Gene methylation endometrial cancer detection kit

Abstract

The present invention relates to the technical field of detection kits, and discloses a gene methylation endometrial cancer detection kit, which comprises PCR reaction liquid for RP4, RP11 and CTD methylation detection, a negative control and a positive control, and the PCR reaction liquid comprises a PCR reaction liquid A and a PCR reaction liquid B; the PCR reaction liquid A comprises dNTP (deoxyribonucleoside triphosphate), a salt ion component, a stabilizer and DNA (deoxyribonucleic acid) polymerase; the PCR reaction liquid B comprises an RP4 primer, an RP11 primer, a CTD primer, a GAPDH primer and a fluorescent probe. The kit is based on the fluorescent quantitative PCR principle, the methylation of RP4, RP11 and CTD genes in a cervical epidermal cell sample is detected by a methylation PCR fluorescent probe method, the detection accuracy is high, the operation is convenient and rapid, and a reliable reference basis can be provided for clinical diagnosis of endometrial cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of detection kits, and in particular to a gene methylation endometrial cancer detection kit. Background Art

[0002] Endometrial cancer is a group of epithelial malignant tumors that occur in the endometrium. It is one of the three major malignant tumors of the female reproductive tract. It is prone to occur in perimenopausal and postmenopausal women. In recent years, it has shown a trend of younger patients. The five-year survival rate of patients with early (stage I and II) endometrial cancer is more than 70%, the five-year survival rate of patients with stage III is about 40-50%, and the five-year survival rate of patients with stage IV is only 15-20%. Therefore, achieving early diagnosis of endometrial cancer is an important measure to improve patient prognosis. Early diagnosis methods for endometrial cancer include diagnostic curettage, cytology, transvaginal ultrasound and hysteroscopy, but these diagnostic methods all have certain drawbacks. Clinical diagnosis mainly relies on diagnostic curettage, which is traumatic for sampling. The uterine wall of lactating women or those with invasive endometrial cancer is weak, and curettage may cause uterine perforation, causing harm to patients. Cytology is closest to the screening strategy, but there is currently a lack of unified cytology standards. Transvaginal ultrasound cannot determine the threshold of endometrial thickness in endometrial lesions. Hysteroscopy is expensive and has limited diagnostic value when used alone. Therefore, a highly accurate, convenient, and less invasive detection method is urgently needed for early detection of endometrial cancer.

[0003] According to current clinical data, prognosis is closely related to the time of diagnosis. The earlier the diagnosis (FIGO stage I-II), the better the prognosis. For example, the 5-year survival rate drops from 85% in stage I to 25% in stage IV. At present, transvaginal ultrasound scan (TVUS) combined with endometrial biopsy is a common method for diagnosing EC. However, there is great heterogeneity in accuracy, and the sensitivity of gynecological tumor diseases is between 0.5 and 0.8. On the other hand, endometrial biopsy is not only invasive and uncomfortable, but also has a large number of technical problems and is limited by the amount of tissue. In addition, personalized treatment after diagnosis also faces great challenges. For example, changes in histological classification increase the complexity of patient care, making it challenging to compare randomized trials, make treatment decisions, and accurately predict prognosis. In addition, many unresolved research issues remain, ranging from treatment toxicity, diagnostic procedures to adjuvant therapy. Therefore, reliable diagnosis is necessary for adequate treatment and reducing EC mortality.

[0004] LncRNAs are considered to be an important part of the epigenetic regulatory network, regulating gene expression and post-transcription by affecting the structure of protoproteins, chromatin, and transcription factors. Many studies have shown that some lncRNAs affect many aspects of human cancer, such as permanent replication, antagonism of cell death, and escape from immune surveillance. Therefore, lncRNAs are used as biomarkers for the diagnosis of many cancers. High methylation of CpG islands in gene promoters can silence genes. Currently, DNA methylation has been shown to be a highly stable molecular feature that can be detected in tumor tissues and cells. In this study, a diagnostic model for endometrial cancer was established based on the combined diagnosis of RP4 / RP11 / CTD multi-indicator methylation sites. Summary of the invention

[0005] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and to provide an endometrial cancer diagnostic kit with high detection accuracy, convenient and fast operation, and the ability to provide a reliable reference for the clinical diagnosis of endometrial cancer. The kit contains primers, probes, reaction reagents, etc. The kit detects cervical epithelial cell samples collected by an endometrial cell collector, and has relatively high screening sensitivity and specificity.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A gene methylation endometrial cancer detection kit comprises: a PCR reaction solution for detecting RP4, RP11 and CTD methylation, as well as a negative control and a positive control, wherein the PCR reaction solution comprises a PCR reaction solution A and a PCR reaction solution B;

[0008] The PCR reaction solution A includes dNTP, salt ion components, stabilizer, and DNA polymerase;

[0009] The PCR reaction solution B includes RP4 primer, RP11 primer, CTD primer and GAPDH primer, as well as a fluorescent probe.

[0010] This kit is based on the principle of fluorescent quantitative PCR and uses the methylation PCR fluorescent probe method to detect the methylation of RP4, RP11 and CTD genes in cervical epidermal cell samples. It has high detection accuracy, convenient and fast operation, and can provide a reliable reference for the clinical diagnosis of endometrial cancer.

[0011] Preferably, the RP4 primers include RP4-616B8.5-F and RP4-616B8.5-R, the forward primer sequence is 5'-ATGAGTGTGGCAGCCTATGT-3', and the reverse primer sequence is 5'-AACTCCTGACCTCGTGATCC-3'.

[0012] Preferably, the RP11 primers include RP11-389G6.3-F and RP11-389G6.3-R, the forward primer sequence is 5'-GGCCTTGAGAGATAGAGGGG-3', and the reverse primer sequence is 5'-ATACGTCCTTCCCATCCTGC-3'.

[0013] Preferably, the CTD primers include CTD-2377D24.6-F and CTD-2377D24.6-R, the forward primer sequence is 5'-TTCCGGTGTCCAGATGTTCA-3', and the reverse primer sequence is 5'-AAGGTGAGTTGGGGAGGATG-3'.

[0014] Preferably, the GAPDH primers include GAPDH-F and GAPDH-R, the forward primer sequence is 5'-GCACAGTCAAGGCTGAGAATG-3', and the reverse primer sequence is 5'-ATGGTGGTGAAGACGCCAGTA-3'.

[0015] The detection method of the gene methylation endometrial cancer detection kit comprises the following steps:

[0016] RNA isolation and qRT-PCR analysis;

[0017] DNA methylation analysis;

[0018] PCR amplification.

[0019] This product detects methylation of RP4, RP11 and CTD. In the CpG regions of the RP4, RP11 and CTD gene promoters that are highly correlated with endometrial cancer, specific primers and fluorescent probes are designed for the sequences after bisulfite conversion, which can specifically detect methylation sequences in PCR reactions.

[0020] Preferably, the specific process of RNA isolation and qRT-PCR analysis is as follows: fresh tissue samples are obtained from the subjects, total tissue RNA is extracted using TRIzol reagent, and then RNA is converted into cDNA using PrimeScript RT reagent kit, and the expression of 3′lncRNAs in RP4, RP11 and CTD is detected in QuantStudio 6 system using Hiff qPCR SYBR Green Master Mix, and the expression amount is normalized to the content of GAPDH mRNA and calculated by ΔCT method.

[0021] Preferably, the specific process of the DNA methylation analysis is as follows: the fresh tissue sample is a cervical epithelial cell sample, which is taken from an endometrial cell collector, and genomic DNA is extracted using a TIANamp genomic DNA kit to obtain 20 DNA eluates, and then bisulfite is treated with 125 μL conversion solution, 500 μL digestion solution and 60 μL BS eluate for bisulfite conversion, and finally, RP4 / RP11 / CTD PCR amplification is performed on an ABI 7500 device.

[0022] Preferably, the specific steps of the PCR amplification include:

[0023] Pre-denaturation;

[0024] transsexual;

[0025] annealing;

[0026] extend;

[0027] Detect fluorescence.

[0028] Preferably, during the pre-denaturation, the temperature is set at 95°C, the duration is 180 seconds, and the number of cycles is 1;

[0029] During the denaturation, the temperature was set at 95°C, the duration was 10 seconds, and the number of cycles was 45 times;

[0030] During the annealing, extension and fluorescence detection, the temperature was set at 60° C., the duration was 32 seconds, and the number of cycles was 45 times.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] This kit is based on the principle of fluorescent quantitative PCR and uses the methylation PCR fluorescent probe method to detect the methylation of RP4, RP11 and CTD genes in cervical epidermal cell samples. It has high detection accuracy, convenient and fast operation, and can provide a reliable reference for the clinical diagnosis of endometrial cancer.

[0033] This product detects methylation of RP4, RP11 and CTD. In the CpG regions of the RP4, RP11 and CTD gene promoters that are highly correlated with endometrial cancer, specific primers and fluorescent probes are designed for the sequences after bisulfite conversion, which can specifically detect methylation sequences in PCR reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram of the components of the gene methylation endometrial cancer detection kit proposed by the present invention;

[0035] Figure 2The clinical performance of primers EC (n=35) and NO-EC (n=14) of the RP4 / RP11 / CTD multi-index methylation detection kit was tested. DETAILED DESCRIPTION

[0036] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0037] Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present invention is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0038] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0039] As used herein, the terms "comprising", "including" and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

[0040] Numerical ranges expressed as endpoints herein include all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0041] The present invention relates to concentration values, and its meaning includes fluctuations within a certain range. For example, it can fluctuate within the corresponding accuracy range. For example, 2% can allow fluctuations within the range of ±0.1%. For values ​​that are large or do not require too fine control, its meaning is also allowed to include greater fluctuations. For example, 100mM can allow fluctuations within the range of ±1%, ±2%, ±5%, etc. Involving molecular weight, its meaning is allowed to include fluctuations of ±10%.

[0042] In the present invention, descriptions such as "plurality" and "multiple" refer to a quantity greater than or equal to 2 unless otherwise specified.

[0043] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0044] In the present invention, "preferably", "better", "more preferably", and "suitably" are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute a limitation on the scope of protection of the present invention. In the present invention, "optionally", "optional", and "optional" refer to being optional, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent of each other.

[0045] The term "diagnosis" as used herein refers to the act or process of identifying or determining a disease or condition in a mammal, or a factor causing a disease or condition, by evaluating the signs and symptoms of a disease or disorder, or genetic analysis, pathological analysis, histological analysis, etc.; the direct purpose is to determine the presence or absence of a disease. Typically, a disease or condition is diagnosed based on an evaluation of one or more factors and / or symptoms that symbolize the disease. That is, a diagnosis can be made based on the presence or absence of a factor or amount that symbolizes the presence or absence of a disease or condition. Each factor or symptom that is considered to symbolize a specific disease and is used for diagnosis does not need to be uniquely related to the specific disease; that is, there may be different diagnoses that can be inferred from diagnostic factors or symptoms. Similarly, there may be a situation where factors or symptoms that symbolize a specific disease are present in an individual who does not have the specific disease.

[0046] The object of diagnosis can be a subject suspected of having endometrial cancer. "Subject suspected of having endometrial cancer" covers individuals who have received a preliminary diagnosis (e.g., a CT scan showing a mass, or elevated levels of markers such as CA199, CA125) but whose cancer stage is not yet clear. The term also includes people who have had endometrial cancer (e.g., individuals in remission). As used herein, the term "stage of cancer" refers to a qualitative or quantitative assessment of the level of cancer development. Criteria for determining the stage of cancer include, but are not limited to, the size of the tumor and the extent of metastasis (e.g., localized or distant). In some embodiments, "subject" is a control subject suspected of having endometrial cancer or diagnosed with endometrial cancer. In some embodiments, the diagnosis is an early diagnosis.

[0047] The basic goal of the term "screening" as used herein is to find early-stage diseases or disease risks in a large number of otherwise healthy people, with the purpose of determining the high or low risk of developing a certain health problem. In some embodiments, the screening is an early screening.

[0048] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of the present application, the cited documents involved in the present invention are cited with all contents and all purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into the present application as references, but are limited to the ability to implement the present invention. It should be understood that when the content of the citation conflicts with the description in the present application, the present application shall prevail or be modified adaptively according to the description of the present application.

[0049] The term "DNA methylation level" is the same as the general understanding, which refers to whether the cytosine in one or more CpG dinucleotides in a DNA sequence is methylated, or the frequency / proportion / percentage of methylation. It represents both qualitative and quantitative concepts. For example, if the cytosine (C) residue in the nucleic acid sequence is methylated, it can be called "highly methylated" or have "increased methylation". In practical applications, different detection indicators can be used to compare DNA methylation levels according to actual conditions. For example, in some cases, the comparison can be based on the Ct value of the sample detection. In some cases, the methylation ratio of the marker in the sample can be calculated, that is, the number of methylated molecules / (the number of methylated molecules + the number of non-methylated molecules) × 100, and then compared. In some cases, it is also necessary to statistically analyze and integrate the various indicators to obtain the final judgment indicator.

[0050] Reference Figure 1 and Figure 2 , a gene methylation endometrial cancer detection kit, comprising: a PCR reaction solution for detecting RP4, RP11 and CTD methylation, and a negative control and a positive control, the negative control being human non-methylated whole genome DNA, the positive control being human methylated whole genome DNA, the PCR reaction solution comprising PCR reaction solution A and PCR reaction solution B;

[0051] Among them, the positive control is a sample that is known to produce the expected results, that is, it is possible to predict what effect it should show in the experiment. In this way, when the positive control is introduced into the experiment and the expected effect is observed, the feasibility and effectiveness of the experiment can be verified.

[0052] Negative control is the concept corresponding to positive control. It is a sample that is not expected to produce the effect being studied. In the experiment, the negative control is usually a sample without the substance to be tested or unrelated to the substance to be tested. Introducing negative control can help us confirm whether the experimental results are really related to the factors we are studying, and exclude erroneous observations caused by irrelevant factors, that is, exclude false positives.

[0053] The PCR reaction solution A includes dNTP, salt ion components, stabilizer, and DNA polymerase;

[0054] The PCR reaction solution B includes RP4 primer, RP11 primer, CTD primer and GAPDH primer, as well as a fluorescent probe.

[0055] In another embodiment, each component is preferably in a freeze-dried form, for example, in the form of one or more so-called freeze-dried beads. Freeze-dried beads can generally be understood to refer to freeze-dried materials that are pressed into a spherical shape after manufacturing (after the manufacturing, the material is usually present as a powder). Therefore, the components required for PCR batches, particularly DNA polymerase, nucleic acid components and reaction buffer components, can be provided in a freeze-dried form, for example. In this way, the PCR process can be directly started in a very user-friendly manner by adding a sample to be quantified and optionally other required components. In particular, the provision of freeze-dried forms is very conducive to automated applications.

[0056] This kit is based on the principle of fluorescent quantitative PCR and uses the methylation PCR fluorescent probe method to detect the methylation of RP4, RP11 and CTD genes in cervical epidermal cell samples. It has high detection accuracy, convenient and fast operation, and can provide a reliable reference for the clinical diagnosis of endometrial cancer.

[0057] In this embodiment, the RP4 primers include RP4-616B8.5-F and RP4-616B8.5-R, the forward primer sequence is 5'-ATGAGTGTGGCAGCCTATGT-3', and the reverse primer sequence is 5'-AACTCCTGACCTCGTGATCC-3'.

[0058] In this embodiment, the RP11 primers include RP11-389G6.3-F and RP11-389G6.3-R, the forward primer sequence is 5'-GGCCTTGAGAGATAGAGGGG-3', and the reverse primer sequence is 5'-ATACGTCCTTCCCATCCTGC-3'.

[0059] In this embodiment, the CTD primers include CTD-2377D24.6-F and CTD-2377D24.6-R, the forward primer sequence is 5'-TTCCGGTGTCCAGATGTTCA-3', and the reverse primer sequence is 5'-AAGGTGAGTTGGGGAGGATG-3'.

[0060] In this embodiment, the GAPDH primers include GAPDH-F and GAPDH-R, the forward primer sequence is 5'-GCACAGTCAAGGCTGAGAATG-3', and the reverse primer sequence is 5'-ATGGTGGTGAAGACGCCAGTA-3'.

[0061] In this embodiment, in the polymerase chain reaction (PCR), the primer is a short single-stranded DNA molecule used to guide the DNA synthesizer to synthesize a specific DNA fragment on the template DNA. Therefore, the 3' end of the primer is very important because it is the position where the polymerase starts to synthesize a new chain. In this embodiment, all the primers are designed with a full T structure (i.e., three consecutive thymine bases) at the 3' end, which can:

[0062] Improve specificity: The all-T structure has a higher solubility in the PCR reaction mixture, which helps to reduce the formation of primer dimers, thereby improving the specific binding of the primer to the target DNA sequence;

[0063] Enhance affinity: The pairing between the T base and the A base on the template DNA is formed by two hydrogen bonds, which is looser than other base pairs. The all-T structure at the 3' end of the primer can increase the contact area with the template, thereby improving the affinity between the primer and the template DNA;

[0064] Promote primer extension: The all-T structure allows the polymerase to more easily recognize and bind to the 3' end, thereby promoting primer extension. This structure helps the polymerase to quickly start the synthesis of new chains and improve PCR efficiency;

[0065] Reduce nonspecific amplification: Since the all-T structure reduces the formation of primer dimers, it can reduce the production of nonspecific amplification products, which is very important for obtaining pure and highly specific PCR products;

[0066] Adapt to template diversity: The all-T structure can adapt to the sequence diversity on the template DNA due to its relatively loose base pairing characteristics. Even if there are some variations in the template sequence near the primer binding region, PCR amplification can be performed well.

[0067] At the same time, in this embodiment, the GC content of the amplified fragment is controlled at 40-50%, because the bases are connected by hydrogen bonds, while the nucleotides are connected by phosphodiester bonds. In the DNA double helix structure, the GC pair has a higher binding energy than the AT pair (containing two hydrogen bonds) because it contains three hydrogen bonds. This means that each GC pair can provide more stabilizing energy relative to each AT pair. Therefore, a DNA fragment containing a higher proportion of GC is usually more stable than a fragment containing a lower proportion of GC.

[0068] In the PCR reaction, a higher GC content may cause the DNA double-stranded strands to be too stable in certain regions, which makes it difficult for the DNA polymerase to separate the double-stranded strands for the synthesis of new strands. On the contrary, if the GC content of the DNA fragment is too low, the double-stranded strands may not be stable enough, and the polymerase may easily slip, affecting the PCR efficiency.

[0069] Therefore, controlling the GC content of the PCR amplified fragment to about 40-50% can provide a moderate stability, which helps the DNA polymerase to effectively bind to the template and synthesize new chains, while avoiding the problem of excessive stability caused by too high a GC content and the double-strand instability caused by too low a GC content. Such a GC content helps to achieve efficient and specific PCR amplification.

[0070] The detection method of the gene methylation endometrial cancer detection kit comprises the following steps:

[0071] RNA isolation and qRT-PCR analysis;

[0072] DNA methylation analysis;

[0073] PCR amplification.

[0074] In this example, the specific process of RNA isolation and qRT-PCR analysis is as follows: fresh tissue samples were obtained from the subjects, total RNA of the tissues was extracted using TRIzol reagent (Life Technologies, NY, USA), and then RNA was converted into cDNA using PrimeScript RT kit (Takara, Dalian, China).

[0075] Hiff qPCR SYBR Green Master Mix (easen Biotech Co., Shanghai, China) was used to detect the expression of 3′ lncRNAs in RP4, RP11 and CTD in the QuantStudio 6 system (Applied Biosystems, Waltham, MA). The expression was normalized to the content of GAPDH mRNA and calculated by the ΔCT method. Fluorescence quantitative PCR, often abbreviated as qPCR in English and daily use, is one of the most common experimental methods in molecular biology. After more than 20 years of development, qPCR data processing methods are very rich. The most commonly used method is the ΔΔCT method in the relative quantification category, which is a method that uses internal references for normalization to obtain the relative expression level of the target gene. The mathematical model is simple, the analysis is also simple, and reliable results can be obtained in most cases.

[0076] Among them, the Ct value represents the cycle threshold of the sample, which is a value fed back to you by the machine when running fluorescent quantitative PCR. The real meaning is the corresponding cycle number after the fluorescence signal of the product amplification in the PCR process reaches the set threshold. For example, Ct=19 means that the product expanded to the set fluorescence threshold at the 19th cycle. It can also indirectly reflect the starting concentration of the template during the PCR. The higher the concentration, the lower the Ct value.

[0077] delta: means the difference between two values

[0078] The ΔCt is obtained by the following formula:

[0079] ΔCt=Ct(target gene)-Ct(housekeeping gene)

[0080] Housekeeping gene, also known as internal reference gene. ΔCt is the difference between the Ct of the target gene and the internal reference gene.

[0081] The calculation method is a prior art, so it will not be described in detail here.

[0082] The subject referred to in the present invention is a mammal, preferably a primate, and more preferably a human.

[0083] In this embodiment, the specific process of the DNA methylation analysis is as follows: the fresh tissue sample is a cervical epithelial cell sample, which is taken from an endometrial cell collector (sap-1, Jingyou, China). The genomic DNA is extracted using the TIANamp genomic DNA kit (DP304, Tiangen, China) to obtain 20 DNA eluates. Then, 125 μL of conversion solution, 500 μL of digestion solution and 60 μL of BS eluate are used to treat bisulfite for bisulfite conversion, wherein the components of the conversion solution (sodium bisulfite NaHSO3 and sodium sulfite Na2SO3) are combined with another buffer (sulfite treatment reagent) to avoid salt crystallization, which can effectively improve the process of converting DNA base C to U. Even if there are few methylated fragments on the nucleic acid molecules after extraction, high concentrations of sulfite can convert the bases 100%. Finally, RP4 / RP11 / CTD PCR amplification is performed on the ABI 7500 device.

[0084] In another embodiment, the method based on nucleic acid sulfurization of uterine exfoliated cells is:

[0085] ① Add 1540ul of the conversion agent to the eluent, vortex and shake (at room temperature) for 10 minutes, and set aside;

[0086] ② Add anhydrous ethanol to the combination solution, digestion solution, wash A and wash B and set aside;

[0087] ③ Add 125ul transformation solution, 5ul protective agent, and 20ul DNA to the eight-tube concatenation, vortex and centrifuge instantly;

[0088] ④ Place in PCR instrument, 98℃, 10min; 64℃, 90min; 4℃, hold;

[0089] ⑤ Add 450ul of binding solution, 20ul of magnetic beads, and 150ul of the above sample to a new 1.5ml EP tube, and mix by inversion for 15min (room temperature);

[0090] ⑥ Place it on a magnetic stand for adsorption and discard the waste liquid;

[0091] ⑦ Add 500ul of wash A, shake and mix, place on a magnetic stand, and discard the waste liquid;

[0092] ⑧ Add 500ul digestion solution, mix by inversion for 15min (room temperature), place on a magnetic stand for adsorption, and discard the waste liquid;

[0093] ⑨Add 500ul of Wash B, shake and mix, place on a magnetic stand, and discard the waste liquid;

[0094] ⑩ Demagnetize, repeat the previous step, suck out the remaining liquid with a small gun tip, open the centrifuge tube and place it in a 56℃ metal bath for 1-2 minutes;

[0095] Add 60ul BS elution buffer, shake and mix, incubate at 56℃ for 5min, centrifuge briefly, place on a magnetic stand for adsorption, and transfer 55ul supernatant to a new 1.5ml EP tube.

[0096] This method has a very high conversion efficiency in treating trace amounts of exfoliated cell nucleic acids, because according to the comparison of sulfite treatment kits currently on the market, salt crystallization after preparing the solution is a key factor in reducing the sulfidation efficiency. In this method, the components of the conversion solution (sodium bisulfite NaHSO3 and sodium sulfite Na2SO3) are combined with another buffer (sulfite treatment reagent) to avoid salt crystallization, which can effectively improve the process of converting DNA base C to U. Even if there are few methylated fragments on the nucleic acid molecules after extraction, high concentrations of sulfite can convert the bases by 100%. The sulfidation efficiency of most sulfite kits on the market in treating trace amounts of cell nucleic acids is between 60-80%, while the conversion rate of this method is above 95%.

[0097] Moreover, the biggest innovation of this method is to deal with the problem of bacterial contaminants in uterine exfoliated cells, avoid contamination caused by non-cellular product nucleic acids, and improve the accuracy of detection.

[0098] In this embodiment, the specific steps of the PCR amplification include:

[0099] Pre-denaturation;

[0100] transsexual;

[0101] annealing;

[0102] extend;

[0103] Detect fluorescence.

[0104] In this embodiment, during the pre-denaturation, the temperature was set at 95°C, the duration was 180 seconds, and the number of cycles was 1;

[0105] During the denaturation, the temperature was set at 95°C, the duration was 10 seconds, and the number of cycles was 45 times;

[0106] During the annealing, extension and fluorescence detection, the temperature was set at 60° C., the duration was 32 seconds, and the number of cycles was 45 times.

[0107] Wherein: in step 3, fluorescence detection is performed at 60°C, the detection channels include: FAM, VIC, CY5, ROX, and the quenching groups are selected as None, None, None, and NFQ-MGB respectively;

[0108] Among them, FAM channel: RP4 gene; VIC channel: RP11 gene; CY5 channel: CTD gene; ROX channel: GAPDH gene.

[0109] Test result determination:

[0110]

[0111] Figure 2 To test the clinical performance of primers EC (n=35) and NO-EC (n=14) of the RP4 / RP11 / CTD multi-index methylation detection kit. Figure 2 It can be seen that this product detects methylation of RP4, RP11 and CTD. In the CpG regions of the RP4, RP11 and CTD gene promoters that are highly correlated with endometrial cancer, specific primers and fluorescent probes are designed for the sequences after bisulfite conversion. The methylation sequences can be specifically detected in the PCR reaction, and the screening sensitivity and specificity are relatively high.

[0112] The reason why the present invention targets RP4, RP11 and CTD methylation detection is that based on the CTC / exfoliated cell methylation / LncRNA triple test, the partial methylation of the four genes CDO1, CELF4, HAND2 and HS3ST2 in endometrial cancer methylation was re-evaluated and defined.

[0113] There are essential differences between vaginal exfoliated cells and serum and body tissues. Due to the instability, solubility and contamination of uterine exfoliated cells, not only an accurate method is needed to judge endometrial cancer, but also a method to distinguish the difference in methylation between exfoliated cells and blood tissues.

[0114] We started with all the methylation sites reported in endometrial cancer in the past 10 years, including BHLHE22, CDO1, CELF4, ADCYAP1, ASCL2, HS3ST2, HTR1B, MME, NPY, SOX1, HAND2, COX2, VEGF, TNF, HIF1A, P14, P16, ER, RASSF1A, etc. We found that some methylation sites are not actually the criteria for judging endometrial cancer, such as HS3ST2, HTR1B, MME, NPY gene methylation, which is only the potential ability to judge the benign or malignant nature of the patient. In addition, the methylation of genes such as P14, P16, ER, and RASSF1A was discovered from whole-genome sequencing of tissues and has not been verified on exfoliated cells of endometrial cancer patients.

[0115] Therefore, the present invention starts with the patient's blood, tissue, and exfoliated cells, redefines the difference between the methylation sites of exfoliated cells and blood tissues, and creatively establishes an endometrial cancer diagnostic model based on the combined diagnosis of RP4 / RP11 / CTD multi-indicator methylation sites.

[0116] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A gene methylation endometrial cancer detection kit, characterized in that: include: A PCR reaction solution for detecting RP4, RP11 and CTD methylation, as well as a negative control and a positive control, wherein the PCR reaction solution includes a PCR reaction solution A and a PCR reaction solution B; The PCR reaction solution A includes dNTP, salt ion components, stabilizer, and DNA polymerase; The PCR reaction solution B includes RP4 primer, RP11 primer, CTD primer, GAPDH primer, and fluorescent probe.

2. The gene methylation endometrial cancer detection kit according to claim 1, characterized in that: The RP4 primers include RP4-616B8.5-F and RP4-616B8.5-R, the forward primer sequence is 5'-ATGAGTGTGGCAGCCTATGT-3', and the reverse primer sequence is 5'-AACTCCTGACCTCGTGATCC-3'.

3. The gene methylation endometrial cancer detection kit according to claim 1, characterized in that: The RP11 primers include RP11-389G6.3-F and RP11-389G6.3-R, the forward primer sequence is 5'-GGCCTTGAGAGATAGAGGGG-3', and the reverse primer sequence is 5'-ATACGTCCTTCCCATCCTGC-3'.

4. The gene methylation endometrial cancer detection kit according to claim 2, characterized in that: The CTD primers include CTD-2377D24.6-F and CTD-2377D24.6-R, the forward primer sequence is 5'-TTCCGGTGTCCAGATGTTCA-3', and the reverse primer sequence is 5'-AAGGTGAGTTGGGGAGGATG-3'.

5. The gene methylation endometrial cancer detection kit according to claim 1, characterized in that: The GAPDH primers include GAPDH-F and GAPDH-R, the forward primer sequence is 5'-GCACAGTCAAGGCTGAGAATG-3', and the reverse primer sequence is 5'-ATGGTGGTGAAGACGCCAGTA-3'.

6. The detection method of the gene methylation endometrial cancer detection kit according to any one of claims 1 to 5, characterized in that: The following steps are involved: RNA isolation and qRT-PCR analysis; DNA methylation analysis; PCR amplification.

7. The detection method according to claim 6, characterized in that: The specific process of RNA isolation and qRT-PCR analysis is as follows: fresh tissue samples are obtained from the subjects, total tissue RNA is extracted using TRIzol reagent, and then RNA is converted into cDNA using PrimeScript RT reagent kit, and Hiff qPCR SYBR Green Master Mix is ​​used to detect the expression of 3′lncRNAs in RP4, RP11 and CTD in the QuantStudio 6 system, and the expression amount is normalized to the content of GAPDHmRNA and calculated by the ΔCT method.

8. The detection method according to claim 7, characterized in that: The specific process of the DNA methylation analysis is as follows: the fresh tissue sample is a cervical epithelial cell sample, which is taken from an endometrial cell collector, and the genomic DNA is extracted using the TIANamp genomic DNA kit to obtain 20 DNA eluates, and then treated with 125 μL conversion solution, 500 μL digestion solution and 60 μL BS eluate for bisulfite conversion, and finally, RP4 / RP11 / CTD PCR amplification is performed on an ABI 7500 device.

9. The detection method according to claim 8, characterized in that: The specific steps of the PCR amplification include: Pre-denaturation; transsexual; annealing; extend; Detect fluorescence.

10. The detection method according to claim 9, characterized in that: During the pre-denaturation, the temperature was set at 95°C, the duration was 180 seconds, and the number of cycles was 1; During the denaturation, the temperature was set at 95°C, the duration was 10 seconds, and the number of cycles was 45 times; During the annealing, extension and fluorescence detection, the temperature was set at 60° C., the duration was 32 seconds, and the number of cycles was 45 times.

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