Method for extracting tumor and infectious disease body fluid free nucleic acid, kit and application

Abstract

The invention discloses a method for extracting tumor and infectious disease body fluid free nucleic acid, a kit and application. The extraction method provided by the invention is high in extraction efficiency; through comparison, the extraction quality is 10 times that of a traditional kit. The kit comprises a specific MOF adsorption reagent, a lysis solution, a separating agent and a nucleic acid storage tube, the specific MOF adsorption reagent can capture free nucleic acid in body fluid of a patient through specific immunomagnetic beads, and then the free nucleic acid is separated and extracted through the lysis solution and the separating agent. According to the kit provided by the invention, free nucleic acid in tumor or infectious disease body fluid can be specifically extracted, interference of other normal cell DNA is eliminated, and the kit can be used for scientific research or clinical diagnosis analysis such as PCR amplification, gene expression, gene sequencing, chip detection, library construction and high-throughput sequencing, so that the subsequent detection result is more accurate, and the detection efficiency is improved. And the detection effect is better.

Description

Technical Field

[0001] The present application relates to the technical field of gene detection of tumors and infectious diseases, and in particular to a method, a kit and an application of extracting free nucleic acids from tumor and infectious body fluids. Background Art

[0002] Tumors and some infectious diseases are diseases caused by gene mutations. Experts believe that early detection, diagnosis and treatment of tumors and infectious diseases can effectively improve patients' survival and quality of life. Nucleic acid testing is the most important way to diagnose tumors and infectious diseases in the early stage, and the content and quality of nucleic acid extracted from the patient's body is an important guarantee for the effective completion of nucleic acid testing.

[0003] Free nucleic acids (cfNA) in human body fluids include free DNA (cfDNA) and free RNA (cfRNA), which are derived from apoptosis, necrosis, and active secretion of cells in different tissues. cfNA molecules reveal the health status of many solid tissues and have the potential to be used as a "liquid biopsy" genome for diagnosing and monitoring malignant diseases. cfNA molecules have outstanding effects as biomarkers for the diagnosis or prognosis of cancer-related point mutations, methylation, and fragmentation patterns. The concentration of free nucleic acids (cfNA) in human blood is low, and the concentration of cfNA in human blood is only about 5 to 10 ng / mL. In addition, the concentration of cfNA in human blood will show personalized differences in patients with different diseases or different patients with the same disease. Too low a concentration of cfNA makes it difficult to extract cfNA and detect cfNA.

[0004] Therefore, it is of great significance to develop methods and personalized products for extracting cfNA from the body fluids of patients with tumors or infectious diseases. Summary of the invention

[0005] In view of this, the purpose of this application is to provide a method, a kit and an application for extracting free nucleic acids from body fluids of tumors and infectious diseases. This method uses MOF materials to extract free nucleic acids from body fluids of patients with various diseases. The extraction quality is 10 times that of traditional methods. The extracted free nucleic acids can be used for disease diagnosis and have clinical significance.

[0006] To achieve the above objectives, this application provides the following technical solutions:

[0007] In a first aspect, the present application provides a method for extracting free nucleic acid from a body fluid sample for disease diagnosis, the method comprising:

[0008] Pretreatment of body fluid samples;

[0009] Using MOF materials to adsorb free nucleic acids in body fluid samples;

[0010] The MOF material is separated from the free nucleic acid to obtain the free nucleic acid in the body fluid sample.

[0011] In a second aspect, the present application provides a kit for free nucleic acid in body fluid samples for disease diagnosis, the kit comprising a specific MOF adsorption reagent, a lysis solution, a separation agent, and a nucleic acid storage tube.

[0012] In a third aspect, the present application provides the use of the aforementioned kit for extracting free nucleic acids from tumors and / or infectious body fluids.

[0013] The present application provides a method, a kit and an application for extracting free nucleic acids from tumor and infectious body fluids. Compared with the prior art, the present application has at least the following beneficial effects:

[0014] (1) The method provided in the present application for extracting circulating free nucleic acids from body fluid samples of tumors or infectious diseases has a very high extraction efficiency; by comparison, its extraction quality is 10 times that of traditional kit extraction.

[0015] (2) The kit for extracting free nucleic acids from tumor or infectious body fluids involved in the present application includes a specific MOF adsorption reagent, a lysate, a separation agent, and a nucleic acid storage tube. The specific MOF adsorption reagent can capture free nucleic acids in the patient's body fluids through specific immunomagnetic beads, and then separate and extract the free nucleic acids through the lysate and separation agent. The kit provided by the present application can specifically extract free nucleic acids from tumor or infectious body fluids, eliminating the interference of other normal cell DNA, and can be used for scientific research or clinical diagnostic analysis such as PCR amplification, gene expression, gene sequencing, chip detection, library construction, high-throughput sequencing, etc., so that subsequent test results are more accurate and the detection effect is better. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A schematic diagram of the process of separating cfDNA / cfRNA from body fluids using the MOF method provided in the embodiments of the present application, wherein (a) is a schematic diagram of separating cfDNA / cfRNA from plasma / serum, and (b) is a schematic diagram of separating cfDNA / cfRNA from bile.

[0017] Figure 2 The PXRD pattern of the Co-IRMOF-74-IV sample provided in the examples of the present application was tested using a Rigaku Intelligent Laboratory 9kW diffractometer under different conditions.

[0018] Figure 3 This is a SEM image of Co-IRMOF-74-IV provided in the examples of the present application.

[0019] Figure 4The examples of this application provide results for comparing the recovery rates of oligonucleotides of different lengths by polyacrylamide gel electrophoresis.

[0020] Figure 5 The MOF method is used to evaluate the total RNA release efficiency and fragment integrity results provided in the examples of this application.

[0021] Figure 6 The embodiment of the present application provides a Qubit TM 1X dsDNA HS method to detect the total mass of cfDNA, and the correlation between MOF and kit methods.

[0022] Figure 7 The embodiment of the present application provides a Qubit TM The total quality results of cfDNA detected by 1X dsDNA HS method.

[0023] Figure 8 The examples of the present application provide a comparison of the integrity results of cfDNA extracted by the MOF method and the kit through gel electrophoresis.

[0024] Fig. 9 The results of qPCR detection of ALU expression in cfDNA provided in the embodiments of the present application.

[0025] Fig.10 The embodiment of the present application provides the detection of the expression of the ALU gene in cfDNA by qPCR.

[0026] Fig.11 The total mass results of cfNA extracted using the MOF method and commercial kit provided in the examples of this application.

[0027] Fig.12 The correlation results of cfRNA expression levels obtained by the kit method and MOF method provided in the examples of the present application.

[0028] Fig.13 The number of RNA species detected by the MOF method and the kit method provided in the examples of this application.

[0029] Fig.14 The proportion distribution of RNA of different types of species in the MOF method and the kit method provided in the examples of this application.

[0030] Fig.15 The embodiment of the present application provides a Qubit TM The microRNA detection kit detects the total quality results of cfRNA.

[0031] Fig.16These are the parallel experimental results of cfRNA expression levels obtained by the kit method and MOF method provided in the examples of this application.

[0032] Fig.17 The length distribution results of cfRNA fragments provided in the examples of this application.

[0033] Fig.18 The length distribution of cfRNA fragments is calculated by sequencing data provided in the embodiments of the present application.

[0034] Fig.19 The MOF method and the kit method provided in the examples of this application are the results of detecting the quantity of different types of RNA.

[0035] Fig. 20 Schematic diagram of the MOF method for extracting and detecting HCV RNA provided in the examples of the present application.

[0036] Fig.21 Analysis of HCV copy number extracted by the MOF method provided in the examples of this application.

[0037] Fig. 22 The MOF method and the kit method provided in the examples of the present application provide a correlation in detecting HCV copy number.

[0038] Fig.23 The linear relationship between the quantitative cycle (Ct) value and the logarithmic HCV RNA copy number is provided in the examples of the present application.

[0039] Fig.24 Schematic diagram of the MOF method for extracting and detecting HBV RNA provided in the embodiments of the present application.

[0040] Fig.25 The Taqman qPCR provided in the examples of this application is used to detect the copy number analysis of HBV DNA and RNA in serum.

[0041] Fig.26 This is the correlation analysis of HBV DNA and RNA copy numbers provided in the examples of this application.

[0042] Figure 27-29 Quality analysis of cfRNA extracted using the MOF method and magnetic bead method (VAHTS) provided in the examples of this application. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described here are only used to explain the present application and are not used to limit the present application.

[0044] The embodiments of the present application separate the nucleic acids from the body fluids of tumor patients or infectious disease patients, treat them with a lysis buffer, denature them to precipitate proteins, thereby leaving the nucleic acids in the solution, and finally add specific MOF materials to adsorb free nucleic acids, maximally adsorb tumor free nucleic acids and infectious pathogen nucleic acids, that is, produce mutated nucleic acid molecules, and then release the adsorbed nucleic acids under acidic conditions. Finally, after purification, they can be used for genetic testing to achieve the purpose of diagnosing tumors or infectious diseases.

[0045] Based on this, the present application provides a method for extracting free nucleic acids from body fluid samples for disease diagnosis, the method comprising: pretreatment of body fluid samples; using MOF materials to adsorb free nucleic acids in body fluid samples; separating the MOF materials from the free nucleic acids to obtain free nucleic acids from whole blood samples.

[0046] In certain embodiments, the body fluid sample is a whole blood sample or bile. Figure 1 Shown is a schematic diagram of the process of separating cfDNA / cfRNA from body fluids using the MOF method, wherein (a) is a schematic diagram of separating cfDNA / cfRNA from plasma / serum, and (b) is a schematic diagram of separating cfDNA / cfRNA from bile.

[0047] In the above embodiment, when the body fluid is bile, the extraction scheme of free nucleic acid includes pre-treatment of bile to release nucleic acid wrapped in protein, adsorbing circulating free nucleic acid from bile by using metal organic framework material MOF material to adsorb nucleic acid, and then separating the MOF material adsorbing circulating free nucleic acid from bile, releasing circulating free nucleic acid from MOF material, and finally purifying the obtained circulating free nucleic acid; the purified nucleic acid can be subjected to downstream analysis and detection, including real-time fluorescence quantitative PCR and second-generation sequencing technology to quantitatively and qualitatively analyze circulating free DNA. The method for extracting cfDNA / cfRNA from bile using MOF material is as follows:

[0048] a. First, the bile needs to be centrifuged to remove cell debris and obtain the supernatant containing free cfDNA / cfRNA; b. Since there are protein / nucleic acid complexes in bile, it is necessary to use a protein denaturing agent to destroy the protein structure to dissociate the cfDNA / cfRNA fragments. Then the protein is salted out, and the protein can be removed by centrifugation to retain the cfDNA / cfRNA fragments; c. Then, the MOF material only needs to be incubated with the bile supernatant solution. The metal and organic ligand parts in the MOF material can form stable ionic bonds and hydrogen bonds with cfDNA / cfRNA, respectively, to achieve effective enrichment of cfDNA / cfRNA in the bile supernatant by the MOF material. The MOF material containing cfDNA / cfRNA can be quickly and efficiently recovered by centrifugation, realizing a fast and simple enrichment process of cfDNA / cfRNA in bile; d. Release cfDNA / cfRNA from the MOF material: The MOF material can selectively enrich cfDNA / cfRNA in bile based on the size exclusion effect and efficient adsorption capacity to obtain a complex of MOF material and cfDNA / cfRNA. In order to obtain cfDNA / cfRNA molecules for downstream analysis, cfDNA / cfRNA needs to be extracted from the MOF material. Phosphates can competitively bind to the sites where MOF materials and nucleic acids interact to release nucleic acids from the material. Finally, the cfDNA / cfRNA is purified. These processes only require simple reagent mixing and ordinary centrifugation, which greatly reduces the difficulty and cost of extracting cfDNA / cfRNA. The purified free nucleic acid can be stored at -80 degrees or used directly for subsequent testing.

[0049] In certain embodiments, the disease is a tumor or an infectious disease, including patients in early, middle or late stages of the disease, wherein early stage patients are patients with no obvious clinical symptoms or mild symptoms.

[0050] In certain embodiments, the tumor is hepatocellular carcinoma (HCC).

[0051] In certain embodiments, the infectious disease is chronic viral hepatitis (CHB), such as hepatitis B, hepatitis C, etc.

[0052] In certain embodiments, the MOF material is at least one of Co-IRMOF-74-II, Co-IRMOF-74-III and Co-IRMOF-74-IV among Co-based MOF materials.

[0053] In certain embodiments, the free nucleic acid comprises free DNA and / or free RNA.

[0054] Based on this, an embodiment of the present application provides a kit for free nucleic acid in body fluid samples for disease diagnosis, wherein the kit includes a specific MOF adsorption reagent, a lysis solution, a separation agent, and a nucleic acid storage tube.

[0055] In certain embodiments, the disease is hepatocellular carcinoma (HCC) or chronic viral hepatitis (CHB).

[0056] In certain embodiments, the MOF adsorption agent includes at least one of the following Co-based MOF materials: Co-IRMOF-74-II, Co-IRMOF-74-III, and Co-IRMOF-74-IV.

[0057] In a third aspect, the present application provides the use of the aforementioned kit for extracting free nucleic acids from tumors and / or infectious body fluids.

[0058] In certain embodiments, the tumor is hepatocellular carcinoma (HCC).

[0059] In certain embodiments, the infectious disease is chronic hepatitis B (CHB). In certain preferred embodiments, the infectious disease is hepatitis B or hepatitis C.

[0060] The following will illustrate the substantial features and significant effects of the present technical solution through more specific embodiments. It should be understood that the enumerated embodiments should not be construed as limiting the technical solution of the present application.

[0061] The present invention uses Co-IRMOF-74-III, Co-IRMOF-74-IV and Co-IRMOF-74-V as free nucleic acid adsorption materials, uses whole blood / plasma / serum as research samples, and compares the effect difference between the cfDNA obtained by the MOF extraction method and the traditional kit enrichment method. Figure 1 (a) is used to extract and verify nucleic acid. The specific method is as follows:

[0062] 1. Blood sample preparation

[0063] Blood samples were collected in vacuum tubes K3 EDTA tubes for plasma processing or vacuum tubes for serum processing. Store at room temperature and process within 2 hours after blood collection. Centrifuge (10 min, 1500 g, 4 ° C); transfer the supernatant to a fresh 1-2 mL tube and centrifuge again (10 min, 3000 g, 4 ° C); the supernatant was used as plasma or serum for subsequent experiments; plasma or serum was fresh or quickly frozen and stored at -80 ° C for a long time; avoid freeze-thaw cycles.

[0064] 2. Extraction of free nucleic acids

[0065] (1) MOF materials adsorb and extract free nucleic acids.

[0066] The steps are as follows: The reaction system of the adsorption process includes the supernatant obtained in step 1, MOF adsorption solution, 10×PBS, and 10×MgCl2. The above reaction is reacted on a rotator at room temperature for 2 hours; after the reaction is completed, the above reaction is centrifuged at 12000g, the supernatant is removed, and the precipitate is collected (the precipitate is a pink solid); destroy the MOF material to release cfDNA: add 40μL of enzyme-free water to the precipitate, then add EDTA solution, and mix with a pipette tip. You can see that the pink solid disappears and white flocculent insoluble matter appears; the purification step obtains cfDNA: add 1uL ribonuclease and 2uL ribonuclease buffer solution, react at 37°C for 1h, and then purify cfRNA through RNA, RNA cleaning and concentrator TM -5 (ZYMO Research, R1013, USA), the operation steps followed the instructions provided by the manufacturer; the purified cfDNA was placed in a -20 degree refrigerator for subsequent experiments.

[0067] (2) Extraction of free nucleic acid using traditional kits.

[0068] The most commonly used commercial kit on the market (Qiagen, catalog number: 55184) was used to free nucleic acid according to the instructions.

[0069] 3. Characterization of MOF materials

[0070] The examples of this application fully characterize the materials, including PXRD, n2 adsorption analysis, stability of MOF materials under different buffer conditions, etc. The examples of this application also perform PXRD characterization on each synthetic material. Figure 2 PXRD patterns of Co-IRMOF-74-IV samples were measured under different conditions using a Rigaku Smart Lab 9kW diffractometer, operating at 45 kV, 200 mA of Cu Kα (λ = 1.5406A). Red line: untreated activated Co-IRMOF-74-IV. Blue line: adsorption of cfNA by Co-IRMOF-74-IV after incubation with lysed plasma for 2 h. Yellow line: adsorption of cfNA by Co-IRMOF-74-IV, treated with HAc for 5 min.

[0071] The characterization results are as follows Figure 2 As shown in the figure, it can be seen that the pattern of activated Co-IRMOF-74-IV is consistent with the simulation model, indicating that the material has the same crystal structure as the simulation model. Figure 2 , blue line) is the same as the peak pattern of the untreated material ( Figure 2, red line). This shows that the crystal structure of the MOF material during cfNA adsorption is the same as that of the untreated material, and it is stable during cfNA adsorption. The next process is to elute cfNA from the MOF material. The present application example utilizes the instability of the MOF material under acidic conditions, uses HAc to destroy the structure of the MOF material, and releases free nucleic acids from the material. Therefore, the present application example tests the stability of the material after incubation with HAc (2M, pH = 2.06), and the PXRD results ( Figure 2 , yellow line) shows that after adding HAc, the crystal structure of the material is completely destroyed. In summary, MOF is stable and maintains its crystal structure during the adsorption of free nucleic acids from lysed plasma. However, during the elution of cfNA, when HAc is added to destroy the structure of MOF, the crystal structure of the material is completely destroyed, allowing cfNA to be released.

[0072] The present invention also uses a scanning electron microscope to characterize the morphology of the material. Figure 3 As shown. The SEM images of Co-IRMOF-74-IV were collected on 2000-460 with an accelerating voltage of 500.0 V directly sprinkled on the sample stage. All Co-IRMOF-74-IV samples have uniform size of micrometers.

[0073] 4. Use MOF method to evaluate nucleic acid recovery efficiency and fragment integrity

[0074] In order to detect the enrichment rate of nucleic acids extracted by the MOF method, nucleic acid fragments of different lengths were added to the serum. The recovery rate of these nucleic acids was then tested and compared with the standard kit method. The recovery rate of oligonucleotides of different lengths added to plasma was compared by polyacrylamide gel electrophoresis using the MOF method and the kit method.

[0075] like Figure 4 As shown, the recovery rate of 33-nt DNA, 22-nt DNA and 22-nt RNA by the MOF method reached 33%, while the recovery rates of the kit method were 27.75%, 0.79% and 3.07%, respectively. In addition, MOF materials were used to enrich total RNA in solution. These MOFs were then opened under acidic conditions to release the adsorbed total RNA-crystalline MOF materials are connected by coordination bonds, so nucleic acids can be released by breaking the coordination bonds under acidic conditions (2M HAc, pH = 2.06). Gel electrophoresis showed that total RNA was efficiently released from MOFs and the integrity of the fragments was maintained relative to the total input RNA (such as Figure 5 These results demonstrate the high recovery rate, versatility for different types of nucleic acids, and non-invasiveness of the MOF method.

[0076] 5. cfDNA testing

[0077] In this example, a large amount of plasma from different populations was mixed, and cfDNA was extracted from 250 μL and 1000 μL of plasma using the MOF enrichment method and a commercial kit, respectively. Finally, all cfDNA was dissolved in 20 μL H2O as a template for qPCR detection and dsDNA Qubit quantification.

[0078] 5.1 Detection and comparison of cfDNA by qPCR

[0079] All qPCR reactions were performed in 10 μL reactions using 2× Hieff qPCR SYBR Green master mix (Yeasen, 11201) and amplified on a CFX-96tm Real-Time System (Bio-Rad, USA). The qPCR primers for the ALU115 sequence used in this study were 5'-3', and 5'-3' (Sangon Biotech, China), see Table S8 (ALU115-F is the forward primer; ALU115-R is the reverse primer). A 10 μL reaction contained 5 μL Hieff qPCR SYBR Green master mix; 1 μL 10 μmol / L ALU115-F; 1 μL 10 μmol / L ALU115-R; and 2 μL template cfDNA. Thermal cycling was performed on a CFX-96 tm. Real-Time System (Bio-Rad, USA) under the following conditions: 95°C, 5 min; 95°C, 20 sec; 55 cycles, 95°C 3 sec, 60°C 30 sec. Fluorescence was measured at 60°C for each cycle, and all assays were performed at least 3 technical replicates.

[0080] First, use Qubit TM 1X dsDNA HS detection kit, the total cfDNA mass of the two methods was highly correlated (r = 0.968, p = 0.002) ( Figure 6 and Figure 7 ). In addition, the integrity of cfDNA was compared by gel electrophoresis and was found to be the same for both methods ( Figure 8 ). It is worth noting that samples A and B are DNA ladders formed by DNA fragments, and the cfDNA content of sample C is lower, concentrated at the 170bp position, indicating significant differences between samples. Therefore, in the comparison process, the embodiment of the present application uses different samples, different operators, and different serum amounts to minimize sample, personnel, and sample amount errors. In addition, the Ct values ​​of the ALU gene in cfDNA by the two methods are similar, indicating that the extraction efficiency is comparable ( Fig. 9 and Fig.10 ). Therefore, the extraction efficiency and integrity of cfDNA extracted by the MOF method were highly similar to those of cfDNA extracted by the kit method.

[0081] In order to illustrate the total mass of cfRNA extracted by the MOF method, the present embodiment compares the results with those obtained by using Qubit TM The results of quantifying cfRNA using a microRNA detection kit were compared. The concentration of cfRNA extracted from the same serum sample by the MOF method was ~300 ng / mL serum, while the kit method was only 1 / 10 of that by the MOF method ( Fig.11 ). In addition, the MOF method generated 5.9 and 5.5 million reads mapped to the human genome, while the kit method, with different input serum volumes, 0.5 mL, 1 mL, and 2 mL, generated 3.7, 2.3, and 3.2 million reads mapped to the human genome. The correlation coefficient of the two replicate experiments of the MOF method was 0.93, and the correlation coefficient of the three replicate experiments of the kit method was above 0.83, but the correlation coefficient between the MOF method and the kit method was less than 0.5 ( Fig.12 ). Therefore, the MOF method has high reproducibility, and the cfRNA extracted by the MOF method is significantly different from that by the kit method.

[0082] The present application embodiment also found the difference of expression level of different RNA biotypes. The gene code V.31 annotation (www.gencodegenes.org) downloaded from the gene code website was used to draw the expression of different RNA biotypes, including protein coding, lncRNA (long non-coding RNA), small RNA ("misc_RNA", snRNA", "miRNA", "siRNA", "snoRNA", "scaRNA" and "mt_tRNA"), pseudogenes and others ("rRNA", "mt_rRNA", "immunoglobulin gene (IG_gene)", "T cell receptor gene (TR_gene)" and "to be confirmed by experiment (TEC)". The gene expression amount was set to 1 fragment per kilobase million> (FPKM>1), and the number of expressed RNAs of each type that met the conditions was calculated. MOF-A identified a total of 18,993 coding RNAs, 15,437 lncRNAs, 5,006 small RNAs, 1,358 and 13,756 pseudogenes, accounting for 95.1%, 91.7%, 71.4%, 88.5% and 90.3% of the annotated transcripts in each RNA biotype, respectively. The same results were observed in the biological replicate (MOF-B), with 95.1% of coding RNAs, 91.9% of lncRNAs, 71.6% of small RNAs, 87.8% of others, and 90.4% of pseudogenes identified. The consistent results of the repeated experiments showed that the MOF method has robust reproducibility. In contrast, the present application example identified a total of 7103 coding RNAs, 1011 lncRNAs, 1094 small RNAs, 127 and 1044 pseudogenes using the 0.5mL-kit, accounting for 35.6%, 6.0%, 15.6%, 8.3% and 6.9% of the annotated transcripts of each RNA biotype, respectively. Similar results were observed for the 1mL kit and the 2.5mL kit (45.0%, 11.5%, 18.2%, 11.8% and 10.8% for the 2.5mL kit; 37.7%, 5.0%, 12.2%, 8.2% and 5.5%, respectively), indicating that the expression of coding RNAs, lncRNAs, small RNAs, pseudogenes and others obtained by the MOF method was about 3 times, 10 times, 4 times and >10 times the number obtained by the kit ( Fig.13). In addition, the number of various types of small RNAs obtained by the MOF method reached more than 60% of the annotations, and "misc_RNA" and "snRNA" were three times that of the tool method, about 11 times that of "miRNA" and 5 times that of "snoRNA". Therefore, the most abundant cfRNA MOF method is protein-coding RNA and lncRNA, which can reach more than 90% of the annotation amount. The total RNA obtained by MOF is on average more than 3 times that obtained by the kit, among which lncRNA and miRNA can reach 10 times that of the kit method. Therefore, the types of RNA obtained by the MOF method are richer, more RNA information can be retrieved, and low-abundance RNA can be detected. In addition, the proportion of lncRNA detected by the MOF method reached ~20%, while the proportion detected by the kit was less than 4% ( Fig.14 ), indicating that the proportion of lncRNA obtained by the kit is small and biased. In addition, to illustrate the authenticity of the results, the same comparative experiment in another batch of plasma samples MOF method extracted cFNA with higher quality ( Fig.15 ), high reproducibility ( Fig.16 ), long fragment length ( Fig.17 and 18 ), and more RNA types ( Fig.19 ).

[0083] The present application example compares the MOF method with a cfDNA / cfRNA extraction kit based on a silica membrane spin column, and finds that the extraction efficiency of cfDNA is not much different. The MOF method is even more effective than the kit method for cfRNA extraction. First, the RNA extracted by the MOF method is much higher than the traditional kit extraction method based on silica membrane adsorption. DNA is much more stable than RNA, so when the MOF method is compared with the kit method, the difference is not obvious. Secondly, the silica membrane spin column-based kit can lead to differences in the adsorption and elution efficiency of DNA and RNA. The structure and properties of DNA and RNA molecules differ significantly, resulting in a wide range of adsorption and elution efficiencies of DNA and RNA. The length of double-stranded cfDNA molecules is generally around 170bp, while single-stranded cfRNA molecules come in many types, such as miRNAs with a length of about 20nt and mRNA fragments with a length of several hundred nt. Simultaneous purification of DNA and RNA of different fragment lengths in the same system will inevitably lead to inefficient purification of more complex cfRNA.

[0084] 5.2 RT-qPCR detection of HCV RNA

[0085] RT-qPCR was performed on the samples extracted by MOF method to detect HCV RNA in serum, and the results were compared with those extracted by clinical kit method ( Fig. 20 shown),

[0086] Fig.21 and Table 1 shows the HCV RNA copy numbers detected in the 30 serum samples.

[0087] Table 1

[0088]

[0089] The kappa statistical test showed that the two methods had a high degree of consistency (Kappa = 0.724, p = 0.000063, Table 2). In the 10 samples that were all positive (r = 0.754, p = 0.011788), the HCV copy number correlation between the two methods was extremely high ( Fig. 22 ), indicating that the MOF method and the clinical detection method have a high degree of consistency in HCV copy number detection. Therefore, the MOF method can extract viral RNA from blood with high accuracy and good reproducibility. In addition, given the linear working curve, the MOF extraction method has high accuracy and stability ( Fig.23 ).

[0090] Table 2

[0091]

[0092] 5.3 RT-qPCR detection of HBV RNA

[0093] The present invention analyzes HBV RNA in CHB patients and healthy individuals by cfRNA transcriptome sequencing. Fig.24 ), counted the reads aligned to the HBV genome, normalized the gene expression of HBV RNA to RPM (per 10 million reads), and converted the log 10 (RPM, HBV RNA reads per 10 million reads) was used to assess HBV and compared with the clinical method detection parameter log 10 (HBV DNA copy number) was compared (Table 3). No HBV RNA reads were detected in samples from healthy individuals, while obvious HBV RNA signals were observed in samples from CHB patients ( Fig.25 In addition, the correlation coefficient r of HBV expression levels between MOF and clinical detection methods was 0.791 (p<0.001) ( Fig.26 ), which is consistent with the correlation coefficient reported in the literature, indicating that serum HBV RNA and HBV DNA have a good correlation (Pearson correlation 0.7). Therefore, the signal detected by our MOF method is as accurate as the clinical method, proving that the MOF extraction method is suitable for downstream detection of large-scale high-throughput sequencing.

[0094] Table 3

[0095]

[0096]

[0097] 6. Comparison of MOF method and magnetic bead method (VAHTS) for extracting cfDNA and cfRNA.

[0098] The present embodiment uses a commercial product for nucleic acid magnetic bead purification to extract free nucleic acid and compares it with the MOF method provided in the present embodiment. The commercial product is the VAHTS serum / plasma free DNA kit (Vazyme TM , catalog number N902).

[0099] The present embodiment adopts the MOF method and the VAHTS method to extract the same mixed plasma sample, and then detects the contents of cfDNA and cfRNA respectively.

[0100] The results are as follows Figure 27-29 As shown in the figure, for the comparison results of cfRNA, the VAHTS method is ineffective for 18SrRNA extracted from plasma (the Ct value is the same as the control group without template). This result is expected because the operating manual of the VAHTS method states that it is only suitable for the extraction of cfDNA. Because the buffer conditions for magnetic bead adsorption and elution of DNA and RNA are different, RNA and DNA cannot be extracted at the same time.

[0101] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A method for extracting free nucleic acids from body fluids for disease diagnosis, characterized in that: include: Pretreatment of body fluid samples; Using MOF materials to adsorb free nucleic acids in body fluid samples; The MOF material is separated from the free nucleic acid to obtain the free nucleic acid in the body fluid sample.

2. The extraction method according to claim 1, characterized in that The body fluid is a whole blood sample or bile.

3. The extraction method according to claim 2, characterized in that When the body fluid is bile, the extraction method comprises the following steps: a. Centrifuge the bile to remove cell debris and obtain the supernatant containing free cfDNA / cfRNA; b. Use a protein denaturing agent to destroy the structure of bile proteins, dissociate cfDNA / cfRNA fragments, salt out proteins, and centrifuge to remove proteins and retain cfDNA / cfRNA fragments; c. incubating the MOF material with the bile supernatant solution, centrifuging, and recovering the MOF material containing cfDNA / cfRNA; d. Release of cfDNA / cfRNA from MOF materials.

4. The extraction method according to claim 1, characterized in that The disease is a tumor or an infectious disease; the tumor is hepatocellular carcinoma, and the infectious disease is chronic viral hepatitis.

5. The extraction method according to claim 1, wherein the MOF material is at least one of Co-IRMOF-74-II, Co-IRMOF-74-III and Co-IRMOF-74-IV among Co-based MOF materials. The extraction method according to claim 1 , wherein the free nucleic acid comprises free DNA and free RNA.

7. A kit for free nucleic acid in body fluid samples for disease diagnosis, comprising a specific MOF adsorption reagent, a lysis solution, a separation agent, and a nucleic acid storage tube.

8. The kit according to claim 7, wherein the disease is hepatocellular carcinoma or chronic hepatitis B.

9. The kit according to claim 7, wherein the MOF adsorption agent comprises at least one of the following Co-based MOF materials: Co-IRMOF-74-II, Co-IRMOF-74-III, and Co-IRMOF-74-IV.

10. Use of any kit according to claims 7-9 for extracting free nucleic acids from tumors and / or infectious body fluids.

Images