Preservation method and application of bile sample

Abstract

The invention provides a bile sample preservation method. The method comprises the following steps: collecting a bile sample; and mixing the bile sample with a nucleic acid protective agent, and preserving at the temperature of 25-80 DEG C. The invention aims to design a plurality of storage conditions for storing bile from the presence / absence of a bile nucleic acid protective agent, different storage temperature gradients, different storage time periods and the like, and further preliminarily explores the stability of the bile cfDNA, so as to determine the condition for stably storing the bile cfDNA, and provide a foundation for subsequent application of the bile cfDNA to liquid biopsy. And corresponding technical support is provided for the aspects of early clinical diagnosis, prognosis monitoring and the like of patients with malignant biliary tract tumors.

Description

Technical Field

[0001] The invention belongs to the technical field of life sciences, and in particular relates to a bile sample preservation method and application thereof. Background Art

[0002] The diagnosis of cancer patients is based on imaging technology combined with tissue biopsy (the gold standard for accurate diagnosis of tumor pathology), but there are still certain disadvantages, such as obvious lag in tissue biopsy, increased risk of promoting tumor metastasis, the obtained tissue samples cannot well reflect the disease status, and some patients cannot obtain tissue or the obtained tissue is insufficient for tumor molecular diagnosis. Liquid biopsy technology is based on body fluid samples that are easier to obtain and is a non-invasive or minimally invasive sampling method. It has now become a powerful supplement to tissue biopsy, which helps to further improve the precision diagnosis and treatment of cancer patients. At present, the types of body fluids for liquid biopsy are diverse, including blood, urine, cerebrospinal fluid, ascites, and various organ lavage fluids. Among them, cell-free DNA (cfDNA) in body fluids is one of the main research directions. In addition to the above body fluid types, bile is also an important type of human body fluid, but there are currently very few studies that apply bile to liquid biopsy, and correspondingly, there are few studies that apply bile cfDNA to the diagnosis, treatment, and recurrence monitoring of tumors. In addition, the application of liquid biopsy is based on the stable preservation of biological fluid samples. Reasonable and efficient preservation of body fluid samples is particularly important in translational medicine and precision medicine related research and clinical applications.

[0003] Biliary tract cancers (BTCs) are malignant tumors of the biliary epithelium. They are the second most common type of liver cancer after hepatocellular carcinoma (the third leading cause of cancer-related deaths worldwide), accounting for 10-15% of all primary liver malignancies, including gallbladder cancer, cholangiocarcinoma, and ampullary carcinoma. Pathologically, they are mainly adenocarcinomas. BTCs are a rare cancer with an incidence of less than 6 cases per 100,000 people. Most patients have no obvious symptoms in the early stage and are difficult to detect before jaundice appears. Generally, they are already malignant when discovered and have generally missed the best time for surgical resection. At the same time, some patients cannot undergo surgical resection. In addition, the incidence of BTCs is continuing to increase, especially in patients with intrahepatic bile duct cancer, and the prognosis is very poor, with a five-year survival rate of less than 20%. In addition, even for patients who receive treatment and resection, the recurrence rate is still high, accounting for about >50%. Therefore, early diagnosis is an important measure to improve the prognosis of patients with malignant biliary tumors.

[0004] At present, the main methods for diagnosing malignant biliary tumors include imaging scans, histocytology, and serum markers. Imaging scans mainly include transabdominal ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), magnetic resonance cholangiography (MRCP), percutaneous transhepatic cholangiography (PTC), [18F] fluorodeoxyglucose positron emission tomography (FDG-PET), endoscopic retrograde cholangiopancreatography (ERCP), and real-time elastography (RTE). Ultrasound, CT, and MRI have limited sensitivity and specificity in diagnosis, and show no characteristic differences in the identification of some malignant biliary tumor lesions; PTC and ERCP are invasive examination methods; (FDG-PET) can reliably detect nodular cholangiocarcinomas ≥1 cm in size, but the accuracy will be significantly reduced when the tumor is less than 1 cm or even smaller. At the same time, due to nonspecific tissue inflammation, FDG uptake can increase, which is prone to false positive results in diagnosis. Brush cytology during transhepatic surgery is the most common technique for tissue sampling in patients with presumed malignant biliary strictures, but the sensitivity for diagnosing cholangiocarcinoma is only 30% to 60%. In addition, puncture biopsy is useful for tumor diagnosis in the case of positive biliary cancer, but a negative result cannot rule out malignant tumors and is prone to cause tumor spread and metastasis in patients. Serum antigens can be used clinically to diagnose malignant biliary tumors, but the commonly used CA19-9 and embryonic antigens are elevated in patients with cholestasis, liver damage, and benign biliary obstruction, resulting in limited diagnostic accuracy in patients with cholangiocarcinoma.

[0005] Compared with tissue biopsy and cytology brushing, liquid biopsy technology uses various types of human body fluids as raw materials, and can be sampled multiple times in a minimally invasive or non-invasive manner. At present, it has important clinical application prospects in early tumor screening, auxiliary diagnosis, postoperative evaluation, drug guidance, efficacy evaluation and prognosis detection. Among them, the body fluid type is mainly blood, and also includes urine, cerebrospinal fluid and pleural lavage fluid. The early diagnosis, prognosis and recurrence monitoring of cancer can be achieved by detecting and analyzing the tumor-related components including circulating tumor cells (CTC), circulating tumor DNA (ctDNA) and exosomes. However, after the blood flows through the human body, it is enriched with various molecular components and metabolites, resulting in further dilution of the already small amount of tumor-related components, making it difficult to make accurate early diagnosis of patients with biliary tract cancer. Bile is a new type of body fluid that is less studied in liquid biopsy. The total amount of bile is sufficient: an adult produces about 800 mL of bile per day; it is continuously secreted by liver cells, transferred to the gallbladder through the bile duct for concentration and storage, and discharged into the duodenum for fat digestion and fat-soluble vitamin absorption when the human body eats and digests. It is in direct contact with the liver, gallbladder and digestive system, and tumor components can be more easily monitored. Studies have reported that bile cfDNA of patients with malignant biliary tumors can be used for liquid biopsy (PMID: 31173267). When comparing bile cfDNA with DNA single nucleotide variations / insertions and deletions in tumor tissue, targeted deep sequencing results showed high sensitivity (94.7%) and specificity (99.9%). Another study showed that the sensitivity and specificity of gene mutations in bile cfDNA of patients with pancreatic ductal carcinoma were better than those in blood (PMID: 33375555). At the same time, studies have shown that the consistency of genomic changes between bile supernatant / granules and tumor tissues is significantly higher than that between plasma and tumor tissues, and more other gene mutations can be detected in bile supernatant and granules than in tissues (PMID: 36091112). Therefore, bile may be a better type of liquid biopsy fluid in studies related to hepatobiliary and digestive system tumors.

[0006] Biological samples are an indispensable resource for humans to discover the causes and characteristics of the occurrence and development of diseases. They are also an important basis for the development of new drugs and diagnostic and treatment methods. Therefore, the stable preservation of biological samples is particularly important in the basic research of translational medicine and precision medicine. Body fluid samples are important biological samples in liquid biopsy, mainly including blood, urine and cerebrospinal fluid. Blood will be separated and processed into plasma or serum and stored at -80℃ or in liquid nitrogen. Urine and cerebrospinal fluid will also be stored at -80℃ or in liquid nitrogen after centrifugation. Bile is an important type of body fluid sample in the human body. It is rich in a variety of biological molecules and can be used for liquid biopsy. As mentioned earlier, bile is a better type of liquid biopsy fluid in studies related to hepatobiliary and digestive system tumors. Therefore, how to well preserve the obtained bile body fluid samples plays a vital role in the early diagnosis of tumors, surgical guidance, efficacy evaluation and prognosis monitoring in liquid biopsy. However, the stable storage conditions of the new body fluid bile are currently unknown. Good bile preservation conditions are urgently needed that do not affect the subsequent extraction and sequencing of bile cfDNA, and can be used in subsequent research on early tumor diagnosis, treatment guidance and prognosis monitoring in liquid biopsy. Summary of the invention

[0007] The purpose of the present invention is to provide a bile sample preservation method and its application. The present invention aims to design a variety of storage conditions for bile preservation, such as the presence or absence of bile nucleic acid protective agents, different storage temperature gradients, and different storage time periods, and then conduct a preliminary exploration of the stability of bile cfDNA, thereby determining the conditions for stable storage of bile cfDNA, and providing corresponding technical support for the subsequent application of bile cfDNA in liquid biopsy, early clinical diagnosis and prognosis monitoring of patients with malignant biliary tumors, etc.

[0008] The technical solution adopted by the present invention to solve the above technical problems is as follows:

[0009] The present invention provides a method for preserving a bile sample, comprising the following steps:

[0010] S1. Collect bile samples;

[0011] S2. Mix the bile sample with the nucleic acid protective agent and store them at a temperature of 25°C to -80°C.

[0012] As a preferred embodiment, the nucleic acid protective agent includes K3-EDTA solution.

[0013] As a preferred embodiment, the nucleic acid protective agent is contained in a free nucleic acid storage tube.

[0014] As a preferred embodiment, the storage temperature is 4°C to -80°C.

[0015] As a preferred embodiment, the storage temperature is -20°C to -80°C.

[0016] As a preferred embodiment, the storage temperature is selected from any one of 4°C, -20°C or -80°C.

[0017] As a preferred embodiment, the storage period is no more than 2 months.

[0018] The present invention also provides application of the preservation method in bile cfDNA storage or bile cfDNA liquid biopsy.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The bile cfDNA extraction method adopted in the present invention can effectively extract bile cfDNA from cancer patients, thereby using bile cfDNA for early clinical diagnosis of cancer patients.

[0021] (2) The bile preservation method established by the present invention uses a preservation tube with a nucleic acid protective agent to store bile more stably, providing corresponding technical support for the subsequent application of bile cfDNA in liquid biopsy, early clinical diagnosis and prognosis monitoring of patients with malignant biliary tumors, etc.

[0022] (3) The bile preservation method established by the present invention uses a preservation tube with a nucleic acid protectant to stably store bile at room temperature for at least 10 days, which provides a certain reference for subsequent studies on how to preserve bile in the short term without affecting bile cfDNA. Bile cfDNA can be used as a new type of liquid biopsy material to provide important clinical indications for early diagnosis, treatment plans and disease progression monitoring of tumor patients.

[0023] (4) The bile preservation method established by the present invention can stably preserve bile at a temperature below 4°C using a preservation tube with a nucleic acid protective agent for at least 2 months. This provides a certain reference for subsequent studies on how to preserve bile for a long time without affecting bile cfDNA. Using bile cfDNA as a new type of liquid biopsy material can provide important clinical indications for early diagnosis, treatment plans, and disease progression monitoring of tumor patients.

[0024] (5) The samples selected in the present invention are derived from bile samples of patients with clinically malignant biliary tract tumors. The isolated cfDNA can accurately reflect the clinical characteristics, which is the basic prerequisite for using bile cfDNA for liquid biopsy, and further provides a solid foundation for the early diagnosis, treatment and monitoring of patients with biliary tract tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1This is a result graph of the total amount of bile cfDNA detected when the bile samples of the present invention were stored at room temperature for 1, 4, 7 and 10 days without a nucleic acid protective agent.

[0026] Figure 2 This is a graph showing the bile cfDNA A260 / A280 ratio results of the bile samples of the present invention when stored at room temperature for 1, 4, 7 and 10 days without a nucleic acid protective agent.

[0027] Figure 3 This is a graph showing the distribution of bile cfDNA fragments detected when the bile samples of the present invention were stored at room temperature for 1, 4, 7 and 10 days without a nucleic acid protective agent.

[0028] Figure 4 This is a result graph of the total amount of bile cfDNA detected when the bile samples of the present invention were stored at different temperatures for 2 months without a nucleic acid protective agent.

[0029] Figure 5 This is a graph showing the bile cfDNA A260 / A280 ratio results of the bile samples of the present invention stored at different temperatures for 2 months without a nucleic acid protective agent.

[0030] Figure 6 This is a graph showing the distribution of bile cfDNA fragments detected when the bile samples of the present invention were stored at different temperatures for 2 months without a nucleic acid protective agent.

[0031] Figure 7 This is a graph showing the distribution percentage of bile cfDNA fragments larger than 3000 bp detected when the bile samples of the present invention were stored at different temperatures for 2 months without a nucleic acid protective agent.

[0032] Figure 8 This is a result graph of the total amount of bile cfDNA detected when the bile sample of the present invention is stored at room temperature in the presence of a nucleic acid protective agent.

[0033] Fig. 9 This is a result graph of the bile cfDNA A260 / A280 ratio detected when the bile sample of the present invention was stored at room temperature in the presence of a nucleic acid protective agent.

[0034] Fig.10 This is a graph showing the distribution of bile cfDNA fragments detected when the bile sample of the present invention is stored at room temperature in the presence of a nucleic acid protective agent.

[0035] Fig.11 This is a result graph of the total amount of bile cfDNA detected when the bile samples of the present invention were stored at different temperatures in the presence of a nucleic acid protective agent for 2 months.

[0036] Fig.12This is a graph showing the bile cfDNA A260 / A280 ratio results of the bile samples of the present invention, which were detected under the condition of being stored at different temperatures in the presence of a nucleic acid protective agent for 2 months.

[0037] Fig.13 This is a graph showing the distribution of bile cfDNA fragments detected when the bile samples of the present invention were stored at different temperatures in the presence of a nucleic acid protective agent for 2 months. DETAILED DESCRIPTION

[0038] The nucleic acid extraction method used in the present invention is an adsorption column-based separation method suitable for bile cfDNA. This method may be replaced by other nucleic acid extraction methods, such as Kangwei Century's free DNA extraction kit (cat: CWY020), EpiGentek's FitAmp TM Plasma / Serum DNA Isolation Kit (cat: P-1004) or other newly established extraction methods suitable for body fluid cfDNA. In the present invention, the nucleic acid protective agent storage tube for collecting bile from patients with malignant biliary tumors is a free nucleic acid storage tube (BEAVER Cell Free DNA Tubes, 43803, China), which may be replaced by nucleic acid storage tubes from other manufacturers, such as Lakebio free DNA room temperature storage tube (cat: 3aB0310.0), Kangwei Century's free DNA storage tube (cat: CW2815M), Shuomei Biological's free DNA storage tube (cat: CF003), etc.; the nucleic acid protective agent-free storage tube for collecting bile from patients with malignant biliary tumors is Aisheng's 50ml collection tube (cat: SCT-50ML-25-s), which may be replaced by collection tubes from other manufacturers, such as Corning's 50ml centrifuge tube (cat: 430828), LABSELECT's 50ml centrifuge tube (cat: CT-002-50A), Biosharp's 50ml screw-mouth pointed bottom stand-up centrifuge tube (cat: BS-500-MJL), etc. The bile storage temperature adopted in the present invention is a common temperature for preserving samples in the laboratory. Other temperature conditions can also be selected to store bile to detect the effect on the stability of bile cfDNA. In order to better preserve the bile sample, it can theoretically be stored in liquid nitrogen; accordingly, other storage time conditions can also be selected to detect the effect on the stability of bile cfDNA.

[0039] This invention selects bile cfDNA from patients with malignant biliary tumors as the research object, and provides certain technical support for the application of bile cfDNA to liquid biopsy and the diagnosis and treatment of cancer patients in subsequent studies. At the same time, in addition to cfDNA, bile is rich in many other important molecular components, including extracellular vesicles, circRNA, miRNA, lncRNA, proteins, lipids, etc. These bile components also have great potential for liquid biopsy, which can assist in the early diagnosis of cancer patients, guide the drug treatment of patients, and accurately monitor the recovery stage from multiple perspectives.

[0040] In order to more precisely illustrate the technical solution and optimization steps of the present invention, the present invention is further described in detail below in conjunction with specific implementation methods.

[0041] Example 1: Preparation of bile cfDNA

[0042] (1) Study cohort and experimental groups

[0043] All patients with malignant biliary tumors enrolled in this study had their bile samples collected before surgery, and each enrolled patient was given an accurate pathological diagnosis after surgery. In order to study the differences in the stability of bile cfDNA after bile was stored under different conditions, the experimental group was divided into a group with a nucleic acid protective agent and a group without a nucleic acid protective agent, and each group was repeated three times. The so-called group with a nucleic acid protective agent is when the patient collects bile, the bile is directly collected in a commercial free nucleic acid protection tube (BEAVER Cell Free DNA Tubes, 43803, China); the present invention uses a beaver free nucleic acid storage tube. The main components of the protective agent are K3-EDTA and a stabilizer (the specific components of the stabilizer are unknown). The so-called group without a nucleic acid protective agent is when the patient collects bile, the bile is directly collected in a commercial ordinary sample collection tube (SCT-50ML-25-s, US). The two groups of patients were further divided into a short-term room temperature storage group and a long-term 2-month storage group; room temperature usually refers to 25°C. In the short-term room temperature storage group, the bile of the same patient was evenly divided into 300 μL / tube and stored at room temperature for 1 day, 4 days, 7 days, and 10 days before cfDNA extraction; the group with nucleic acid protective agent included bile from 6 patients with malignant biliary tumors, and the group without nucleic acid protective agent included bile from 1 patient with malignant biliary tumors. In the long-term 2-month storage group, the bile of the same patient was evenly divided into 300 μL / tube and stored at 4°C, -20°C, and -80°C for 2 months before cfDNA extraction, and the fresh bile of the same patient was used as the control (fresh bile was directly extracted from the bile cfDNA after the bile sample was pre-treated after receiving the patient's bile). The group with nucleic acid protective agent included bile from 5 patients with malignant biliary tumors, and the group without nucleic acid protective agent included bile from 1 patient with malignant biliary tumors.

[0044] (2) Pretreatment of bile samples from patients with malignant biliary tumors

[0045] The bile samples collected from patients with malignant biliary tumors before surgery were transported at room temperature in 10 mL free nucleic acid storage tubes or 50 mL ordinary collection tubes without nucleic acid protective agents. After receiving the samples, the bile was first centrifuged at 1,600 g for 10 min at 4 ° C. After centrifugation, the supernatant was aspirated into a new 1.5 mL centrifuge tube, and the cell debris precipitate was discarded. Then, it was centrifuged again at 16,000 g for 15 min at 4 ° C, and the supernatant was aspirated again. After collecting the supernatant, it was mixed again and divided into 1.5 mL centrifuge tubes at 300 μL per tube for subsequent experiments. Then, the divided bile was stored under different storage conditions set. After reaching the extraction conditions, bile cfDNA was separated. If there was still sample left after aliquoting, it was stored in a -80 ° C refrigerator.

[0046] (3) Extraction of cfDNA from bile samples of patients with malignant biliary tumors (3D-BCF method)

[0047] The experimental groups were divided into a nucleic acid protective agent group and a non-nucleic acid protective agent group according to step (1); cfDNA was extracted after the bile samples were stored at room temperature for 1, 4, 7 and 10 days and after they were stored at 4°C, -20°C and -80°C for 2 months; after the bile samples stored under the above-mentioned different storage conditions reached the time point for extracting bile cfDNA, the bile was taken out from the corresponding conditions and placed in a 37°C water bath, among which the samples in the short-term room temperature group and the samples in the 4°C condition did not need to be heated in a water bath to melt; after the above bile samples were completely melted, they were centrifuged at 4°C, 16,000g for 5 minutes, and the supernatant was aspirated into a new 1.5mL centrifuge tube. After mixing 240 μL of Buffer ACL (939017, Qiagen, Shanghai, China) and 1.68 μL of Carrier RNA (1017647, Qiagen, Shanghai, China), 240 μL of the mixture was added to 300 μL of the above bile and mixed, and then 30 μL of proteinase K (19133, Qiagen, Shanghai, China) was added. The mixture was mixed by inversion and incubated at 60°C for 30 minutes, and the mixture was mixed by inversion every 10 minutes. After the incubation, 540 μL Buffer ACB (1069275, Qiagen, Shanghai, China) was added and vortexed to mix, and finally placed in a -20°C refrigerator for 5 minutes. After the end, 700 μL of bile mixture was added to a QIA-quick adsorption column (28115, Qiagen, Shanghai, China) and the column was left at room temperature for 5 minutes, and then the centrifugation operation was repeated until all the mixture passed through the adsorption column. 500 μL Buffer PE (19065, Qiagen, Shanghai, China) was added to the adsorption column and washed twice at 6,000 rpm, and then centrifuged at 13,000 rpm to remove the remaining liquid in the adsorption column and place the adsorption column in a new 1.5 mL centrifuge tube. After the adsorption membrane was dried, 30 μL EB (19086, Qiagen, Shanghai, China) was added to the adsorption column and incubated at room temperature for 3 minutes. Finally, cfDNA was eluted and collected by centrifugation at 13,000 rpm for 1 minute, and then quality control operations were performed after bile cfDNA extraction.

[0048] (4) Quality control after bile cfDNA extraction

[0049] 1) Concentration detection

[0050] The bile cfDNA concentration was detected using the Qubit 3.0 fluorescence quantification instrument and supporting reagents (Q32854, Thermo Fisher, USA). For specific steps, please refer to the reagent instructions. If the test result concentration is too high, the sample needs to be diluted for further testing.

[0051] 2) Purity testing

[0052] Bile cfDNA purity was measured using a micro-spectrophotometer NanoDrop 2000, with nucleic acid detection selected as the mode. Before measuring the concentration, the probe was cleaned with triple-distilled water or sample diluent, and after drying, 1 μL of sample diluent was used to click "blank" for blank detection. Subsequently, 1 μL of DNA sample was taken for detection, and the A260 / A280 value was recorded.

[0053] 3) Fragment distribution detection

[0054] Bile cfDNA fragment distribution was tested using the 2100 analyzer and supporting chips and reagents (5067-4626, Agilent, USA). Samples with too high concentrations needed to be diluted before testing. For specific steps, refer to the reagent instructions.

[0055] Example 2: Effect of the absence of nucleic acid protective agent on the total amount, purity and fragment distribution of bile cfDNA

[0056] 1) To determine the effect of storing bile from patients with malignant biliary tumors at room temperature for different days without a nucleic acid protective agent on the stability of bile cfDNA, the bile samples without a nucleic acid protective agent after packaging as described in Example 1 were stored for 1, 4, 7 and 10 days, respectively. After extraction using the 3D-BCF method, the total amount, purity and fragment distribution of bile cfDNA were tested. Figures 1 to 3 They are respectively the total amount of bile cfDNA, the A260 / A280 ratio of bile cfDNA and the distribution of bile cfDNA fragments detected when the bile samples of the present invention were stored at room temperature for 1, 4, 7 and 10 days without a nucleic acid protective agent. Figure 1 The results showed that when bile samples were stored at room temperature without nucleic acid protective agents for 1, 4, 7, and 10 days, the total amount of bile cfDNA decreased significantly with the extension of storage days; Figure 2 The results showed that when bile samples were stored at room temperature for 1, 4, 7, and 10 days without nucleic acid protective agents, the A260 / A280 ratio of bile cfDNA did not change significantly with the extension of storage days; Figure 3 The results showed that when bile samples were stored at room temperature for 1, 4, 7 and 10 days without nucleic acid protective agents, the distribution of bile cfDNA fragments changed significantly with the extension of storage days. The proportion of large fragments of cfDNA gradually decreased, while the proportion of small fragments of cfDNA gradually increased. Figures 1 to 3 The results show that bile samples without nucleic acid protective agents are not suitable for long-term protection at room temperature.

[0057] 2) To determine the effect of different storage temperatures on the stability of bile cfDNA in bile of patients with malignant biliary tumors stored for 2 months without a nucleic acid protective agent, the bile samples without a nucleic acid protective agent described in Example 1 were stored at 4°C, -20°C and -80°C for 2 months, respectively, and cfDNA was extracted after extraction using the 3D-BCF method. The total amount, purity and fragment distribution of bile cfDNA were tested, and the cfDNA extraction results of the corresponding fresh bile without a nucleic acid protective agent that had only been pretreated and not stored were used as a control. Figures 4 to 7 They are respectively a graph showing the total amount of bile cfDNA detected when the bile samples of the present invention are stored at different temperatures for 2 months without a nucleic acid protective agent, a graph showing the A260 / A280 ratio of bile cfDNA, a graph showing the distribution of bile cfDNA fragments, and a graph showing the distribution percentage of bile cfDNA fragments larger than 3000 bp. Figure 4 The results showed that after bile samples without nucleic acid protective agents were stored at 4°C, -20°C and -80°C for 2 months, the changes in the total amount of bile cfDNA did not differ significantly under each temperature condition, but there was a significant downward trend at 4°C. Figure 5 The results showed that there was no significant difference in the A260 / A280 ratio of bile cfDNA after bile samples without nucleic acid protective agents were stored at 4°C, -20°C, and -80°C for 2 months; Figure 6 The results showed that after bile samples without nucleic acid protective agents were stored at 4°C, -20°C, and -80°C for 2 months, there was no significant difference in the fragment distribution of bile cfDNA; Figure 7 The results showed that after bile samples without nucleic acid protective agents were stored at 4°C, -20°C and -80°C for 2 months, it can be seen that the proportion of bile cfDNA larger than 3000bp at 4°C also showed a downward trend compared with fresh bile cfDNA, indicating the degradation of large fragments of cfDNA.

[0058] Example 3: Effects of nucleic acid protectants on the total amount, purity and fragment distribution of bile cfDNA

[0059] 1) To determine the effect of storing bile from patients with malignant biliary tumors at room temperature in the presence of a nucleic acid protective agent for different days on the stability of bile cfDNA, the bile samples with a nucleic acid protective agent after packaging as described in Example 1 were stored for 1, 4, 7 and 10 days, respectively. After extraction using the 3D-BCF method, the total amount, purity and fragment distribution of bile cfDNA were tested. Figures 8 to 10They are respectively the total amount of bile cfDNA, the A260 / A280 ratio of bile cfDNA and the distribution of bile cfDNA fragments detected when the bile samples of the present invention were stored at room temperature in the presence of a nucleic acid protective agent for 1, 4, 7 and 10 days. Figure 8 The results showed that when bile samples were stored at room temperature with a nucleic acid protectant for 1, 4, 7, and 10 days, the total amount of bile cfDNA varied greatly between patients with malignant biliary tract cancer, but there was no significant difference in the bile cfDNA of the same patient over time. Fig. 9 The results showed that when bile samples were stored at room temperature for 1, 4, 7, and 10 days in the presence of a nucleic acid protectant, the A260 / A280 ratio of bile cfDNA did not change significantly with the extension of storage days; Fig.10 The results showed that when bile samples were stored at room temperature in the presence of a nucleic acid protective agent for 1, 4, 7 and 10 days, the distribution of bile cfDNA fragments at each time point was almost the same as the storage days increased.

[0060] 2) To determine the effect of different storage temperatures in the presence of nucleic acid protective agents on the stability of bile cfDNA in patients with malignant biliary tumors after 2 months of storage in the presence of nucleic acid protective agents, the bile samples with nucleic acid protective agents described in Example 1 were stored at 4°C, -20°C, and -80°C for 2 months, respectively, and cfDNA was extracted after that. After extraction using the 3D-BCF method, the total amount, purity, and fragment distribution of bile cfDNA were tested, and the cfDNA extraction results of the corresponding fresh bile with nucleic acid protective agents that had only been pretreated and not yet stored were used as a control. Figures 11 to 13 They are respectively a graph showing the total amount of bile cfDNA, a graph showing the A260 / A280 ratio of bile cfDNA, and a graph showing the distribution of bile cfDNA fragments when the bile samples of the present invention are stored at different temperatures for 2 months without a nucleic acid protective agent. Fig.11 The results showed that after bile samples with nucleic acid protectants were stored at 4°C, -20°C, and -80°C for 2 months, there was no significant difference in the total amount of bile cfDNA under each temperature condition; Fig.12 The results showed that there was no significant difference in the A260 / A280 ratio of bile cfDNA after bile samples with nucleic acid protectants were stored at 4°C, -20°C, and -80°C for 2 months; Fig.13 The results showed that there was no significant difference in the fragment distribution of bile cfDNA after bile samples with nucleic acid protectants were stored at 4°C, -20°C and -80°C for 2 months.

[0061] The above are only some preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the technical solution of the present invention, and any changes and modifications made are within the protection scope of the present invention.

Claims

1. A method for preserving a bile sample, characterized in that: The following steps are involved: S1. Collect bile samples; S2. Mix the bile sample with the nucleic acid protective agent and store them at a temperature of 25°C to -80°C.

2. The storage method according to claim 1, characterized in that: The nucleic acid protective agent includes K3-EDTA solution.

3. The storage method according to claim 1, characterized in that: The nucleic acid protective agent is contained in a free nucleic acid storage tube.

4. The storage method according to claim 1, characterized in that: The storage temperature is 4°C to -80°C.

5. The storage method according to claim 4, characterized in that: The storage temperature is -20°C to -80°C.

6. The storage method according to claim 4, characterized in that: The storage temperature is selected from any one of 4°C, -20°C or -80°C.

7. The storage method according to claim 1, characterized in that: The storage period is no more than 2 months.

8. Use of the preservation method according to any one of claims 1 to 7 in bile cfDNA storage or bile cfDNA liquid biopsy.

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