to start eating liquid or less slag and semi-liquid diet 3 days before the examination, check the fasting in the morning, and start taking mannitol the day before the examination. The cathartic cleaner cleans the intestines, and the amount of fluid is usually as high as 2L. The enema is cleaned to ensure the cleanliness of the intestines, and then can no longer be eaten.
  • the doctor will inject a certain amount of gas into the intestinal lumen through the enteroscopy to make the intestines expand for observation. Due to the twists and turns of the colon structure, the examinee may have varying degrees of pain or pulling feeling during the examination. For subjects who are overly stressed or highly intestinal, a sedative or antispasmodic drug is required. For children who cannot cooperate, it needs to be performed under anesthesia. Secondly, it has high requirements for the doctor's proficiency in operation, and it is easy for beginners to miss the diagnosis of missing lesions under the colonoscopy. Due to the need to inject gas during the inspection process, the pressure in the intestine is increased, which easily causes perforation. Again, the patient is laborious, time consuming, and painful during the examination. And the inspection cost is high, and it is difficult to promote it on a large scale.
  • the molecular diagnostic method is a rapidly developing cancer diagnosis method in recent years.
  • Currently reported (Wu Yong et al) molecular diagnostic method for colorectal cancer there is a method using fecal DNA detection combined with FOBT combined diagnosis, the method needs to first extract the DNA in the feces, and then detect the markers APC, K- Ras, p53.
  • Feces are food residue excretions of humans or animals. A quarter of the feces is water, and the rest are mostly protein, inorganic, fat, undigested dietary fiber, dehydrated digestive residue, and cells that have fallen from the intestines and dead bacteria, as well as vitamin K. , vitamin B, fecal pigment and urobilin. Under normal circumstances, the human digestive tract epithelium will also shed some cells. These normal detached cells undergo programmed necrosis, and the nucleic acids contained in the cells are naturally degraded. Therefore, human nucleic acid is not detected in normal human feces. .
  • Li Jiansheng et al studied the significance of quantitative detection of long DNA fragments in feces in the diagnosis of colorectal cancer. It discloses the detection of long DNA fragments in feces by fluorescence quantitative Alu PCR, which can be used as a potential screening method for colorectal cancer.
  • a typical human genome Alu sequence is 282 bp long and consists of two homologous but distinct subunits.
  • the restriction cleavage enzyme AluI can be cleaved into two segments of 130 bp and 170 bp, so it is named as the Alu sequence, and there is one Alu sequence per 5 kb of DNA.
  • the Alu sequences are generally scattered and a few are clustered.
  • the Alu repeats are concentrated in the most active chromosomal segments of the gene. Almost all of the known gene introns are found in the Alu sequence. Due to the specificity of the Alu sequence, amplification of the sequence using specific primers can distinguish human genomic DNA from non-human genomic DNA.
  • the method disclosed by Li Jiansheng et al first extracts the DNA in the feces and then tests the Alu therein. The total sensitivity of the diagnosis of colorectal cancer is 46.67%.
  • nucleic acid extraction first using mechanical force to fully mix and fragment the feces, and then using organic solvents or magnetic beads to capture all or the need
  • the nucleic acids are enriched and then purified before PCR amplification experiments are cumbersome and time consuming.
  • the object of the present invention is to provide a colorectal cancer detecting reagent which is convenient to use and simple in pretreatment.
  • the present invention provides the following technical solutions:
  • the present invention provides a colorectal cancer detecting kit comprising Alu (Arthrobacterluteus) detecting reagent and PvPP.
  • the present invention also provides a colorectal cancer detecting kit comprising Alu (Arthrobacterluteus) detecting reagent and PvPP.
  • the invention finds a suitable sample which is a stool sample.
  • the sample obtained by this non-invasive sampling method can detect the intestinal cancer marker by the simple method first discovered by the present invention.
  • the phenol extraction method first break the cells with egg enzyme K, SDS, digest the protein, and then use phenol and phenol-chloro DNA size of 100-150kb;
  • isopropanol precipitation method replace the ethanol with two volumes of isopropanol, precipitate DNA, and at the same time remove small RNA (soluble state in isopropanol);
  • the surfactant rapid preparation method TritonX-100A or NP40 surfactant to break the cells, and then proteinase K or phenol to remove protein, ethanol precipitation or dialysis; and so on.
  • the present invention finds that it is possible to carry out colorectal cancer markers without performing DNA extraction of feces, but by suspending the supernatant of feces or feces without DNA extraction for simple pretreatment and direct PCR amplification. Method of stool detection. Therefore, the detection kit provided by the present invention does not contain a DNA extraction reagent such as an enzyme or an organic solvent. This makes it an environmentally friendly kit while being easy to handle.
  • a suitable pretreatment method (adding PvPP) can further promote the smooth expansion of Alu.
  • the inventors successfully screened the Alu marker, which can be directly PCR-amplified and detected by PvPP pretreatment without proper fecal DNA extraction under appropriate conditions. .
  • a colorectal cancer detection kit which can directly perform PCR amplification using feces as a test sample, which contains an Alu detection reagent, and particularly preferably, It is also required to be provided with crosslinked polyvinylpyrrolidone (PvPP, polyvinyl-polypyrrolidone).
  • PvPP polyvinyl-polypyrrolidone
  • the kit can optionally be equipped with a dilution directly diluted with the stool sample to facilitate the use of the operator, but of course due to dilution
  • the acquisition of a liquid such as water is simple, and therefore, the diluent is not a fixed component of the kit.
  • PvPP Cross-linked polyvinylpyrrolidone
  • crospovidone Cross-linked polyvinylpyrrolidone
  • the inventors can remove the PCR inhibitor by directly diluting the stool sample and adding PvPP, and Alu can directly perform PCR amplification and detection in the PvPP-treated stool sample, and can be smoothly realized without any DNA extraction. Alu detection.
  • the mass ratio of the volume of the diluent to the stool sample is at least 20:1; more preferably 20:1 to 8000:1; more preferably 20:1 to 4000:1; more preferably 40:1 to 4000:1.
  • the ratio of the PvPP content to the stool sample mass ratio is 1:10-10:1.
  • the PvPP may be a suspension of water or loaded in a centrifuge tube.
  • the PvPP employed is a suspension of 1% to 50% concentration (w/w). It is preferably 5% to 20%.
  • the PvPP is loaded on the centrifuge tube and can be obtained by adding a proper amount of the PvPP suspension of 1%-50% concentration to the centrifuge tube. Centrifuge at 750g-17000g for 30s-15min, and then absorb the upper layer of liquid after centrifugation.
  • the reagent/kit provided by the present invention can easily realize the diagnosis of colorectal cancer by the following sample processing and detection methods.
  • the sample used in the method is fresh or a stool sample to which a buffer (such as a saline, TE, PBS, etc. solution containing no DNA degradation component) is added;
  • a buffer such as a saline, TE, PBS, etc. solution containing no DNA degradation component
  • the amount of sample used is preferably at least 10 mg of feces
  • the sample is subjected to PvPP treatment, and the PvPP used may be in a dry powder state or a shape mixed with other plasticizers, and may be filled in a specific vessel or in a water suspension state.
  • the amount of the PvPP is related to the amount of feces: the mass ratio of PvPP to feces is 1:10 or more, more preferably 1:5 or more.
  • the sample is diluted with a diluent (water or TE buffer, etc.): the sample is subjected to a high dilution of at least 20 times; more preferably 100-2000 times, more preferably 200-800 times. That is to say, the amount of diluent per g of feces is at least 20 ml.
  • a diluent water or TE buffer, etc.
  • Detection is performed using a general fluorescent quantitative PCR system (using a polymerase, a saturated or unsaturated fluorescent dye, and compatible Mg 2+ , dNTPs or other additives) and a corresponding fluorescent quantitative PCR machine.
  • a general fluorescent quantitative PCR system using a polymerase, a saturated or unsaturated fluorescent dye, and compatible Mg 2+ , dNTPs or other additives
  • a corresponding fluorescent quantitative PCR machine using a general fluorescent quantitative PCR system (using a polymerase, a saturated or unsaturated fluorescent dye, and compatible Mg 2+ , dNTPs or other additives) and a corresponding fluorescent quantitative PCR machine.
  • Amplification is carried out according to a PCR reaction system common to those skilled in the art.
  • the reaction temperature rise and fall program is widely adapted, and is amplified according to a 2-step method or a 3-step method commonly used by those skilled in the art.
  • the primer used in the invention may be a primer designed by a conventional method.
  • a variety of primers for Alu colon cancer markers in fecal samples can be amplified using the simple sample processing method of the present invention.
  • SEQ ID NO. 1 GCCTGTAATCCCAGCACTTT
  • SEQ ID NO. 2 CTCACTGCAACCTCCACCTC; or,
  • SEQ ID NO. 3 TCGCCCAGGCTGGAGTGCA
  • SEQ ID NO. 4 ACGCCTGTAATCCCAGCACTT; or,
  • SEQ ID NO. 6 ATCAGCACGGGAGTTTTGAC; or,
  • SEQ ID NO. 7 CCAGGAGTTCTGGGCTGTAG
  • SEQ ID NO. 6 ATCAGCACGGGAGTTTTGAC; or,
  • SEQ ID NO. 8 AAGAGACGGGGTCTCGCTAT; or
  • SEQ ID NO. 9 CTGATCAGCACGGGAGTTTT; or
  • SEQ ID NO. 10 GTGCCTGTAGTCCCAGCTA
  • SEQ ID NO. 11 ACTCCTGGACTCAAGCGATC; or
  • SEQ ID NO. 12 CTACTCGGGAGGCTGAGGT
  • SEQ ID NO. 13 CTGGACTCAAGCGATCCTCC.
  • the primers employed in the amplification system are:
  • SEQ ID NO. 1 GCCTGTAATCCCAGCACTTT
  • SEQ ID NO. 2 CTCACTGCAACCTCCACCTC.
  • the invention also provides a method for detecting colorectal cancer, which uses feces as a test sample, and the test sample is mixed with the detection reagent in the colorectal cancer detection kit, and then amplified, and the detection result is obtained according to the amplification result. result.
  • the target of amplification is a colorectal cancer marker; the colorectal cancer marker is selected from the group consisting of Alu, Kras, P53, APC.
  • the obtaining the detection result is comparing the amplification result of the sample to be tested with the normal sample, and when the amplification result of the sample to be tested is significantly different or significantly different from the amplification result of the normal sample, The donor of the sample to be tested is positive.
  • the present invention has the following advantages:
  • the solution of the invention does not require cumbersome sample processing, saving time and labor.
  • the method is directly used after dilution of the sample, preferably PvPP treatment.
  • the whole process is simple to operate and takes a short time. It takes only 2 minutes to process a sample on average.
  • the traditional method to detect human nucleic acid in feces is to use nucleic acid extraction method, first use mechanical force to make the feces fully mixed and fragmented, and then use organic solvent or magnetic bead capture to remove all Or the required nucleic acids are enriched and then purified before PCR amplification In the experiment, it takes about 4 hours for the entire process to process a single sample.
  • the conventional method is not only time consuming and labor intensive, but also causes contamination and errors between samples due to cumbersome operation steps.
  • Amplification by fluorescence quantitative PCR instrument direct detection of DNA fragments in feces, easy to operate, high sensitivity.
  • Traditional colorectal cancer screening method is to detect the content of hemoglobin in feces by chemical method or immunological method. This method can only be detected in feces after intestinal bleeding, and early detection of intestinal lesions and lesions cannot be detected. The case of bleeding is low, and the sensitivity of detection by chemical method is low.
  • the detection by immunoassay can obtain high sensitivity, the detection method takes a long time, and generally takes about 3 hours.
  • the use of real-time PCR to amplify nucleic acids allows the amount of nucleic acid in the original feces to be magnified by hundreds of millions of times, producing a very pronounced signal. Moreover, the entire PCR amplification process is short, and nearly 100 samples can be amplified in 40 minutes at the earliest.
  • the test sample used in the present invention is a stool sample of a patient. Samples are very easy to obtain and do not cause any pain or inconvenience to the patient.
  • the sample size is very small, and the sampling process is simple and convenient. At the same time, the samples are easy to mail or take them to the hospital for inspection.
  • the method is low in cost, the operation process is very simple, the requirements on personnel and equipment are low, the detection speed is fast, the predictability of the lesion is good, and the promotion is favorable.
  • the invention uses stool as a test sample, and contains Alu detection reagent and PvPP.
  • the invention has been explored many times in many colorectal cancer markers, and it is found that Alu can directly dilute the sample for PCR amplification without complicated steps such as cell disruption and DNA extraction under suitable simple pretreatment conditions. And get good sensitivity and specificity.
  • Figure 1 (a) is a graph showing the results of amplification at 5, 10, 20, 40, 80, 250, 500, 1000, 2000, 4000-fold dilutions of samples not treated with PvPP;
  • Figure 1 (b) is a graph showing the results of amplification at 5, 10, 20, 40, 80, 250, 500, 1000, 2000, 4000-fold dilutions of samples treated with PvPP;
  • Figure 1 (c) is a graph showing the relationship between the sample dilution number and the post-amplification fluorescence value
  • Figure 1 (d) is a graph showing the relationship between the sample dilution number and the post-amplification Ct value
  • Figure 2 (a) is a graph showing the amplification results of 5, 10, 20, 40, 80, 250, 500, 1000, 2000, 4000 using the same sample treated with PvPP, and then performing amplification of the Alu sequence;
  • Figure 2(b) is a graph showing the results of amplification of the ACTB sequence using the same sample of PvPP treated for 5, 10, 20, 40, 80, 250, 500, 1000, 2000, 4000 different dilutions;
  • Figure 3 is a graph showing the results of Alu detection according to the above method in 146 stool samples, 73 cases of colorectal cancer (Ca) and 73 cases of normal (Norm);
  • Figure 4 shows a sample of 146 feces, including 73 cases of colorectal cancer (Ca) and 73 cases of normal (Norm). Alu was measured according to the above method, and the colonoscopy results of patients from which fecal samples were derived were used as criteria. Receiver operating characteristic curve analysis (ROC analysis);
  • Figure 5 (A) is the same sample processed according to the above method, respectively, Alu sequence and KRAS sequence detection, PCR amplification results;
  • Figure 5 (B) is the same sample processed according to the above method, respectively, Alu sequence and p53 sequence detection, PCR amplification results;
  • Figure 5 (C) is the same sample processed according to the above method, respectively, Alu sequence and APC sequence detection, PCR amplification results;
  • Figure 6 is an embodiment of a centrifuge tube used when PvPP is loaded in a centrifuge tube.
  • the invention discloses a colorectal cancer detection kit, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.
  • the raw materials and reagents used in the colorectal cancer detection kit provided by the present invention are commercially available.
  • the colorectal cancer diagnostic reagent provided by the invention can conveniently use feces as a test sample. Diagnosis of colorectal cancer.
  • a phenol-free water was used to prepare a PvPP suspension having a mass fraction of 10%. Shake the PvPP suspension thoroughly.
  • the PvPP suspension charged into the centrifuge tube will be divided into upper and lower layers. Using a pipette, the liquid on the PvPP precipitation layer is carefully sucked out to obtain a relatively dry PvPP.
  • a phenol-free water was used to prepare a PvPP suspension having a mass fraction of 10%. Shake the PvPP suspension thoroughly.
  • the PvPP suspension charged into the centrifuge tube will be divided into upper and lower layers. Using a pipette, the liquid on the PvPP precipitation layer is carefully sucked out to obtain a relatively dry PvPP.
  • the diluted sample in this example was subjected to the amplification experiment and the result analysis as in Example 3.
  • Figure 1 (a) amplification curve shows the PCR amplification curve of the same sample that was not treated with PvPP at 5, 10, 20, 40, 80, 250, 500, 1000, 2000, 4000 times dilution, the sample can be seen
  • the dilution can be directly diluted and subjected to PCR amplification to obtain an ideal amplification map. To achieve the detection of Alu.
  • the amplification line in Fig. 1(b) shows the PCR amplification curves of samples treated with PvPP at dilutions of 5, 10, 20, 40, 80, 250, 500, 1000, 2000, 4000 times, respectively, and the dilution can be seen in the figure.
  • the PCR amplification curve of the sample treated with PvPP is higher than the Ct value when the PvPP is not used, and the fluorescence value is increased; in the case of relatively high dilution, the sample is treated with PvPP and not passed.
  • the Ct value of the PCR amplification curve after PvPP treatment did not change, and the fluorescence value increased slightly, indicating that the inhibitor in the sample was less after treatment with PvPP, and the Alu detection was more desirable to some extent.
  • Figure 1 (c) compares the final fluorescence values of the amplification curves between different dilutions of the same treatment sample (treated by PvPP). It can be seen from the figure that as the dilution increases, the end of the amplification curve The fluorescence value is continuously increased; in Figure 1(d), as the dilution increases, the Ct value of the PCR amplification of the sample increases; the above two images show that the ideal PCR amplification can be achieved within a certain dilution range.
  • the sample was processed according to the method described in Example 2.
  • Sample Processing Method 2 and a series of templates of different dilutions were obtained, and then according to the quantitative quantitative PCR amplification system described in Example 3, different dilution templates for the same sample.
  • the Alu gene and the ACTB gene were determined using the respective primers, respectively.
  • the result is shown in Figure 2.
  • the results of Figure 2(a) show amplification of the Alu gene, and the results indicate that the Alu fragment can be amplified using this method at a certain dilution.
  • the results of Figure 2(b) show the effect of using this method on the amplification of the ACTB gene. From the results, no matter the dilution of the template, there is no amplification curve for the detection of ACTB using this method.
  • Example 6 Test for detecting the effect of Alu levels in feces on colorectal cancer
  • Example 1 Sample Processing Method 1 The 146 samples were processed, and then subjected to QPCR quantitative detection according to the QPCR reaction system and the reaction procedure described in Example 3. The results are shown in FIG. 3 and FIG. .
  • the Alu level in the colorectal cancer samples was significantly higher than that in the normal samples, and was significantly different from the normal group.
  • the early screening of colorectal cancer was performed according to the method. The method can distinguish between normal and colorectal cancer patients, and the detection sensitivity is 71.5%, the specificity is 90.3%, and the area under the subject curve. It is 0.875.
  • samples diluted with PvPP are diluted by 5, 10, 20, 40, 80, 250, 500, 1000, 2000, 4000, respectively, and samples having different dilutions are respectively used to determine Alu.
  • some genes related to the prediction of colorectal cancer such as Kras, P53, APC and other genes.
  • SEQ ID NO. 14 Fp: TGAGATTTTGGGGTGGTGGT
  • SEQ ID NO.17 Rp:GAAGGACTCGGATTTCACGC
  • SEQ ID NO. 18 Fp: CTCTCCCCAGCCAAAGAAGA
  • the PCR system is as follows:
  • the PCR reaction procedure is as follows:
  • Figure 5A is a comparison of amplification results of Alu and Kras genes in samples after treatment of fecal samples from colon cancer patients. From the measured results, the method has a very good effect on the determination of Alu, and an amplification curve starts at a 20-fold dilution, and the amplification curve moves backward as the dilution number increases. The use of the same template using the primers for detecting the Kras gene did not produce efficient amplification at any dilution.
  • Fig. 5B is a comparison of amplification results of Alu and P53 genes in a sample after treating a stool sample of a colon cancer patient. From the results of the measurement, the method was very effective for the determination of Alu, and the amplification curve was started at a dilution of 20 times, and the amplification curve was moved backward as the dilution pair increased, and P53 was detected. The primers of the gene amplify the same template, and no effective amplification can be produced at any dilution.
  • Figure 5C is a comparison of amplification results of Alu and APC genes in samples after treatment of fecal samples from colon cancer patients. From the results of the measurement, the method was very effective for the determination of Alu, and the amplification curve was started at a dilution of 20 times, and the amplification curve was amplified with the dilution pair. The line moves backwards, but the same template is amplified with primers that detect the APC gene, and no effective amplification can be produced at any dilution.

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Abstract

La présente invention concerne un kit de détection du cancer colorectal, qui est plus particulièrement un kit de détection du cancer colorectal utilisant des excréments en tant qu'échantillon de détection, comprenant un réactif de détection Alu et de la PVPP. Après plusieurs explorations, on a découvert qu'Alu, parmi de nombreux marqueurs du cancer colorectal, peut être détecté directement en diluant l'échantillon pour réaliser une amplification par PCR sur la base d'un prétraitement correct des échantillons, sans subir les étapes complexes telles que la rupture cellulaire et l'extraction d'ADN, et une bonne sensibilité et une grande spécificité sont obtenues.
PCT/CN2016/109627 2015-12-14 2016-12-13 Kit de détection du cancer colorectal WO2017101756A1 (fr)

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CN105349686A (zh) * 2015-12-14 2016-02-24 广州市康立明生物科技有限责任公司 一种结直肠癌检测试剂盒
CN111197073A (zh) * 2019-12-19 2020-05-26 武汉艾米森生命科技有限公司 从粪便中提取dna样本的方法和结直肠癌相关基因的甲基化检测方法
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