CN116334221A - Pancreatic cancer diagnosis or screening biomarker and application thereof - Google Patents

Abstract

The invention discloses a pancreatic cancer diagnosis or screening biomarker and application thereof. The invention relates to an application of a reagent for detecting ctDNA-CLDN18.2 methylation level in preparing a product for diagnosing or screening pancreatic cancer, wherein the ctDNA-CLDN18.2 methylation level is down-regulated in the tissue or sample of pancreatic cancer. Pancreatic cancer can be conveniently and accurately diagnosed or screened by detecting the ctDNA-CLDN18.2 methylation level.

Description

Pancreatic cancer diagnosis or screening biomarker and application thereof

Technical Field

The invention relates to a tumor marker, relates to a pancreatic cancer diagnosis or screening biomarker and application thereof, and belongs to the field of pancreatic cancer diagnosis or screening biomarkers.

Background

Pancreatic cancer (PDAC) is regarded as "king in cancer", and has extremely high malignancy, 3% of its incidence, 10% of five-year survival rate, and 8% of its mortality rate (mortality rate is the 4 th place of malignant tumor mortality rate, and is next to colorectal cancer). In recent years, the incidence of pancreatic cancer has been on the rise due to changes in the human living environment, eating habits, etc., such as smoking, high calorie foods, decreased exercise amount, etc.

Gemcitabine is used as a first-line clinical medicine for pancreatic cancer, and the remission rate of the gemcitabine to the pancreatic cancer is lower than 12%, so that the combined medicine becomes a main means of pancreatic cancer chemotherapy at present. However, chemotherapy resistance also becomes a troublesome problem in current pancreatic cancer treatment procedures. Pancreatic cancer is so high in malignancy, mainly because of hidden onset, difficult early diagnosis, and high metastatic potential, with greater than 50% of patients undergoing distant metastasis at or after diagnosis, losing the opportunity for radical treatment, and extremely poor prognosis. Recurrence and metastasis of pancreatic cancer are thus a difficult problem affecting patient prognosis and afflicting the clinic. Therefore, accurate diagnosis means, accurate treatment schemes and high-sensitivity prognosis diagnosis indexes become main measures for improving prognosis of pancreatic cancer at present. And the high risk group of pancreatic cancer must be kept vigilant, necessary measures are taken to prevent pancreatic cancer from happening, and regular screening is performed. In particular, once some symptoms related to pancreatic cancer appear, the pancreatic cancer must be checked in time so as to prevent the occurrence of pancreatic cancer, and once the pancreatic cancer is generated, early diagnosis and early treatment can be performed so as to obtain good treatment effect.

Currently, abdominal B-ultrasound, CT/enhanced CT, magnetic resonance MRI, PET-CT are the most commonly used diagnostic methods for pancreatic cancer. However, the manner in which a technician operates, the physical condition of the patient, such as obesity, intestinal gas, and other factors, can affect the accuracy of the diagnosis, both in the experience and in the experience of the diagnostician. Furthermore, early diagnosis is not easy due to the special location of the pancreas, compared to other digestive tract tumors. Thus, the identification of sensitive and specific non-invasive biomarkers would be of great value for early diagnosis of pancreatic cancer.

Since the 21 st century, the development of tumor biomarkers has been continuously optimized and perfected with the development of molecular biology techniques. Among the serum tumor biomarkers that have been incorporated into routine tumor examinations, the carbohydrate antigens CA199, CA125 and carcinoembryonic antigen CEA are auxiliary diagnostic indicators of pancreatic cancer, while some retrospective studies indicate that they are also predictive risk factors for poor prognosis of pancreatic cancer. In the prior art, diagnosis of pancreatic cancer is reported by detecting tumor biomarker (CA 199, CA125, CEA) changes in serum and combining clinical symptoms, imaging diagnosis and pathology examination, but the tumor biomarker level changes cannot directly represent the change of tumor tissue molecular biology, and the specificity of the tumor biomarker for early diagnosis and treatment prognosis evaluation of pancreatic cancer is low, so that large data support is required, and a large amount of labor and financial support is required.

In recent years, circulating DNA of tumor origin (ctDNA), referred to as tumor-derived genetic material in the blood of cancer patients, has attracted more attention in the medical community due to its clinical advantages. As a small double-stranded DNA fragment, ctDNA is released by tumor cells and circulates in peripheral blood. During tumorigenesis, increased cell necrosis and apoptosis lead to accumulation of ctDNA, which can be detected at a relatively early stage of the tumor. In addition, ctDNA not only contains the same mutations as tumor cells, but also has the same methylation pattern, thereby providing the possibility and convenience for early cancer diagnosis, even for diagnosis of those hidden organs (such as pancreas and bile duct).

Whereas methylation DNA immunoprecipitation combined with high throughput sequencing (MeDIP-seq) is a sensitive technique for detecting DNA methylation, even initial amounts of DNA as low as 1ng can be detected. Therefore, whole genome detection of ctDNA methylation profiles using the MeDIP-seq method has recently been developed to screen for potential biomarkers of early cancer.

In addition, the Claudins (CLDNs) family, which is a key protein for a tight junction structure between cells, is widely distributed in various epithelial tissues, contains at least 27 family members, and the structure including 4 transmembrane regions and 2 extracellular loops, which are transmembrane proteins located on the surface of a cell membrane, has high consistency, and this consistency is particularly remarkable in the transmembrane regions. The CLDNs protein was found to be closely related to the maintenance of osmotic pressure, barrier function, and cell polarity by epithelial cells and to be involved in the immune defense process against pathogens. In addition, CLDNs have been demonstrated to have altered expression patterns during the development of many tumors, and targeted therapeutic studies using CLDNs lineages as specific marker proteins have been of great interest.

Disclosure of Invention

The main object of the present invention is to provide pancreatic cancer diagnosis or prognosis refers to biomarkers;

it is another object of the present invention to apply said biomarker in the preparation of a product for diagnosing or screening pancreatic cancer.

In order to achieve the above purpose, the technical scheme adopted by the invention comprises the following steps:

the invention provides the use of an agent for detecting ctDNA-CLDN18.2 methylation level in the manufacture of a product for diagnosing or screening pancreatic cancer, wherein the ctDNA-CLDN18.2 methylation level is down-regulated in the tissue or sample of pancreatic cancer.

The product comprises a kit, a chip, a detection test strip or a pharmaceutical composition and the like.

The reagent comprises a reagent for detecting the methylation level of ctDNA-CLDN18.2 by PCR detection reagent, immunoassay reagent, in situ hybridization or chip detection;

the PCR detection can be fluorescence quantitative PCR or RT-PCR, and the amplified fragment sequence of the PCR detection contains CpG island nucleic acid sequence of SEQ ID NO. 1.

When amplified using fluorescent quantitative PCR, the amplification primers include a pair of forward and reverse primers and a fluorescein probe that specifically binds to ctDNA-CLDN 18.2.

The principle of the detection kit for pancreatic cancer diagnosis or screening is as follows:

the applicant's research found that: 1) The abnormal methylation of the CLDN18.2 occurs in early pancreatic cancer, and the abnormal methylation level of the CLDN18.2 in blood plasma is detected because the expression level of the CLDN18.2 in tumor tissues is obviously increased, so that the detection and evaluation of early pancreatic cancer can be used; 2) The abnormal high expression of the CLDN18.2 is closely related to prognosis indexes such as the recurrence-free survival time, the tumor differentiation degree and the like of pancreatic cancer patients, and the poor prognosis of the CLDN18.2 high expression can evaluate the clinical treatment progress and judge the prognosis trend of pancreatic cancer patients.

Based on the above-described findings of the applicant, the present invention extracts free DNA in peripheral blood, then converts unmethylated cytosines with sulfite, and generates uracil sulfonate by deamination, whereas methylated cytosines are not converted with sulfite. The DNA converted by bisulfite (BisDNA) is subjected to multiplex PCR amplification, and the primer and the probe in the PCR reaction can distinguish methylated sequences and unmethylated sequences, so that the methylated sequences are amplified preferentially. The methylated sequence can be detected specifically in a PCR reaction by a fluorescein probe that specifically binds to the methylated CLDN18.2 gene sequence. The reference control ACTB (beta-actin) gene can be used to assess whether the amount of DNA in the assay is sufficient.

In a preferred embodiment, the invention provides the use of a ctDNA-CLDN18.2 methylation level detection kit for the preparation of a pancreatic cancer diagnosis or screening kit, wherein the primers have 3 pairs, the forward primer of the first pair has a sequence as shown in SEQ ID No.2, and the reverse primer has a sequence as shown in SEQ ID No. 3; the sequence of the forward primer of the second pair is shown as SEQ ID NO.4, and the sequence of the reverse primer is shown as SEQ ID NO. 5; the sequence of the third pair of forward primers is shown as SEQ ID NO.6, and the sequence of the reverse primers is shown as SEQ ID NO. 7.

In a preferred embodiment, the present invention provides the use of a ctDNA-CLDN18.2 methylation level detection kit for the preparation of a pancreatic cancer diagnosis or screening kit, wherein said PCR detection reagent further comprises a fluorescein probe that specifically binds to said ctDNA-CLDN 18.2.

In a preferred embodiment, the invention provides the use of a ctDNA-CLDN18.2 methylation level detection kit for preparing a pancreatic cancer diagnosis or screening kit, wherein the sequence of the fluorescein probe is shown as SEQ ID No. 8.

In a preferred embodiment, the present invention provides the use of a ctDNA-CLDN18.2 methylation level detection kit for the manufacture of a pancreatic cancer diagnosis or screening kit, wherein said detection kit further comprises reagents for converting unmethylated cytosines in DNA.

In a preferred embodiment, the present invention provides the use of a ctDNA-CLDN18.2 methylation level detection kit for the preparation of a pancreatic cancer diagnosis or screening kit, wherein the reagent that converts unmethylated cytosines in DNA is bisulfite.

The ctDNA-CLDN18.2 methylation level detection kit has the beneficial effects that pancreatic cancer can be diagnosed or screened conveniently and accurately.

The detection kit is used for detecting the methylation level of ctDNA-CLDN18.2, so that when the ctDNA-CLDN18.2 is used as a biomarker for pancreatic cancer diagnosis or screening, the accuracy (positive coincidence rate, negative coincidence rate), precision, detection limit, analysis specificity and the like are verified, and the method and the result are as follows:

(1) The accuracy study uses negative/positive reference and a negative/positive/weak positive sample pool from clinical samples to detect respectively, and the detection results show that the positive coincidence rate is 100% and the negative coincidence rate is 100%. Meanwhile, the detection kit and the similar comparison reagent are used for respectively detecting clinical samples, including 120 cases of cancers and corresponding normal tissue samples in pancreatic cancer samples in each period, and the result of FIG. 3 shows that the detection kit has the accuracy of 91.7% for monitoring cancer tissues/normal tissue samples of patients with pancreatic cancer in a definite way (the specificity and the sensitivity of the primer are more favorable for judgment and calculation by directly detecting the tissue samples, and a foundation is laid for the subsequent detection of ctDNA with low peripheral blood concentration).

(2) The detection result shows that the variation coefficients of the CLDN18.2 and the ACTB genes detected by the detection kit are less than 10 percent. The second sample is verified by adding a cell line DNA analog precision enterprise reference with a certain concentration into a clinical normal human plasma sample, and the detection result shows that the variation coefficients of the CLDN18.2 and the ACTB genes are less than 10 percent. Meanwhile, the precision of the matched nucleic acid extraction reagent is also researched, and the result shows that the variation coefficient of the DNA extraction amount in the sample collection process and the nucleic acid extraction process is less than 10%; in the process of nucleic acid transformation, the variation coefficient of Ct value of ACTB gene is less than 10%.

(3) And (3) detecting limit research, namely detecting plasma samples containing methylated DNA in different proportions under different free DNA concentrations by taking a negative plasma sample as a matrix to obtain the detecting limit range of the plasma samples. The result shows that the lowest concentration of the positive DNA in the detectable plasma of the detection kit is 0.1pg/mL; the detection limit of the positive detection P value of the detection kit is 0.5% when the concentration of the plasma DNA is 0.02 ng/mL. The cross reaction experimental result shows that the comprehensive specificity of the detection kit for detecting the interference sample exceeds 85%.

Drawings

FIG. 1 is a schematic representation of the methylation of CLDN18.2 in pancreatic cancer and a sequence position map.

FIG. 2 is a graph showing the change in expression level of CLDN18.2 in pancreatic cancer and survival prognosis.

Fig. 3 is an ROC diagram of CLDN18.2 detection of cancer and corresponding normal tissue in pancreatic cancer.

Fig. 4 is a graph showing ROC values of CLDN18.2 in peripheral blood detection in pancreatic and non-pancreatic cancer patients.

Detailed Description

The following describes the invention in more detail with reference to examples.

Example 1: peripheral blood free DNA extraction

The patients extracted from the 80-exception peripheral blood free DNA are diagnosed by pathological diagnosis in Tianjin tumor hospitals whether pancreatic cancer patients or not, and other diseases are diagnosed by comprehensive diagnosis such as pathological diagnosis or CT and the like according to relevant diagnosis and treatment guidelines. The group-entering cases are suspected pancreatic cancer cases, and the sample type is that the peripheral blood free DNA is extracted to obtain plasma. The specific method for plasma extraction of peripheral blood free DNA is as follows (materials methods from TIANGENBIOTECH (BEIJING) co., LTD):

the peripheral blood DNA extraction experimental steps:

the purpose of the experiment is as follows: extracting DNA from blood samples or body fluids such as saliva and serum.

The experimental steps are as follows:

1. treatment material

If the extraction material is blood, 200 μl of fresh, frozen or blood with various anticoagulants can be directly used, and less than 200 μl of buffer GA can be added for supplementing;

note that: if a larger volume of blood, such as 300. Mu.l-1 ml, is to be treated, the following steps are performed: add 3 volumes of red blood cell lysate (e.g., 300. Mu.l blood to 900. Mu.l red blood cell lysate) to the sample, mix upside down, and leave at room temperature for 5min, during which time mix upside down again several times. 10000rpm (-11,500 Xg) for 1min (3000 if the maximum rotation speed of the centrifuge is not allowed)

Centrifugation at rpm (3,400 Xg) for 5 min) was performed, the supernatant was aspirated, the white blood cell pellet was left, 200. Mu.l of buffer GA was added and shaken until thoroughly mixed. The red blood cell lysate is available from Hematocrit (catalog number: RT 122) and can be purchased as desired.

2. Add 20. Mu.l ProteinaseK solution and mix well.

a. When extracting blood genome, only adding protein aseK and mixing uniformly, and then proceeding to the next step.

b. When the genome of the cell is extracted, the next step can be continued by only adding the ProteinaseK and uniformly mixing.

c. When extracting tissue genome, adding ProteinaseK, mixing, standing at 56 deg.C until tissue is dissolved, and briefly isolating

The core is used for removing the water drops on the inner wall of the pipe cover, and then the next step is carried out.

Note that: the lysis time of different tissues is different, and the lysis time is usually 1-3 hours (the tail needs to be digested overnight). And the subsequent operation is not affected. The samples were mixed 2-3 times per hour with the water bath shaker.

3. 200 μl of buffer GB was added, mixed well by inversion, left at 70deg.C for 10min, the solution was clear and centrifuged briefly to remove water droplets from the inner wall of the tube cap.

Note that: white precipitate may be generated when buffer GB is added, and generally disappears when the buffer GB is placed at 70 ℃, so that subsequent experiments are not affected. If the solution is not clear, indicating that the cell lysis is not complete, it may result in a small amount of extracted DNA and an impure extracted DNA. When the blood volume is less than or equal to 200 mu l and no erythrocyte lysis treatment is adopted, or the sample storage condition is poor, the color after water bath may be dark brown, and the solution is not precipitated with lumps and the like.

4. 200 μl of absolute ethanol was added, and mixed well for 15sec with shaking, at which time flocculent precipitate was likely to appear, and centrifuged briefly to remove water droplets on the inner wall of the tube cap.

5. The solution obtained in the previous step and the flocculent precipitate were both added to an adsorption column CB3 (the adsorption column was placed in a collection tube), centrifuged at 12,000rpm (13,400Xg) for 30sec, the waste liquid was poured off, and the adsorption column CB3 was placed back in the collection tube.

6. To the adsorption column CB3, 500. Mu.l of a buffer solution GD (whether or not absolute ethanol had been added was checked before use) was added, and the mixture was centrifuged at 12,000rpm (13,400Xg) for 30sec, and the waste liquid was poured off, and the adsorption column CB3 was placed in a collection tube.

7. 600 μl of rinse solution PW (which was checked for the presence of absolute ethanol before use) was added to the column CB3, centrifuged at 12,000rpm (13,400Xg) for 30sec, the waste liquid was poured off, and the column CB3 was placed in a collection tube.

8. And repeating the operation step 7.

9. The adsorption column CB3 was put back into the collection tube and centrifuged at 12,000rpm (13,400Xg) for 2min, and the waste liquid was discarded. The adsorption column CB3 was left at room temperature for several minutes to thoroughly dry the residual rinse solution in the adsorption material.

Note that: the purpose of this step is to remove the residual rinse solution from the column, which residual ethanol can affect subsequent enzymatic reactions (digestion, PCR, etc.).

10. Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50-200 μl of elution buffer TE into the middle part of the adsorption film, standing at room temperature for 2-5min, centrifuging at 12,000rpm (13,400×g) for 2min, and collecting the solution into the centrifuge tube.

Note that: the volume of the elution buffer should not be less than 50. Mu.l, and too small a volume affects recovery efficiency. The pH of the eluate has a great influence on the elution efficiency. If ddH2O is used as eluent, the pH value of the eluent is ensured to be in the range of 7.0-8.5, and the eluting efficiency is reduced when the pH value is lower than 7.0; and the DNA product should be kept at-20℃to prevent DNA degradation. To increase the yield of genomic DNA, the solution obtained by centrifugation may be added to an adsorption column CB3, and left at room temperature for 2min at 12,000rpm (13,400Xg) for 2min.

Clinical sensitivity and specificity evaluations were incorporated into 80 clinical cases, 60 of which were pancreatic cancer cases (covering multiple pathological genotypes), and 20 of which were normal (including other, interference-prone tumors and various benign disease cases).

Example 2: sulfite conversion and purification of each extracted peripheral blood free DNA sample

1) The genomic DNA to be treated (extracted peripheral blood episomal DNA) was taken out of the refrigerator and thawed for use.

2) According to experimental requirements, a BisulfiteMix is prepared. In the preparation process, 850 μl of buffer BM is added into each tube of dry powder, and the mixture is stirred and mixed until the dry powder is completely dissolved, and the whole process takes about 5min. Note that: the volume of the dissolved BisulfiteMix was about 950. Mu.l, which was used to process 10 samples, and the unused BisulfiteMix was stored at-20℃for 1 month.

3) The bisulfite reaction system was prepared in a 200. Mu.l centrifuge tube (EP tube) as follows:

4) After the reaction system is prepared, the bisulfite conversion is carried out on a PCR instrument or other instrument capable of setting a temperature change program, the whole process takes about 1 hour, and the specific program is as follows:

the PCR reaction liquid was as follows:

note that: if the initial DNA amount is less than 500ng, the DNA is treated for 30min at 64 ℃; if the amount of the initially treated DNA is more than 500ng, the treatment is carried out at 64℃for 60 minutes. In addition, the cap of the EP tube should be ensured to be tightly covered during the treatment process, and the heat cover treatment should be performed.

5) DNA purification after bisulfite treatment

Column balancing: 500 μl of balance liquid BL is added into the adsorption column CB1 (spinColumNSCB 1) (the adsorption column is placed in the collecting tube), the mixture is centrifuged at 12,000rpm (13,400 Xg) for 1min, the waste liquid in the collecting tube is poured out, the adsorption column is replaced in the collecting tube, and the balanced CB1 column is used for the same day.

After completion of the bisulfite treatment procedure, the reaction system in the EP tube was transferred to a clean 1.5ml centrifuge tube by brief centrifugation. After transfer, 5 volumes (600. Mu.l) of the binding solution PB was added to EP, and after thoroughly mixing, the mixture was added to the adsorption column CB1 (the adsorption column was placed in the collection tube), and the mixture was centrifuged at 12,000rpm (13,400 Xg) for 30-60 seconds at room temperature, and the waste liquid in the collection tube was discarded, and the adsorption column CB1 was placed in the collection tube. Note that: the volume of the adsorption column was 800. Mu.l, and if the sample volume was greater than 800. Mu.l, the column was added in portions.

600 μl of rinse solution PW (absolute ethanol was confirmed before use) was added to the adsorption column CB1, and the mixture was centrifuged at 12,000rpm (13,400Xg) for 30-60 seconds, and the waste liquid in the collection tube was poured off, and the adsorption column CB1 was placed in the collection tube. 600 μl of buffer DB was added to the adsorption column CB1, and the mixture was left at room temperature (15-25deg.C) for 15min, centrifuged at 12,000rpm (13,400Xg) for 30-60 seconds, and the waste liquid in the collection tube was poured off, and the adsorption column CB1 was placed in the collection tube.

600 μl of the rinse PW was added to the adsorption column CB1, centrifuged at 12,000rpm (13,400Xg) for 30-60 seconds, and the waste liquid in the collection tube was discarded. The rinsing and centrifuging operations were repeated once.

The adsorption column CB1 was placed in a collection tube, and centrifuged at 12,000rpm (13,400Xg) for 2 minutes to remove the rinse solution as much as possible. The adsorption column is left at room temperature for a few minutes and is thoroughly dried to prevent the residual rinsing liquid from affecting the next experiment. Note that: the residual ethanol in the rinse solution can affect subsequent PCR experiments.

Taking out the adsorption column CB1, putting into a clean centrifuge tube, suspending and dripping 20 μl of elution buffer EB into the middle of the adsorption film, and standing at room temperature for 2min. The DNA solution was collected by centrifugation at 12,000rpm (13,400Xg) for 2min.

Example 3: multiplex PCR amplification of sulfite-converted DNA (BisDNA)

After the DNA is subjected to sulfite treatment, the methylation of CpG island base guanine (C) is unchanged, namely Cm-Cm; whereas the unmethylated CpG island base guanine is changed to the base uracil, C.fwdarw.U. In primer design for multiplex PCR amplification, uracil in the treated DNA sequence is replaced with thymine, U.fwdarw.T, to design a methylated MSP primer.

Principle of MSP primer design:

(1) At least 1 CpG island should be contained in the primer sequence, and the CpG island should be near the 3' -end.

(2) The primer sequence should contain a plurality of "C" s that are not CpG islands.

(3) The number and positions of CpG islands should be identical for both DNA methylated and unmethylated primers.

(4) Other principles should be as consistent as possible with ordinary PCR.

Entering a website of a free online MSP primer design program 'MethPrimer': http:// www.urogene.org/methprimer/index. Html, designed to give the following 3 pairs of methylated primers for CLDN 18:

F:TTAGGTTGGAGTGTAGTGGC

R:AAAAACCTTAAAAACCGAATACGATA(1)

F:GTTTAGGTTGGAGTGTAGTGGC

R:ACCTTAAAAACCGAATACGATA(2)

F:GTTTAGGTTGGAGTGTAGTGGC

R:AACCTTAAAAACCGAATACGATA(3)

multiplex PCR amplification was performed on sulfite-converted DNA (BisDNA) using the 3 pairs of methylated primers for CLDN18 as described above, with the following amplification method:

PCR amplification required the following cycles:

1. and initializing. This is only essential for hot start PCR. This step heats the solution to 94-98℃to activate the DNA polymerase. The time of this step depends on the polymerase used.

2. A denaturation step. DNA is a double-stranded molecule and DNA amplification requires the interaction of a primer with a single-stranded DNA template. In this step, the reaction mixture is heated to 94-98℃for 20-30 seconds to break the hydrogen bonds between the two strands and produce a single-stranded DNA molecule. At this point, a PCR cycle was entered.

3. And (3) an annealing step. After denaturation, the DNA template in the reaction mixture is single stranded. Because the primer is complementary to the DNA template, when the reaction temperature is reduced to 50-65 ℃, the primer will match the template sequence, and hydrogen bonds are formed between complementary bases. The annealing temperature depends on the Tm of the primer used, and is generally about 3-5℃lower than the Tm of the primer. This step will last for about 20-40 seconds to anneal completely, and then the polymerase will localize to the primer-template hybrid to begin DNA assembly.

4. An elongation step. In this step, the DNA polymerase starts to synthesize DNA, and therefore the temperature should be the optimum temperature of the DNA polymerase. Typically 72℃is chosen, but some enzymes work better at 68 ℃. This step is very similar to in vivo DNA replication, and DNA polymerase adds dNTPs to the primer, complementing the template in the 5 'to 3' direction, ultimately producing a new double stranded DNA fragment. The extension time depends on the length of the target DNA fragment and the ability of the DNA polymerase. In general, DNA polymerase produces one kilobase every 60 seconds.

Steps 5.2-4 are referred to as a cycle, once per cycle, the target fragment amount doubles. One PCR procedure used 30-35 cycles. In the early part of the PCR cycle, the PCR product accumulates at an exponential rate, while in the later part of the PCR cycle, the reaction slows down and the PCR rate gradually decreases as dNTPs, primers decrease and DNA polymerase becomes inactive at the denaturation temperature.

6. Final elongation. After 30-35 cycles, the final extension is performed at a temperature of 68-74℃for about 5-10 minutes to fully extend the remaining single stranded DNA.

7. And (5) storing. The final product may be maintained at a temperature of 4-10C in a PCR machine.

In addition, the specificity and sensitivity of multiplex PCR amplification was verified for the 3 pairs of methylated primers for CLDN18 as follows:

reverse transcription PCR (RT-PCR) verifies the specificity of the primer and then carries out methylation quantitative and qualitative detection judgment.

(1) Total RNA was extracted from PDAC cell lines or tissues using TRizol reagent (Invitrogen).

a. First, it is necessary to culture the cell line to 10X 10 ⁵ Then after the large dish is completely full, counting cells, and evaluating the cell number;

b. depending on the number of cells in the dish, TRIZOL1ml or so was added, and the reagent was allowed to come into sufficient contact with the cells so that all the cells were digested, and then the product was collected using a 1.5ml EP tube.

c. After adding TRIZOL, after waiting for collection, 1ml of chloroform was added, and the mixture was left to stand at room temperature for about 2.5 minutes with uniform stirring, followed by slow centrifugation in a pre-cooled centrifuge at 4℃at 12000rpm for 15-20 minutes.

d. After low-speed centrifugation, taking a cool and transparent water phase substance at the upper layer, namely 400-500 mu l, transferring the water phase substance into an enzyme-free tube by using a 1ml gun head, continuously adding 500 mu l of isopropanol, uniformly stirring, standing at room temperature for about 10 minutes, and centrifuging at 4 DEG for about 10 minutes at the same rotating speed;

e. after the centrifugation, the precipitate at the bottom of the EP tube was found to be the target product, so after the supernatant was removed, 80% ethanol was added to the tube to blow and mix the mixture uniformly, and then centrifugation was performed at 4℃at 12000rpm for about 10 minutes.

f. Finally, the product was allowed to stand at 4℃for about 15 minutes, whereupon RNA was dissolved with DEPC water, and the volume of the solution was determined by the total amount of the product. And the solution is preferably preheated in a water bath kettle with the temperature of 60 degrees before being dissolved;

in order to avoid degradation, the RNA is extremely unstable, the operation is needed to be carried out on ice in the subsequent operation process, the concentration of the extracted product is needed to be measured, a NANODROP instrument is used for detection, and the zeroing setting is needed to be carried out by using dissolving liquid DEPC water before each test;

(2) Reverse transcription of RNA

First, a water bath at two temperatures of 70℃and 42℃was prepared, and preparation of RNA template and important primer deforming solution was performed.

a, preparing a denatured solution, adding 1 microliter of oligo primer/1ng-1ug template to a 1.5ml EP tube without enzyme and finally replenishing with DEPC water; then placing the mixture prepared in advance in a constant-temperature water bath kettle at 70 ℃ for heat preservation for about 15 minutes; and then rapidly cooling on ice for more than 2-3 minutes. Then centrifuging the template and the denatured solution for a few seconds and concentrating at the bottom of the tube part;

design of reverse transcription System

To the resulting RNA template and denatured solution was added 0.5. Mu.l DNTP, 2. Mu.l 5X (MLV-M-BUFFER), 0.25. Mu.l M-RNAase-MLV and 0.25. Mu.l RNAase inhibitor, which eventually required supplementation with enzyme-free water to 10. Mu.l. After the system was completely mixed, incubation was performed in a water bath at 42℃for about 1 hour. Then, the temperature was kept at 70℃for about 15 minutes. Then rapidly transferring to an ice brick at 4 ℃ for low-temperature cooling treatment, and finally obtaining a product which is a target cDNA product, wherein the final product can be stored at-20 degrees or-80 degrees on one hand, and subsequent experimental arrangement is needed on the other hand.

PCR reaction procedure of the polymeric chain

The primer sequence is designed through NCBI on-line website and sent to primer custom company for synthesis, the primer is generally divided into dry powder or solution after coming, the recommended annealing temperature range is also provided in the specification, and the optimal annealing temperature is required to be searched in the later period. Subsequently, 25. Mu.l of the reaction solution was prepared, and 2. Mu.l of cDNA template +12.5. Mu.l of premix +1. Mu.l of F 'primer +1. Mu.l of R' primer +8.5. Mu.l of DEPC water were added in sequence to a 200. Mu.l EP tube rack, followed by centrifugation to ensure uniform mixing. The Primer selected for real-time quantitative PCR is typically SYBERprimer with a fluorescent group.

The next step is to amplify the PCR system: 94-3 min-pre-denaturation; 94 ° +30s+ denaturation; 56-30 seconds-anneal; 72-30 seconds-extension; the number of cycles is generally around 34, which is kept for 10min, mainly according to the length of the target fragment and then at 72 °, for a total of about 2 hours. The data analysis method comprises the following steps: the data calculated by the equipment are mainly derived through EXCEL, the cycle number of the target genes is set as a CT value, the experimental group is CT0, the reference control genes of each group are marked as CT1, and the method can be used for obtaining:

qct=ct 0-CT1; qqct=qct experimental group-QCT control group, whereby 2 can be obtained ^-QQCT The relative expression level of the target gene with respect to the reference gene is represented. And carrying out homogenization treatment subsequently.

d, genome DNA gel electrophoresis

The concentration of the prepared gel electrophoresis liquid is mainly based on the length of the target molecular fragment, and generally 1% agarose gel is used; the preparation of 1 xTAE solution is started, 100ml of the solution is measured by a measuring cylinder, 1g of agarose powder is weighed and dissolved in the TAE solution, and the solution is immediately put into a microwave oven for high-temperature airtight heating and dissolution after shaking. Heating with high power, taking out all liquid after cooling and transparence, adding 2 microlitres of nucleic acid binding agent, rapidly placing into a gel frame prepared in advance, and inserting into a comb; the whole process prevents scalding, avoids generating bubbles, and also needs to pay attention to protecting experimental staff, which are toxic reagents. After the gel is successfully prepared, the PCR product is treated by DNAloading buffer solution and is loaded, a proper DNA marker is selected according to the molecular weight of the fragment, overflow is avoided in the loading process, and 120v/30min is selected for electrophoresis. Finally, the exposure instrument is used for image acquisition and preservation.

The mRNA was then used for first strand cDNA synthesis using a reverse transcription PCR system (bimake) according to the manufacturer's instructions. RT-PCR detects cDNA expression level of target gene. Each RT-PCR experiment was independently repeated at least three times. Actin was used as load control.

Firstly, detecting peripheral blood of 60 pancreatic cancer patients by using a kit, and recording the results; in order to further evaluate the specificity and sensitivity of the detection method, peripheral blood detection was also performed in further patient cases that were negative results (non-pancreatic cancer). The samples of peripheral blood of 20 pancreatic benign diseases are continuously detected by adopting a test in-vitro diagnostic reagent, and the methylation level of the CLDN18 is increased in 18 samples, which shows that the methylation level of the CLDN18 in the peripheral blood DNA of normal 20 patients is obviously higher than that of 60 pancreatic cancer patients; and methylation detection values were plotted on ROC curves to give AUC values=0.814 of fig. 4 (detection of ctDNA in peripheral blood was similar to the above method). Table 1 below is the clinical pathology parameter statistics for CLDN18.2 in pancreatic cancer.

Tab 1CLDN18.2 clinical pathological parameter statistics in pancreatic cancer

The test results show that: the primer PCR amplification was performed in the ctDNA test of peripheral blood of 60 patients with pancreatic cancer, and the designed methylation primer had an AUC value of 0.814. The results show that the in-vitro diagnostic reagent for the test has better clinical sensitivity and specificity and meets the clinical use requirement. To further quantify the methylation level differences of CLDN18.2 in pancreatic cancer tissue specimens (cancer and paracancerous), we performed single cell multigang high throughput sequencing using 14 surgically excised pancreatic cancer specimens from this center. By methylation sequencing analysis, CLDN18.2 had significantly lower methylation levels in cancer tissues than in paracancerous tissues, fold differences were demonstrated by visualization of heat and volcanic patterns.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof. The foregoing examples or embodiments are merely illustrative of the invention, which may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The described embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. The scope of the invention should be indicated by the appended claims, and any changes that are equivalent to the intent and scope of the claims are intended to be encompassed within the scope of the invention.

Claims (7)

1. Use of a reagent for detecting ctDNA-CLDN18.2 methylation level in the preparation of a product for diagnosing or screening pancreatic cancer.

2. The use according to claim 1, wherein the ctDNA-CLDN18.2 methylation level is down-regulated in the tissue or sample of pancreatic cancer.

3. The use of claim 1, wherein the reagent for detecting ctDNA-CLDN18.2 methylation level comprises a reagent for detecting ctDNA-CLDN18.2 methylation level by PCR, immunoassay, in situ hybridization or chip.

4. The use according to claim 3, wherein the reagent for PCR detection of ctDNA-CLDN18.2 methylation level comprises CpG island nucleic acid sequence comprising the sequence shown in SEQ ID NO.1 in the sequence of the amplified fragment.

5. The use according to claim 1, wherein said product comprises a test kit, a chip, a test strip or a pharmaceutical composition.

6. The use according to claim 3, wherein the PCR assay is a fluorescent quantitative PCR assay.

7. The use according to claim 6, wherein the amplification primers for fluorescent quantitative PCR detection comprise a pair of forward and reverse primers and a fluorescein probe that specifically binds to ctDNA-CLDN 18.2.

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