Liquid biopsy method based on white vinegar induced secreted pancreatic juice

Abstract

The invention discloses a liquid biopsy method based on white vinegar induced secreted pancreatic juice, and belongs to the technical field of molecular diagnosis and liquid biopsy. In order to solve the problems that a pancreatic juice sample is difficult to obtain and free DNA is digested by nuclease in the pancreatic juice, a trypsin inhibitor and a whole-course ice bath protection technology are mainly adopted, and a liquid biopsy method for inducing secretion of the pancreatic juice by white vinegar is combined. According to the method, free DNA in pancreatic juice can be effectively extracted, target gene mutation detection is carried out, and the detection sensitivity and accuracy are remarkably improved. The method is easy and convenient to operate, safe, low in cost and suitable for obtaining samples through multiple upper gastrointestinal endoscopy examinations, has high repeatability, detection precision and personalized customization capacity, and can provide important data support for early diagnosis of diseases, treatment response monitoring and targeted therapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular diagnosis and liquid biopsy, and specifically relates to a liquid biopsy method based on pancreatic juice secreted by edible white vinegar. Background Art

[0002] With the development of modern medical technology, the incidence and mortality of various solid tumors, including lung cancer, have been significantly improved. However, data reported by the World Health Organization / International Agency for Research on Cancer show that the incidence of pancreatic cancer is still on the rise worldwide (Klein AP. Pancreatic cancer epidemiology: understanding the role of lifestyle and inherited risk factors. Nat Rev Gastroenterol Hepatol. 2021; 18(7): 493-502. doi: 10.1038 / s41575-021-00457-x). According to the statistics of the National Cancer Center of my country in 2017, pancreatic cancer ranks 13th in the incidence of malignant tumors, ranking 7th in men and 11th in women; at the same time, pancreatic cancer is extremely malignant, ranking 6th in the mortality rate related to malignant tumors in my country. When patients are diagnosed, most of them have lost the opportunity for surgery and have a very poor prognosis, with a 5-year survival rate of only 8% to 10%. The difficulty in early diagnosis is considered to be the main reason (Yang Yinmo, Tian Xiaodong. Guidelines for the diagnosis and treatment of pancreatic cancer in China (2021). Chinese Journal of Practical Surgery, 2021, 41(07): 725-738.doi:10.19538 / j.cjps.issn1005-2208.2021.07.02.).

[0003] At present, pancreatic cancer screening in most countries or regions, including my country, mainly relies on serology and imaging. In terms of serological examinations, serum tumor markers represented by CA19-9 and CEA are positively correlated with tumor burden in the body, which can indicate potential metastasis risk and even evaluate treatment efficacy and patient prognosis (Zhao Z, Liu W. Pancreatic Cancer: A Review of Risk Factors, Diagnosis, and Treatment. Technol Cancer Res Treat. 2020; 19: 1533033820962117. doi: 10.1177 / 1533033820962117). In addition, this examination is convenient and easy to promote, with the dual advantages of timeliness and low cost, and can achieve long-term continuous follow-up. It has been included in the routine diagnosis and treatment of pancreatic cancer by medical institutions at all levels in my country. However, this indicator is closely related to common complications of pancreatic cancer patients, such as biliary obstruction and infection status, and there are risks of false negatives or false positives that cannot be ignored. In addition, the value fluctuates greatly, making it difficult to meet the needs of precision medicine. In terms of imaging examinations, although transabdominal ultrasound is simple, flexible, intuitive, non-invasive, radiation-free, and can be viewed in multiple axes, and is available in medical institutions at all levels, its visible field of view is small, and it is greatly affected by the patient's intestinal gas and body position, and the examination results are highly subjective. Currently, both domestic and foreign countries mainly use multi-phase enhanced CT and multi-modal enhanced MRI, which involve preoperative staging of pancreatic cancer, surgical resectability assessment or evaluation of the effect of neoadjuvant therapy, as well as long-term monitoring and follow-up after treatment. In addition, with the innovation and development of nuclear medicine technology, PET / CT and PET / MRI technologies are used to evaluate the metabolic load of tumor tissue in patients because they can show the metabolic activity of tumor cells, in order to discover potential metastatic lesions and conduct systematic evaluations to evaluate the whole-body tumor load. Especially when the diagnosis of CT and MRI is unclear, this technology can provide additional information to assist clinicians in distinguishing benign and malignant lesions (Kobi M, Veillette G, Narurkar R, et al. Imaging and Management of Pancreatic Cancer. Semin Ultrasound CT MR. 2020; 41(2): 139-151. doi: 10.1053 / j.sult.2019.12.003). However, the above technologies are not sensitive to small pancreatic cancers with no obvious space-occupying effect. In addition, pancreatic cancers are often poorly supplied with blood, so there is a high risk of missed diagnosis of early metastatic lesions and local recurrences. In addition, this type of technology is limited by the inability to obtain lesion tissue and cannot meet the needs of clear diagnosis and individual evaluation.With the development of optical and ultrasonic technology, digestive endoscopy technology represented by ultrasound endoscopy has been widely used in the diagnosis and treatment of pancreatic and biliary diseases because of its dual functions of scanning and biopsy. On the one hand, ultrasound endoscopy can achieve close observation of adjacent pancreatic tissue through the stomach wall and duodenum, clearly displaying anatomical structures such as pancreatic parenchyma and pancreatic duct. Not only is the sensitivity and specificity for space-occupying lesions significantly better than other traditional imaging technologies, but it can also simultaneously scan peripancreatic lymph nodes and organs for accurate staging; on the other hand, with the help of a special puncture needle, this technology can obtain cells or tissues to determine the pathological nature of the lesion and identify the source of the tumor for precise treatment. However, as an advanced endoscopic technique, the quality of scanning and biopsy is highly dependent on the operator's experience and skills, and it has a long learning curve and high cost, making it difficult to promote on a large scale as a screening project (Yang J, Xu R, Wang C, Qiu J, Ren B, You L. Early screening and diagnosis strategies of pancreatic cancer: a comprehensive review. Cancer Commun (Lond). 2021; 41(12): 1257-1274. doi: 10.1002 / cac2.12204).

[0004] As people's understanding of the mechanism of tumor occurrence and development continues to deepen, a large number of studies in recent years have confirmed that there are a variety of substances closely related to tumors in biological samples represented by peripheral blood and tissue secretions. By detecting metabolites, biomarkers or genomic components, it has important clinical significance and application prospects for early diagnosis, biological evaluation, medication guidance, efficacy monitoring and prognosis evaluation of pancreatic cancer (Kuwatani M, Sakamoto N. Pathological and molecular diagnoses of early cancer with bile and pancreatic juice. Dig Endosc. 2022; 34 (7): 1340-1355. doi: 10.1111 / den.14348). However, it is limited by the complexity of pancreatic juice contents. For example, highly active pancreatic enzymes can digest nucleic acids in a short time, resulting in the inability to detect meaningful mutations, while the rich connective tissue can cause non-negligible interference in the extraction of trace pancreatic cancer DNA. In addition, the pancreatic juice collection methods proposed by different teams at home and abroad are quite different at this stage. There is no widely recognized safe, effective and standardized scheme, and the collected pancreatic juice samples lack meaningful quality control methods. Furthermore, secretin, which is widely used internationally to promote pancreatic juice secretion, has not yet been approved by CFDA for use in humans in my country. Therefore, there is currently no means of promoting pancreatic juice secretion confirmed by high-quality research in my country, resulting in the inability to obtain sufficient pancreatic juice for subsequent analysis in a short period of time; based on this, the team of the Endoscopy Center of the Department of Gastroenterology of Peking Union Medical College Hospital took the physiological basis of gastric acid stimulating pancreatic juice secretion as the starting point, and designed a study to find that perfusing my country's commercial edible white vinegar (the active ingredient is 5% acetic acid solution) at the duodenal bulb-descending junction for several minutes can achieve a similar effect to obtain a large amount of high-quality pancreatic juice in a short period of time, which has a very high application prospect. However, there is currently a lack of effective liquid biopsy schemes for pancreatic juice induced by white vinegar, and this problem needs to be solved urgently. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, especially the difficulty in obtaining pancreatic juice samples and the digestion of free DNA by nucleases in pancreatic juice, the present invention proposes a liquid biopsy method based on pancreatic juice induced by edible white vinegar, which aims to simply, safely and efficiently extract and analyze free DNA in pancreatic juice, and detect target gene mutations of diseases.

[0006] A liquid biopsy method based on pancreatic juice secreted by white vinegar, comprising the following steps:

[0007] (1) Add trypsin inhibitor mixture and EDTA solution to a sterile collection bottle and shake thoroughly;

[0008] (2) collecting the pancreatic juice induced by white vinegar, centrifuging it twice, and collecting the supernatant after the centrifugation;

[0009] (3) extracting free DNA from the pancreatic juice supernatant after centrifugation;

[0010] (4) The KRAS mutation sites in the extracted free DNA were detected using droplet digital polymerase chain reaction, the mutation frequency was calculated and the test results were obtained.

[0011] Furthermore, in step (1), 100x trypsin inhibitor mixture and 0.5M EDTA solution with a pH of 8.0 and no RNase are added to the sterile collection bottle. After mixing, the final concentration of the trypsin inhibitor mixture is 1x.

[0012] Furthermore, the solution prepared in step (1) is stored at 4°C for future use.

[0013] Furthermore, in step (2), the mixed sample is centrifuged twice within 5 minutes, the first centrifugation condition is low-speed centrifugation at 4°C for 15 minutes, and the relative centrifugal force is 1600-1900g; the second centrifugation condition is high-speed centrifugation at 4°C for 15 minutes, and the relative centrifugal force is 16000-17000g.

[0014] Furthermore, the residual precipitate from the first centrifugation in step (2) is frozen and stored in an environment of -80°C to -196°C for future use, and the supernatant obtained from the second centrifugation is stored in an environment of -80°C to -196°C for future use.

[0015] Furthermore, in step (3), a free nucleic acid cfDNA extraction kit and a matching HF24 fully automatic nucleic acid purification instrument are used to extract free DNA, and an ultra-micro spectrophotometer is used to determine the concentration and purity of the extracted free DNA.

[0016] Furthermore, the free DNA extracted in step (3) is stored in an environment of -80°C to -196°C for future use.

[0017] Furthermore, the step of using droplet digital polymerase chain reaction to detect KRAS mutation sites in the extracted free DNA in step (4) includes:

[0018] Prepare a PCR amplification reagent Mix solution, vortex mix the Mix solution in a centrifuge tube, centrifuge it instantaneously, and add it to a PCR reaction tube;

[0019] Take the negative control, positive control and free DNA sample to be tested and add them to the Mix solution in the reaction tube, mix well and centrifuge to remove bubbles;

[0020] The solution without bubbles is formed into microdroplets, and PCR amplification is performed in the form of microdroplets;

[0021] During the PCR amplification process, the kit is used to perform microdroplet detection to obtain the 2D scatter plot and the detection results of the positive control;

[0022] According to the detection results of the 2D scatter plot and the positive control, the positive area was delineated to obtain the copy number of each channel, and then the results were interpreted according to the positive judgment value to obtain the detection results of the gene KRAS mutation site.

[0023] Furthermore, the PCR amplification conditions in step (4) include:

[0024] 1) Pre-denaturation: temperature 93-97°C, duration 8-12 minutes, cycle number 1-3 times;

[0025] 2) Denaturation: temperature 92-96°C, duration about 20-40 seconds; annealing and extension: temperature about 58-62°C, duration about 30-90 seconds; cycle number 40-60 times;

[0026] 3) Cool together: temperature is about 10-14°C, duration is about 3-7 minutes, and the number of cycles is 1-3 times.

[0027] Furthermore, the storage temperature of the PCR amplification product in step (4) is 2 to 8°C.

[0028] The core innovation of the present invention is to add trypsin inhibitor and protect it in an ice bath throughout the process, which effectively inhibits the digestion of free DNA by trypsin, thereby protecting free DNA to the greatest extent and significantly improving the sensitivity and reliability of subsequent detection and analysis. Compared with other analysis methods, the present invention has the following significant advantages:

[0029] (1) Wide application scope: The pancreatic juice sample induced by edible white vinegar can be obtained through a variety of upper gastrointestinal endoscopic examinations, such as gastroscopy, ultrasonic endoscopy, duodenoscope, etc. Compared with the traditional endoscopic pancreatic juice collection scheme that relies on intravenous injection of secretin or pancreatic duct cannulation under endoscopic retrograde pancreaticobiliary examination, it is simpler, more economical and safer. In addition, the amount of pancreatic juice secreted after stimulation by edible white vinegar is large. The present invention innovatively uses trypsin inhibitors to inhibit the digestion of free DNA by pancreatic enzymes, maintains the integrity of free DNA from the pancreas, and can meet more research and analysis needs.

[0030] (2) Strong repeatability: The pancreatic juice sample induced by edible white vinegar involved in the present invention will not cause damage to the patient's duodenal papilla during the collection process. Compared with traditional endoscopic pancreatic juice collection schemes such as endoscopic retrograde pancreaticobiliary angiography, it can achieve rapid sampling and processing of multiple time points in the course of a single patient's disease, and obtain continuous results that match the patient's disease course, providing important data support for patient condition assessment, treatment response detection and discovery of potential targets.

[0031] (3) Flexible customization: The present invention relies on the processing of pancreatic juice samples induced by edible white vinegar and the enrichment of free DNA to detect gene mutations through microdroplet digital polymerase chain reaction. This method can perform corresponding mutation detection for different genes according to the specific clinical diagnostic needs of patients, providing personalized and customized detection information.

[0032] (4) High detection accuracy: The present invention uses the whole exome sequencing results of pancreatic lesion puncture tissue as the gold standard, and the detection results of pancreatic juice samples induced by edible white vinegar have a matching degree of more than 88.89%. In addition, for some patients with negative puncture results, this method can also provide rescue diagnosis, effectively improving the comprehensiveness and accuracy of the test.

[0033] (5) Low testing cost: The test consumables and equipment involved in the present invention are cheap. Except for the fixed costs of endoscopic examination and puncture, the consumption of the whole process including edible white vinegar, free DNA extraction, and micro-droplet digital polymerase chain reaction can be controlled at around RMB 1,000. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The figure shows a simplified flow chart of the liquid biopsy method based on pancreatic juice secretion induced by white vinegar. DETAILED DESCRIPTION

[0035] In order to make the various technical features and advantages or technical effects in the above technical solutions of the present invention more obvious and easy to understand, they are described in detail below in conjunction with embodiments.

[0036] The present invention provides a liquid biopsy method based on pancreatic juice secreted by white vinegar, which includes key steps such as pretreatment before pancreatic juice collection, pancreatic juice treatment and free DNA extraction, and detection of KRAS gene mutation sites in pancreatic juice free DNA samples based on digital polymerase chain reaction, and can efficiently capture and detect target gene mutations of diseases. The process is as follows Figure 1 The gene names used in the present invention are all official names in NCBI-Gene (https: / / www.ncbi.nlm.nih.gov). The specific processing steps of this method are as follows:

[0037] 1. Pretreatment of pancreatic juice before collection

[0038] Before the pancreatic juice is formally collected, add 100x trypsin inhibitor mixture and 0.5M EDTA solution (pH 8.0, RNase-free) to the sterile collection bottle to ensure that the final concentration of the trypsin inhibitor mixture is about 1x. After thorough shaking, store it at 4°C for future use.

[0039] 2. Pancreatic juice processing and free DNA extraction

[0040] 2.1. Pancreatic juice separation

[0041] After the pancreatic juice is collected, the pancreatic juice in the sterile collection bottle needs to be transferred into 1.5 ml centrifuge tubes in batches within 5 minutes. First, the supernatant is transferred into a 1.5 ml centrifuge tube after centrifugation at 1600g for 15 minutes at 4°C, and the remaining precipitate is frozen at -80°C for future use. Then, centrifuge at 16000g for 15 minutes at 4°C, discard the precipitate, collect the supernatant into a 4 ml centrifuge tube, and store it at -80°C for future use.

[0042] 2.2. Enrichment and extraction of free DNA in pancreatic juice

[0043] The free DNA in the pancreatic juice supernatant was enriched and extracted using the Kaishuo Biotechnology RC1006-4 free nucleic acid cfDNA extraction kit and the matching HF24 fully automatic nucleic acid purification instrument. A pancreatic juice free DNA sample with a volume of 60 μL was obtained. The concentration and purity of the free DNA in the extracted sample were determined using the Thermo NanoDrop Lite ultra-micro spectrophotometer and then stored at -80°C for future use.

[0044] 3. Detection of KRAS gene mutation sites in pancreatic juice free DNA samples based on digital polymerase chain reaction (PCR)

[0045] (1) Reagent preparation: Prepare the PCR amplification reagent Mix solution according to Table 1 based on the number of negative control (DEPC water), positive control and samples to be tested (n).

[0046] Table 1 KRAS reagent composition ratio

[0047] Reagent components Mix 1 Mix 2 KRAS Reagent A 7.5μL×(n+2) 7.5μL×(n+2) KRAS Reagent B 7.5μL×(n+2) KRAS Reagent C 7.5μL×(n+2)

[0048] Vortex the Mix solution in a 1.5 ml centrifuge tube for 10 seconds, centrifuge briefly, and dispense 15 μL of the PCR reaction solution into a 0.2 ml PCR reaction tube.

[0049] (2) Template addition: Take 15 μL of negative control, positive control and test sample (nucleic acid concentration must be 0.5-10 ng / μL) and add them to the PCR amplification Mix solution, mix well and centrifuge to remove bubbles.

[0050] (3) Microdroplet generation: Use the Xinyi Manufacturing Technology sample preparation instrument (DropMakerM1) and microdroplet generation chip and adapters to add 180μL of generated oil to the oil phase solution well, add 30μL of aqueous system to the aqueous phase solution well, run the instrument, and generate droplets. After the droplets are generated, PCR amplification must be performed immediately or within 1 hour at 2-8°C to ensure the quality of the test.

[0051] (4) PCR amplification: The sealed 8-tube strip containing microdroplets was placed on a PCR instrument for amplification. The amplification conditions were set as shown in Table 2. The heating and cooling rate was set to 1.5°C / s, and the amplification volume was set to 100 μL.

[0052] Table 2 Amplification conditions

[0053]

[0054] The PCR product can be tested immediately. If it cannot be tested immediately, it can be stored at 2-8°C overnight.

[0055] (5) Microdroplet detection: Use the microdroplet detection universal kit (10002) provided with the Xinyi Manufacturing Technology Biochip Analyzer (ChipReader R2) for detection. After the chip detection is completed, obtain the 2D scatter plot and positive control test results.

[0056] (6) Result analysis: After the chip test is completed, the appropriate positive area is delineated according to the 2D scatter plot and the test results of the positive control (generally, the positive area is delineated at the center of the area where the positive signal is concentrated), and the copy number of each channel is obtained. Then, the result is interpreted according to the positive judgment value, see Table 3.

[0057] Table 3 Mutation detection results of pancreatic juice samples

[0058]

[0059]

[0060] A specific embodiment is provided below:

[0061] Taking 23 patients with pancreatic ductal adenocarcinoma (PDAC) who were pathologically diagnosed by endoscopic ultrasound-guided fine needle aspiration biopsy (EUS-FNA) or endoscopic ultrasound-guided fine needle biopsy (EUS-FNB) as an example, the whole exome sequencing results of their lesion tissues were used as the gold standard, and the present invention was used to detect the KRAS gene mutation in free DNA in the pancreatic juice samples induced by edible white vinegar. At the same time, 11 patients with pancreatic ductal adenocarcinoma were matched, but the case in which edible white vinegar was replaced with saline (0.9% sodium chloride) during the pancreatic juice acquisition process was used as a control to illustrate the present invention. It is worth noting that this embodiment is only to illustrate that the scheme and specific steps of the invention are feasible, and the accuracy of the results is relatively impressive, but it cannot be interpreted as a limitation or negation of the present invention in any way. All the above 34 patients with pancreatic ductal adenocarcinoma were voluntary subjects, and the relevant research plan was reviewed by the Ethics Committee of Peking Union Medical College Hospital, Chinese Academy of Medical Sciences. The relevant clinical experiments were registered on ClinicalTrials.gov, and the informed consent signed by the patients was obtained before the start of the study. All of the above patients were clinically diagnosed with pancreatic solid masses with a malignant tendency after imaging examinations, had indications for endoscopic ultrasound examination and puncture, were older than 18 years old, had random gender, and had no other active malignant tumors.

[0062] 1. Pretreatment of pancreatic juice induced by edible white vinegar before collection:

[0063] The patient agreed to participate in the pancreatic juice-related research conducted by the Endoscopy Center Team of the Department of Gastroenterology of Peking Union Medical College Hospital. After completing the informed consent, three sterile collection bottles and matching disposable connecting tubes were prepared for each patient one day before the patient's operation. 153.2 μL of 100x trypsin inhibitor mixture and 153.2 μL of 0.5 M EDTA solution were added to each sterile collection bottle to ensure that the final concentration of the trypsin inhibitor mixture was 1x. After being fully shaken, it was stored at 4°C for future use.

[0064] 2. Treatment of pancreatic juice induced by edible white vinegar and extraction of free DNA:

[0065] During the ultrasound endoscopy, pancreatic juice was collected three times in 0-5 minutes, 5-10 minutes and 10-15 minutes by stimulating pancreatic secretion with edible white vinegar infusion in the duodenum and collecting pancreatic juice through ultrasound endoscopy. During the collection process, the sterile collection bottle was placed on ice to ensure that the environment was about 4°C, and the sterile collection bottle and disposable connecting vessel were replaced every 5 minutes.

[0066] 2.1. Pancreatic juice separation process

[0067] The collected pancreatic juice was divided into multiple 1.5 ml centrifuge tubes according to the patient information and collection time, and the supernatant and precipitate were separated after centrifugation at 1600g for 15 minutes, and the precipitate was frozen and stored at -80°C for future use. The supernatant was centrifuged again at 16000g for 15 minutes, the precipitate was discarded, and the supernatant was transferred to a 4 ml centrifuge tube and stored at -80°C for future use.

[0068] 2.2. Enrichment and extraction process of free DNA in pancreatic juice

[0069] The free DNA was enriched and extracted from the supernatant obtained after the second centrifugation of the pancreatic juice using the Kaishuo Biotechnology RC1006-4 free nucleic acid cfDNA extraction kit and the matching HF24 fully automatic nucleic acid purification instrument. A final volume of 60 μL of pancreatic juice free DNA sample was obtained for each pancreatic juice. The concentration and purity of the free DNA in the sample were determined by the Thermo ultra-micro nucleic acid protein analyzer and then stored at -80°C for future use.

[0070] 2.3. Digital polymerase chain reaction detection of KRAS gene mutation sites in pancreatic juice free DNA samples:

[0071] (1) Prepare PCR amplification reagents according to the negative control (DEPC water), positive control and samples to be tested (n = 3 samples per patient) (see Table 4 for details). Mix the solution thoroughly in a 1.5 ml centrifuge tube and centrifuge it instantaneously. Dispense 15 μL of the PCR reaction solution into a 0.2 ml PCR reaction tube.

[0072] Table 4 KRAS reagent composition ratio

[0073] Reagent components Mix 1 Mix 2 KRAS Reagent A 37.5μL 37.5μL KRAS Reagent B 37.5μL KRAS Reagent C 37.5μL

[0074] (2) Template addition: Take 15 μL of negative control, positive control and test sample (according to the above DNA quantitative test results, ensure that the concentration of free DNA in pancreatic juice is in the range of 0.5-10 ng / μL) and add them to the PCR amplification Mix solution, mix well and centrifuge to remove bubbles.

[0075] (3) Microdroplet generation: Use the Xinyi Manufacturing Technology sample preparation instrument (DropMakerM1) and microdroplet generation chip and adapters to generate droplets according to the operating procedures and perform PCR amplification immediately.

[0076] (4) PCR amplification: The sealed 8-tube strip containing microdroplets was placed on a PCR instrument for amplification. The amplification conditions were set to a temperature ramp rate of 1.5°C / s and an amplification volume of 100 μL (see Table 5 for the cycling conditions). Microdroplet detection was performed immediately after the PCR amplification was completed.

[0077] Table 5 Amplification conditions

[0078]

[0079] (5) Microdroplet detection: Use the Microdroplet Detection Universal Kit (10002) provided by the Xinyi Manufacturing Technology Biochip Analyzer (ChipReader R2) to complete the microdroplet detection according to the operating procedures, and obtain the data and scatter plots of negative and positive controls and the samples to be tested. Samples that produce ≤12,000 droplets are considered unqualified samples and are excluded from subsequent analysis.

[0080] (6) Result analysis: After the chip test, the positive area determined according to the previous research results of the Endoscopy Center Team of the Department of Gastroenterology of Peking Union Medical College Hospital (i.e., samples with a total of ≤2 positive mutation droplets or a variant allele frequency of <0.07%) was considered to have no intentional mutation detected.

[0081] 3. Comparison of whole exome sequencing results of pancreatic lesion biopsy tissue with KRAS mutation results of pancreatic juice free DNA samples

[0082] During the endoscopic ultrasound examination and puncture, the above patients retained as much lesion tissue as possible for whole exome sequencing on the basis of meeting the needs of clinical pathological diagnosis. Before submission, senior pathologists were required to confirm under the microscope that the planned samples contained definite tumor cells. hg38 was selected as the reference genome, and the germline snp / indel analysis software was GATK, and the somatic snv / indel analysis software was muTect2. After the sequencing results were reported, the KRAS gene mutation status in the whole exome sequencing of pancreatic lesion tissue was used as the gold standard to judge the KRAS mutation results of pancreatic juice free DNA samples. If the results of the two were consistent, that is, both negative or both positive, the pancreatic juice sample was judged to be meaningful. If the results of the two were inconsistent, that is, the KRAS mutation results of the pancreatic juice free DNA sample were consistent with the pancreatic puncture tissue, the pancreatic juice sample was judged to be meaningless.

[0083] The results of KRAS mutation detection in 34 patients are shown in Table 6 below:

[0084] Table 6 Mutation detection results of pancreatic tissue and pancreatic juice samples from patients with pancreatic ductal adenocarcinoma

[0085]

[0086]

[0087] In summary, for all patients with pancreatic ductal adenocarcinoma, the pancreatic juice induced by eating white vinegar is processed, enriched and detected by the invention scheme. The whole exome sequencing results of pancreatic lesion puncture tissue are used as the gold standard, which can effectively detect the KRAS gene mutation status of about 70% of patients, and compared with the control group replaced by duodenal injection of normal saline (the consistency rate is only 18%), there is a significant statistical difference between the two (P value = 0.00498). The results show that the method of the present invention has a high consistency between the pancreatic juice induced by vinegar and the detection of free DNA gene mutations and puncture tissue. In view of the fact that this method can be built into a number of upper gastrointestinal endoscopic examinations including gastroscopy, and is simple, easy and low-cost, it has the potential to be used as a screening scheme in actual clinical scenarios. In addition, for some patients with difficult puncture paths, who have not obtained satisfactory samples after multiple punctures, or who have lost the opportunity for surgical surgery, this invention is expected to become an alternative to preliminarily explore the possible gene mutations of patients and provide a reference for subsequent targeted therapy.

[0088] Although the present invention has been disclosed as above by way of embodiments, it is not intended to limit the present invention. Appropriate modifications or equivalent substitutions of the technical solutions of the present invention made by ordinary technicians in the field should all be included in the protection scope of the present invention. The protection scope of the present invention shall be based on what is defined in the claims.

Claims

1. A liquid biopsy method based on pancreatic juice secreted by white vinegar, characterized in that: The following steps are involved: (1) Add trypsin inhibitor mixture and EDTA solution to a sterile collection bottle and shake thoroughly; (2) collecting the pancreatic juice induced by white vinegar, centrifuging it twice, and collecting the supernatant after the centrifugation; (3) extracting free DNA from the pancreatic juice supernatant after centrifugation; (4) The KRAS mutation sites in the extracted free DNA were detected using droplet digital polymerase chain reaction, the mutation frequency was calculated and the test results were obtained.

2. The method according to claim 1, characterized in that In step (1), 100x trypsin inhibitor mixture and 0.5M EDTA solution with a pH of 8.0 and no RNase are added to the sterile collection bottle. After mixing, the final concentration of the trypsin inhibitor mixture is 1x.

3. The method according to claim 1 or 2, characterized in that The solution prepared in step (1) is stored at 4°C for future use.

4. The method according to claim 1, characterized in that In step (2), the mixed sample is centrifuged twice within 5 minutes. The first centrifugation condition is low-speed centrifugation at 4°C for 15 minutes with a relative centrifugal force of 1600-1900g; the second centrifugation condition is high-speed centrifugation at 4°C for 15 minutes with a relative centrifugal force of 16000-17000g.

5. The method according to claim 1 or 4, characterized in that The remaining precipitate from the first centrifugation in step (2) is frozen and stored in an environment of -80°C to -196°C for future use, and the supernatant obtained from the second centrifugation is stored in an environment of -80°C to -196°C for future use.

6. The method according to claim 1, characterized in that In step (3), a free nucleic acid cfDNA extraction kit and a matching HF24 fully automatic nucleic acid purification instrument are used to extract free DNA, and an ultra-micro spectrophotometer is used to determine the concentration and purity of the extracted free DNA.

7. The method according to claim 1, characterized in that The free DNA extracted in step (3) is stored in an environment of -80°C to -196°C for future use.

8. The method according to claim 1, characterized in that The step of using droplet digital polymerase chain reaction to detect KRAS mutation sites in the extracted free DNA in step (4) includes: Prepare a PCR amplification reagent Mix solution, vortex mix the Mix solution in a centrifuge tube, centrifuge it instantaneously, and add it to a PCR reaction tube; Take the negative control, positive control and free DNA sample to be tested and add them to the Mix solution in the reaction tube, mix well and centrifuge to remove bubbles; The solution without bubbles is formed into microdroplets, and PCR amplification is performed in the form of microdroplets; During the PCR amplification process, the kit is used to perform microdroplet detection to obtain the 2D scatter plot and the detection results of the positive control; According to the detection results of the 2D scatter plot and the positive control, the positive area was delineated to obtain the copy number of each channel, and then the results were interpreted according to the positive judgment value to obtain the detection results of the gene KRAS mutation site.

9. The method according to claim 8, characterized in that The PCR amplification conditions in step (4) include: 1) Pre-denaturation: temperature 93-97°C, duration 8-12 minutes, cycle number 1-3 times; 2) Denaturation: temperature 92-96°C, duration about 20-40 seconds; annealing and extension: temperature about 58-62°C, duration about 30-90 seconds; cycle number 40-60 times; 3) Cool together: temperature is about 10-14°C, duration is about 3-7 minutes, and the number of cycles is 1-3 times.

10. The method according to claim 8, characterized in that The storage temperature of the PCR amplification product in step (4) is 2 to 8°C.

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