CN116640846A - Micro residual focus ctDNA quality control product and preparation method and application thereof - Google Patents
Micro residual focus ctDNA quality control product and preparation method and application thereof Download PDFInfo
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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Abstract
The invention discloses a ctDNA quality control product for tiny residual focus, a preparation method and application thereof. The invention prepares the quality control product of the MRD detection ctDNA by screening cells containing clinical MRD common detection gene mutation sites, mixing and diluting the fragmented DNA obtained by cell culture, enzyme digestion and purification, and accurately quantifying by ultra-low frequency digital PCR detection and high-depth sequencing methods. The quality control product comprises 37 clinical solid tumor frequent mutations and more than 3000 endogenous gene mutations. In addition, the gene mutation raw material of the quality control product is obtained by treating cells in an enzyme-sectional segmentation mode, and the distribution characteristics of DNA fragments are consistent with clinical ctDNA. The quality control product prepared by the invention overcomes the defects of MRD ctDNA quality control products in the current market, and can be applied to the development of MRD detection technology, performance verification, the establishment of performance confirmation and quality control of LDT flow, the quality control in the clinical research process of the MRD detection technology, and the like.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a ctDNA quality control product with tiny residual focus, a preparation method and application thereof.
Background
Minimal residual disease (Minimal Residual Disease, MRD) refers to a hidden micrometastasis or minimal residual disease of a tumor in the body after radical treatment (e.g., surgical excision). Circulating tumor DNA (Circulating tumor DNA, ctDNA) is the main test subject for minimal residual lesions. MRD real-time monitoring and evaluation based on ctDNA can provide valuable reference information for clinical accurate medication, molecular typing, prognosis evaluation, curative effect recurrence monitoring and other diagnosis and treatment processes because the convenience and sensitivity of the method are superior to those of the traditional clinical pathology biopsy and imaging detection.
The detection of low frequency ctDNA currently used for MRD is mainly based on both PCR and new generation sequencing (Next Generation Sequencing, NGS). PCR-based assays include digital droplet polymerase chain reaction (ddPCR), digital PCR-flow BEAMing, and mutant amplification blocking systems (ARMS), which have lower overall detection capability than the NGS-based MRD assay platform due to the disadvantage of being able to detect only known types of gene mutations and low detection throughput. NGS has the advantage of high throughput and can detect a large amount of known and unknown genetic mutation information in a short time. Marker amplification deep sequencing (Tam-Seq), cancer personalized deep sequencing (CAPP-Seq), integrated digital error suppression sequencing (iDES), directed mutation enrichment sequencing (phasED-Seq) are MRD ctDNA detection methods developed based on NGS derivatization. The methods are in a research and development stage at present, and no hatching and maturing MRD detection platform is applied to clinic at home and abroad, and the limitation is mainly that cfDNA has the content of about 7ng/ml in peripheral blood of common healthy people, the content is small, the ctDNA accounts for only 0.1-5% in proportion due to individual differences of patients, disease differences and different disease processes, and the amount of ctDNA released from tumor cells after operation of cancer patients to peripheral blood is very little. In 2021, "expert consensus on non-small cell lung cancer molecular residue focus", it is explicitly proposed that the recommended detection technique requires stable detection of genetic variation with abundance greater than or equal to 0.02% in ctDNA of peripheral blood for lung cancer molecular abnormalities. The ultra-low detection sensitivity is a great challenge for the development of the current MRD mainstream detection technology-tumor informed sequencing analysis and tumor unknowing sequencing analysis platform. Due to the requirement of ultra-low sensitivity, false positive and false negative risks caused by insufficient sensitivity of a detection platform are extremely easy to occur, and the detected result cannot be accurately judged due to the fact that a reliable reference standard is not available. Therefore, a scientific and reliable quality control method and system are established through quality control products, and accurate performance evaluation and subsequent clinical detection quality control are performed on the established platform, so that the reliability and the repeatability of a detection result are improved, and the key of the rapid development of the health of the assisted MRD detection is realized.
MRD ctDNA quality control was introduced by Seracare and domestic otto (LDT) biotechnology in recent two years. The quality control product developed in China is prepared by mixing 1 pair of paired cell DNA derived from lung cancer according to different proportions, the quality control product only selects 1 pair of paired cells of a single cancer species, contains only 1-3 normal detection sites with clinical detection significance, cannot meet the performance inspection requirement of a multi-cancer MRD multi-site detection platform, is difficult to popularize in the clinical daily quality control of MRD detection, and in addition, the fragment size of ctDNA is concentrated in the range of 100 bp-200 bp. The Seracare quality control product is prepared by mixing 1 pair of lung cancer paired cell DNA and exogenously synthesized 22 gene mutation site fragments. Compared with the quality control product of LDT company, the product expands the number of pathogenic mutation gene loci. However, the Seracare quality control product has the defect that most site fragments with clinical detection significance are prepared by a chemical synthesis process, and the synthesized fragments tend to be immobilized in sequence, namely the positions of mutation targets are fixed, and the characteristics that ctDNA mutation targets are random in the positions of the fragments are not achieved, so that the fragments cannot be consistent with clinical samples.
In summary, the current methods for MRD ctDNA quality control at home and abroad are mainly prepared by preparing paired cells or doping paired cells into exogenous synthetic fragments. The quality control product has the defect that the quality control product contains few gene mutation sites in clinical significance or exogenous introduced fragments cannot simulate clinical samples. Therefore, there is a need for an MRD ctDNA quality control product that includes the genetic mutation sites frequently detected by various solid tumors as much as possible, and is consistent with the characteristics of clinical samples, so as to adapt to the requirements of single cancer or multiple solid tumor MRD detection platforms for quality control in the development process, LDT process construction quality control, and clinical MRD detection daily quality control.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a micro residual focus ctDNA quality control product and a preparation method and application thereof. The invention prepares the quality control product of the MRD detection ctDNA by screening cells containing clinical MRD common detection gene mutation sites, mixing and diluting the fragmented DNA obtained by cell culture, enzyme digestion and purification, and accurately quantifying by ultra-low frequency digital PCR detection and high-depth sequencing methods. The quality control product comprises 37 clinical solid tumor frequent mutations and more than 3000 endogenous gene mutations. In addition, the gene mutation raw material of the quality control product is obtained by treating cells in an enzyme-sectional segmentation mode, and the distribution characteristics of DNA fragments are consistent with clinical ctDNA. The invention discloses a quality control product of ctDNA of tiny residual focus, which comprises fragmented DNA obtained by enzyme digestion and purification of 38 cells, wherein each fragmented DNA contains gene mutation shown in the following table, and the mass parts of each fragmented DNA are shown in the following table:
furthermore, the gene mutation sites shown in DNA-1 to DNA-37 are the gene mutation sites carried by tumor cells themselves or obtained by gene editing.
Further, cells used with DNA-1-DNA-37 include, but are not limited to, MDA-MB-134-VI, SNU-C1, SW1116, SW48, MDA-MB-468, MCF7, KATOIII, SW620, SK-LMS-1, A-253, SH-4, SNU-449, SW1088, SK-OV-3, A549, BXPC-3, NCI-H1975, NCI-N87, ME-180, RKO, HCT116, HEK293T, GM12878, and KBM-7; cells used for DNA-38 include, but are not limited to, GM12878, KBM-7, HEK293A, HEK293T, and HCT116.
Further, the fragmented DNA of the DNA-1 to DNA-38 is obtained by Mnase digestion, the fragments are concentrated at 100-200bp, the main band size is 144-176 bp, and the small fragment DNA (< 100 bp) accounts for less. Namely, the fragment DNA obtained by Mnase enzyme digestion is consistent with the distribution characteristics of clinical ctDNA fragments, so that the ctDNA can be better simulated.
Further, the mass parts of each fragmented DNA are shown in the following table:
further, the quality control product comprises a positive quality control product with the mutation frequency of 0.5%, a positive quality control product with the mutation frequency of 0.05%, a positive quality control product with the mutation frequency of 0.005% and a negative quality control product; the negative quality control product is fragmented DNA obtained by enzyme digestion and purification of cells without the gene mutation sites of DNA-1 to DNA-37.
The invention also provides a preparation method of the ctDNA quality control product for the tiny residual focus, which comprises the following steps:
s1: selecting cells for gene editing; the method specifically comprises the following steps:
(1) Designing specific guide RNA aiming at a target site of a target gene;
(2) Co-transfecting the expression vector of the guide RNA and the single-stranded DNA into a target cell;
(3) Performing single cell cloning;
(4) After cell clone formation, sanger sequencing is carried out to determine that gene editing is successful;
s2: mnase enzyme cutting;
(1) Performing enzyme digestion on the cell nucleus corpuscles by using Mnase enzyme, and then screening and purifying by using magnetic beads;
(2) Measuring the purity;
OD260/280: x is more than or equal to 1.8 and less than or equal to 2.0, and the qualification is judged;
OD260/230: x is more than or equal to 1.5 and less than or equal to 5.0, and the qualification is judged;
(3) Detecting the distribution of DNA fragments;
the main band size is in the range of 144bp-176bp, and the main band size is judged to be qualified;
s3: mixing and diluting fragmented DNA;
the DNA obtained by enzyme slicing, sectioning and purifying is required to be mixed according to the mass parts, so that a positive quality control product with the gene mutation frequency of 0.5% of each gene mutation target point is obtained; verifying mutation site variation information through digital PCR;
diluting the positive quality control product with the gene mutation frequency of 0.5% by 10 times to obtain a positive quality control product with the gene mutation frequency of 0.05%, and diluting the positive quality control product with the gene mutation frequency of 0.05% by 10 times to obtain a positive quality control product with the gene mutation frequency of 0.005%; the DNA fragment obtained by the enzyme digestion and purification of the cells of the DNA-38 is a negative quality control product after the concentration adjustment; determining the gene frequency by digital PCR, and calculating the ratio of the gene mutation frequency;
s4: new Generation Sequencing (NGS) verification.
Further, the sequence of the guide RNA in step S1 is shown in the following table:
further, the digital PCR in step S3 verifies mutation site variation information as follows:
| gene name | Mutation type | Gene mutation site | COSMICv96 |
| AKT1 | SNV | AKT1 E17K | COSM33765 |
| ALK | Fusion | EML4(13)-ALK(20)FUSION | COSF408 |
| ALK | SNV | ALK F1174L | COSM28055 |
| BRAF | SNV | BRAF V600K | COSM473 |
| BRAF | SNV | BRAF V600E | COSM476 |
| BRCA1 | Stop Gain | BRCA1 R1443* | COSM979730 |
| BRCA2 | Frame Shift | BRCA2 K1691fs | COSM6048456 |
| BRCA2 | SNV | BRCA2 D1420Y | COSM3736087 |
| BRCA2 | Frame Shift | BRCA2 A1784fs | COSM18607 |
| CDK12 | SNV | CDK12 P250H | COSM4266369 |
| EGFR | SNV | EGFR L858R | COSM6224 |
| EGFR | del | EGFR E746_A750del | COSM6223 |
| EGFR | SNV | EGFR T790M | COSM6240 |
| EGFR | SNV | EGFR S768I | COSM6241 |
| EGFR | SNV | EGFR L861Q | COSM6213 |
| EGFR | indel | EGFR L747_P753>S | COSM12370 |
| EGFR | ins | EGFR D770_N771insG | COSM12378 |
| EGFR | SNV | EGFR G719C | COSM6253 |
| FGFR3 | Fusion | FGFR3(17)-TACC3(4)FUSION | COSF1350 |
| Her2 | ins | HER2 A775_G776insYVMA | COSM20959 |
| Her2 | ins | HER2 Y772_A775dup | COSM12558 |
| IDH1 | SNV | IDH1 R132G | COSM28749 |
| IDH2 | SNV | IDH2 R172M | COSM33732 |
| KIT | SNV | KIT D816V | COSM1314 |
| KIT | ins | KIT Y503_F504insAY | COSM12444 |
| KRAS | SNV | KRAS G12C | COSM516 |
| KRAS | SNV | KRAS G12D | COSM521 |
| KRAS | SNV | KRAS Q61H | COSM554 |
| KRAS | SNV | KRAS G13D | COSM532 |
| NRAS | SNV | NRAS Q61R | COSM584 |
| NRAS | SNV | NRAS G12A | COSM565 |
| NTRK1 | Fusion | TPM3(7)-NTRK1(10)FUSION | COSF1329 |
| PDGFRA | SNV | PDGFRA D842V | COSM736 |
| PIK3CA | SNV | PIK3CA H1047R | COSM775 |
| RET | Fusion | CCDC6(1)-RET(12)FUSION | COSF1271 |
| ROS1 | Fusion | CD74(6)-ROS1(34)FUSION | COSF1200 |
| TP53 | SNV | TP53 R273H | COSM10660 |
Further, in step S4, high-depth sequencing of the polygene panel is performed, mutation frequency analysis is performed on sequencing data, and the detected genes are shown in the following table:
the invention also provides a quality control product of the micro residual focus ctDNA, or a micro residual focus ctDNA detection kit of the quality control product of the micro residual focus ctDNA prepared by the method.
The invention also provides the micro residual focus ctDNA quality control product, or the micro residual focus ctDNA quality control product prepared by the method, or the application of the micro residual focus ctDNA detection kit, wherein the application is selected from any one or more of the following:
(1) Preparing a micro residual focus ctDNA quality control product;
(2) The application of the reagent for preparing and evaluating the product or platform for detecting the ctDNA of the tiny residual focus;
(3) Preparing a reagent for calibrating the ctDNA result of the tiny residual focus;
(4) The application of the reagent for preparing and optimally calibrating the ctDNA detection method or system of the tiny residual focus is provided.
In conclusion, compared with the prior art, the invention achieves the following technical effects:
the MRD ctDNA quality control product for detecting the minimal residual focus is a combination set with multiple gradient gene mutation frequencies, and comprises a negative quality control product and positive quality control products with the gene mutation frequencies AF of 0.005%,0.05% and 0.5%. The MRD ctDNA quality control product DNA fragment is concentrated in 100-200bp, the main band size is 144-176 bp, the small fragment (< 100 bp) is less in proportion, namely the quality control product is consistent with the distribution characteristics of clinical ctDNA fragments, and can highly simulate ctDNA. The positive quality control contains 37 mutation sites verified by digital PCR, and the multi-gene panel 35000X deep sequencing verification site information contains more than 3000 mutation sites containing 288 genes and common structural mutation of cancer genome such as SNV, fusion, base insertion deletion and the like. The MRD ctDNA quality control product has various application scenes, and can be used for the development of MRD detection technology based on PCR and NGS platforms, performance verification, the establishment of performance confirmation and quality control of LDT flow, the quality control in clinical research of the MRD detection technology and other processes. The use method of the quality control sample is based on different used kits and platforms, and the quality control sample is used as one sample in specific operation and is treated with other experimental samples by the same technology and experimental operation flow. The difference between the experimental result of the quality control product and the theoretical expected value can reflect whether the whole operation flow and other experimental sample results are credible or not; can be used for evaluating the stability, the specificity and the sensitivity of a workflow from sample extraction to bioinformatic analysis, evaluating the processing method of each sample and detecting the performance difference between platforms.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 shows a DRAGEN TruSight according to an embodiment of the present invention TM Oncology 500ctDNA analysis flow chart.
FIG. 2 is a graph showing comparison of fragment distributions of ctDNA prepared by different methods according to examples of the present invention.
FIG. 3 is a graph showing the distribution of MRD ctDNA negative quality control fragment according to an embodiment of the present invention.
FIG. 4 is a distribution diagram of MRD ctDNA gene mutation AF0.5% quality control fragment according to an embodiment of the present invention.
FIG. 5 is a distribution diagram of MRD ctDNA gene mutation AF 0.05% quality control fragment according to an embodiment of the present invention.
FIG. 6 is a distribution diagram of MRD ctDNA gene mutation AF 0.005% quality control substance fragments according to an embodiment of the present invention.
Detailed Description
In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, shall fall within the scope of the invention.
The invention prepares the quality control product of the MRD detection ctDNA by screening cells containing clinical MRD common detection gene mutation sites, mixing and diluting the fragmented DNA obtained by cell culture, enzyme digestion and purification, and quantifies the quality control product by ultra-low frequency digital PCR detection and high-depth sequencing methods. The quality control product comprises 37 clinical solid tumor frequent mutations and more than 3000 endogenous gene mutations. In addition, the gene mutation raw materials of the quality control product are all obtained by processing different cells in an enzyme-sectional manner, so that the clinical ctDNA can be simulated to the greatest extent. The quality control product prepared by the invention overcomes the defects of MRD ctDNA quality control products in the current market, and can be applied to the development of MRD detection technology, performance verification, the establishment of performance confirmation and quality control of LDT flow, the quality control in the clinical research process of the MRD detection technology, and the like.
EXAMPLE 1MRD ctDNA quality control cell Material screening
The MRD ctDNA quality control product is derived from cells specially cultured in American type culture Collection (American type culture collection, ATCC) and cells obtained by gene editing, and the MRD ctDNA quality control product with different mutation frequency gradients is obtained by respectively culturing the various cells, and then mixing and diluting the DNA obtained by enzyme digestion and purification. The prepared MRD ctDNA was verified by digital PCR and NGS sequencing. The non-gene editing cell raw material is derived from ATCC cells, and most of tumor gene mutation sites detected by clinical hot NGS can be obtained through mixing of a plurality of cells.
Cells mentioned above include, but are not limited to, MDA-MB-134-VI, SNU-C1, SW1116, SW48, MDA-MB-468, MCF7, KATOIII, SW620, SK-LMS-1, A-253, SH-4, SNU-449, SW1088, SK-OV-3, A549, BXPC-3, NCI-H1975, NCI-N87, ME-180, RKO, HCT116, HEK293T, GM12878, and KBM-7.
The tumor mutant gene mutation sites mentioned above include, but are not limited to EGFR, KRAS, NRAS, BRAF, human epidermal growth factor receptor 2 (HER 2), anaplastic Lymphoma Kinase (ALK), and the like.
Example 2 Gene-editing cells
The gene editing method described in this example can obtain most of hot tumor-related mutations except the gene mutation contained in the selected blast cell, so as to expand the tumor gene mutation sites contained in the MRD ctDNA quality control. Such as the epidermal growth factor receptor gene (EGFR), etc. The specific method is as follows:
A. specific guide RNAs (grnas) are designed for target sites of target genes, and expression vectors for the guide RNAs are constructed. Meanwhile, single-strand DNA (ssDNA) is designed and synthesized for use as a template for gene editing repair.
B. The expression vector for the guide RNA and the single-stranded DNA are co-transfected into the target cells.
C. When the cell state is good, it is subjected to single cell cloning.
D. After cell clone formation, a portion of the clone was sequenced by sanger to confirm successful gene editing.
In the gene editing step a, the relevant genes include, but are not limited to, tumor genes including, but not limited to EGFR,
KRAS, NRAS, BRAF HER2, anaplastic Lymphoma Kinase (ALK), etc.
The specific information on the gene editing gRNA and ssDNA in this example is shown in Table 1.
TABLE 1 Gene editing gRNA and ssDNA information
In the step C, monoclonal technique and sanger sequencing technique are adopted to screen out monoclonal cell strain with target gene mutation and genetic stability. The specific method is as follows:
C1. and digesting and collecting the cells when the confluence of the cells reaches 70-90%.
C2. The cells were counted accurately, the living cells were diluted to 5 cells/mL with medium according to the method of double dilution, and the cells were mixed well.
C3. 10mL of the cell suspension was evenly distributed into a 96-well plate.
C4. When cell clones were formed, wells were labeled with clones, and cells were digested with a small amount of trypsin (. About.20. Mu.L): taking part of the cells for PCR (polymerase chain reaction) and using the part of the cells as a sanger sequencing; the remaining cells were continued to be cultured.
Example 3 Mnase cleavage method
(1) Preparation of Mnase enzyme cutting product
The fragmented DNA mentioned in this example was obtained by subjecting the nucleosome to cleavage using Mnase enzyme and purifying it by magnetic bead screening. The fragmented DNA product obtained by the method can simulate the clinical real ctDNA, and the DNA structure and fragment distribution characteristics are consistent with those of the ctDNA. The specific method is as follows:
A. cell pellet was collected by centrifugation and resuspended in PBS.
B. Adding appropriate amount of Protease inhibitor cocktail and surfactant, standing for a period of time, and centrifuging.
C. Removing the supernatant, adding Mnase reaction solution, fully resuspending, and reacting for a period of time at 37 ℃.
D. Proteinase K digest was added and incubated at 65℃for a period of time to obtain a crude DNA fragment.
E. Adding a proper amount of magnetic beads, uniformly mixing, incubating at room temperature for a period of time, placing a sample on a magnetic rack, and removing the supernatant after the magnetic beads are completely adsorbed. The 80% ethanol solution is used for suspending the magnetic beads again, after the magnetic beads are fully and uniformly mixed, the sample is placed in a magnetic rack, and the supernatant is removed after the magnetic beads are completely adsorbed. Taking down the magnetic beads, adding the eluent, uniformly mixing, and placing the mixture on a magnetic rack to obtain a supernatant.
In the step A, the cell quantity for single Mnase enzyme digestion is 10-9 cells; the PBS re-suspension volume was 100-900. Mu.L.
Protease inhibitor cocktail in the step B is 5-80 mu L; the surfactant is 0.1% Tween 80 10-50 mu L or 1% TritonX 5-80 mu L; standing for 5-30 min.
Optionally, the Mnase reaction solution in step C is: 89.2. Mu.L distilled water+10. Mu.L 10 XTC buffer+0.8. Mu. L Micrococcal Nuclease; the reaction time is 5-30 min at 37 ℃.
The proteinase K digestion solution in the step D is as follows: 198. Mu.L of water+2. Mu.L of Proteinase K (20 mg/mL). Incubation time at 65℃was 30min.
In step E, the magnetic beads are mainly used for purifying DNA. The magnetic beads are added according to the proportion of 0.5:1-3:1 of the sample volume. The eluent can be enzyme-free water, tris-EDTA buffer solution, PBS buffer solution and the like.
(2) Concentration measurement
A. The purified product was subjected to DNA concentration measurement using a spectrophotometer.
B. The concentration measurement should be continuously carried out for 3 times, and the following conditions should be satisfied:
a) OD260/280: and the measured value x is more than or equal to 1.8 and less than or equal to 2.0, and the qualification is judged.
b) OD260/230: and the measured value x is more than or equal to 1.5 and less than or equal to 5.0, and the qualification is judged.
(3) DNA fragment distribution detection
A. Detecting the size of the DNA fragment using Agilent TapeStation 4150;
B. DNA fragment detection should satisfy the following conditions:
a) The main band size is in the range of 144bp-176bp, and the main band size is judged to be qualified.
In this example, KAPA enzyme and ultrasonic method are used as comparison experiments to prepare fragmented DNA, and the obtained fragmented DNA and a comparison with real ctDNA are shown in fig. 2, and it can be seen that the fragments obtained by Mnase cleavage in this example have a narrow distribution and are mainly concentrated in the range of 100 bp-200 bp, and the fragments with less than 100bp have a ratio similar to that of ctDNA of about 17%, so that the fragmented DNA obtained by Mnase cleavage is closest to the real ctDNA. The main band size of the fragment obtained by ultrasonic method and KAPA enzyme cutting is larger than that of the real ctDNA, the distribution range of the fragment length is wide, the fragment ratio smaller than 100bp is 30.2%, the difference with the characteristics of the ctDNA fragment is large, and the ctDNA cannot be simulated.
EXAMPLE 4 dilution and mixing of fragmented DNA
(one) fragmenting DNA mixing
The DNA obtained by the enzyme slicing segmentation and purification of the invention is mixed according to the specific parts of the following table 2, and the mixture with the gene mutation frequency AF of 0.5% of each gene mutation target point is obtained. The gene mutation frequency 0.5% expressed here is the expected value of all mutation target sites, and for single mutation sites, the target cell DNA and non-target cell DNA are mixed by adjusting the mass ratio of the target cell DNA to the non-target cell DNA, and finally, the target cell DNA and the non-target cell DNA are determined by digital PCR detection.
The specific method is as follows:
A. the same DNA fragments of the coded and batch cells, which are subjected to enzyme digestion and purification, are mixed, and before mixing, confirmation is needed:
the DNA to be mixed is subjected to enzyme digestion and purification on cells with the same codes and lot numbers;
the results of OD260/280 and OD260/230 of the concentration measurement of the DNA to be mixed are qualified.
B. The appropriate volume of tubing was chosen for mixing (. Gtoreq.1.5 mL should be mixed with a 50mL LBD tube).
C. The formulated mixture was subjected to gene frequency detection by digital PCR at the sites in table 3. Primer probe sequence information is shown in Table 4.
E. The mixture qualified by digital PCR verification is adjusted to 20 ng/. Mu.L, and the concentration is measured.
Alternatively, the specific part mix information of table 2 is as follows:
TABLE 2 raw material mixing ratio
| Raw material code | Parts by weight | Raw material code | Parts by weight |
| DNA-1 | 0.1-2 parts | DNA-20 | 0.1-2 parts |
| DNA-2 | 0.1-2 parts | DNA-21 | 0.1-2 parts |
| DNA-3 | 0.1-2 parts | DNA-22 | 0.1-2 parts |
| DNA-4 | 0.1-2 parts | DNA-23 | 0.1-2 parts |
| DNA-5 | 0.1-2 parts | DNA-24 | 0.1-2 parts |
| DNA-6 | 0.1-2 parts | DNA-25 | 0.1-2 parts |
| DNA-7 | 0.1-2 parts | DNA-26 | 0.1-2 parts |
| DNA-8 | 0.1-2 parts | DNA-27 | 0.1-2 parts |
| DNA-9 | 0.1-2 parts | DNA-28 | 0.1-2 parts |
| DNA-10 | 0.1-2 parts | DNA-29 | 0.1-2 parts |
| DNA-11 | 0.1-2 parts | DNA-30 | 0.1-2 parts |
| DNA-12 | 0.1-2 parts | DNA-31 | 0.1-2 parts |
| DNA-13 | 0.1-2 parts | DNA-32 | 0.1-2 parts |
| DNA-14 | 0.1-2 parts | DNA-33 | 0.1-2 parts |
| DNA-15 | 0.1-2 parts | DNA-34 | 0.1-2 parts |
| DNA-16 | 0.1-2 parts | DNA-35 | 0.1-2 parts |
| DNA-17 | 0.1-2 parts | DNA-36 | 0.1-2 parts |
| DNA-18 | 0.1-2 parts | DNA-37 | 0.1-2 parts |
| DNA-19 | 0.1-2 parts | DNA-38 | 26-96.3 parts |
Alternatively, in step C, the genetic locus information verified by digital PCR of table 3 is as follows:
TABLE 3 digital PCR validation of mutation site variation information
In step C, the primer probe information used for detecting the mutation site of the gene by digital PCR in Table 4 is as follows:
TABLE 4 primer probe information
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In the step C, the digital PCR is used for detecting mutation sites of various genes of the mixed intermediate, and the specific method is as follows:
c1, preparing a reaction premix according to the reaction components in the following table, and preparing two to four compound holes for each sample at each site, wherein the pipetting loss is considered, so that 1.1 reaction amounts are prepared for each amplification hole at each site. The detailed configuration components and the addition amounts thereof are shown in the following table:
| Stock Con. | Final Con. | 1x Volume(μL) | |
| ddPCR supermix | 2x | 1x | 10 |
| Primer mix | 40x(36μM) | 1x | 0.5 |
| Probe mix | 40x(10μM) | 1x | 0.5 |
| H 2 O | - | - | 0 |
| DNA template | 50ng | - | 9 |
| Final Volume | 20 |
c2, droplet generation. The premix solution of the above configuration was added to the middle well of the droplet-generating card, 20 μl of each well was added, 70 μl of the droplet-generating oil was added to the lower well of the card, rubber sleeves were placed on both sides of the droplet-generating card, the droplet-generating card removal compartment was placed in the droplet-generating apparatus, and the lid was closed, waiting for the droplet generation to be completed.
C3, droplet transfer. The appropriate range of pipette was adjusted to 40 μl and the resulting droplets were transferred very carefully into a 96-well PCR reaction plate. Maintaining the position of the gun head at an oblique angle of 4 degrees prevents the bottom of the card from blocking the gun head.
And C4, sealing the film. After the droplet transfer of all samples was completed, a piece of aluminum film was placed on the surface of the 96-well plate, and the film was sealed with a heat sealer.
C5, PCR reaction. The 96-well plate is placed on a PCR instrument, the cover is closed, the operation is performed according to the following procedure, and the temperature rise and reduction speed of the instrument is regulated to 2-3 ℃/s.
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And C6, collecting signals. When the PCR procedure was completed, the 96-well plate was transferred to a QX200 microdroplet reader (half an hour in advance to start preheating), on a computer, "QuantaSoft" software was turned on, wells containing samples were selected, the name of each well was set in the corresponding position, supermix for experiments, the experiment type was set to ABS, the "Assay 1" was set to "Mut", "type" was set to "Ch1 unown", the "Assay 2" was set to "WT", "type" was set to "Ch2 unown", and "RUN" was selected.
And C7, data analysis. After the data reading is completed, the experimental data to be analyzed is opened, and the result can be analyzed by selecting "analysis".
Optionally, in the step C, the mutation sites of the genes of the mixed intermediate product are detected by digital PCR, and the detection result meets the following conditions:
a) Gene mutation frequency (AF): x is more than or equal to 3 and less than or equal to 0.1, and the qualification is judged.
Optionally, in step D, the mixture concentration that is qualified in quality inspection is diluted to 20 ng/. Mu.L, and the concentration is determined by a spectrophotometer. The concentration measurement should be continuously carried out for 3 times, and the following conditions should be satisfied:
a) The concentration range is as follows: x is more than or equal to 17 ng/mu L and less than or equal to 23 ng/mu L, and the qualification is judged
b) OD260/280: and the measured value x is more than or equal to 1.8 and less than or equal to 2.0, and the qualification is judged.
c) OD260/230: and the measured value x is more than or equal to 1.5 and less than or equal to 5.0, and the qualification is judged.
(II) dilution of fragmented DNA mixture
The dilution of the fragment DNA mixture described in this example means that intermediate products with the mutation frequencies of AF of 0.05% and 0.005% were obtained by subjecting the prepared mixture with the mutation frequency of AF of 0.5% to two-time gradient dilutions. The DNA fragment obtained by the DNA-38 cells through enzyme digestion and purification is a negative quality control product after the concentration is adjusted to a proper range. The negative quality control material is DNA-38, the ratio of which is 100 percent, and the negative quality control material does not contain DNA-1 to DNA-37.
The specific method is as follows:
A. the purified DNA fragment of the DNA-38 cells after enzyme digestion needs to be confirmed before use, and the results of OD260/280 and OD260/230 to be measured in purity are qualified.
B. The DNA fragment purified by the digestion of the DNA-38 cells was used to detect the mutation site of the detected gene in the positive intermediate by digital PCR.
The detection result needs to meet the following conditions:
a) And the detected gene mutation AF is less than 0.005 percent, and is judged to be qualified.
C. The concentration of the DNA fragment purified by digestion of the DNA-38 cells was adjusted to 20 ng/. Mu.L and measured by a spectrophotometer. Concentration determination should be performed 3 times in succession, and the following conditions should be satisfied:
a) The concentration range is as follows: and (3) judging whether the sample is qualified by 17 ng/mu L, wherein x is less than or equal to 23 ng/mu L.
b) OD260/280: and the measured value x is more than or equal to 1.8 and less than or equal to 2.0, and the qualification is judged.
c) OD260/230: and the measured value x is more than or equal to 1.5 and less than or equal to 5.0, and the qualification is judged.
D. The DNA product obtained after the enzyme digestion of the DNA-38 cells is taken as a matrix, and the mixture with the mutation frequency AF of 0.5 percent which is required to be prepared is diluted 10 times, so that the AF quality control product with the concentration of 0.05 percent is obtained. Diluting the required AF 0.05% mixture by 10 times to obtain AF 0.005% quality control product. The concentration was adjusted to 20 ng/. Mu.L.
E. Screening 1-2 sites, and performing digital PCR on all prepared gradient quality control products.
F. The concentration of DNA-38 cell cleavage purification was adjusted to 20 ng/. Mu.L, and the resultant product was used as a negative quality control (mutation frequency AF: 0). The prepared gene mutation frequencies AF0.5%,0.05%,0.005% and 0.4 quality control substances were subjected to concentration measurement and fragment size detection.
F1, concentration measurement should be continuously carried out for 3 times, and the following conditions should be satisfied:
a) The concentration range is as follows: and (3) judging whether the sample is qualified by 17 ng/mu L, wherein x is less than or equal to 23 ng/mu L.
b) OD260/280: and the measured value x is more than or equal to 1.8 and less than or equal to 2.0, and the qualification is judged.
c) OD260/230: and the measured value x is more than or equal to 1.5 and less than or equal to 5.0, and the qualification is judged.
F2, DNA fragment distribution detection:
A. detecting the size of the DNA fragment using Agilent TapeStation 4150;
B. DNA fragment detection should satisfy the following conditions:
a) The main band size is in the range of 144bp-176bp, and the main band size is judged to be qualified.
Alternatively, in step E, all gradient cationic controls are subjected to digital PCR. The aim is to verify the accuracy of the dilution. To improve the low frequency detection accuracy. The number of the compound holes can be increased to 8-16 holes.
The ratio of the mutation frequencies of the genes which are measured and calculated should satisfy the following conditions:
a) The AF 0.5%/AF 0.05% range is: and X is more than or equal to 7 and less than or equal to 13, and judging that the product is qualified.
b) AF 0.05%/AF 0.005% range is: and X is more than or equal to 7 and less than or equal to 13, and judging that the product is qualified.
Example 5 New Generation Sequencing (NGS) validation
The quality control product provided by the invention passes the NGS verification. Negative quality control and Gene mutation frequency AF obtained by referring to the preparation method
0.5% positive quality control, polygenic panel high depth sequencing using 30ng of starting DNA, and mutation frequency analysis of sequencing results. The sequencing information is shown in Table 5.TruSight Oncology 500ctDNA combined with unique molecular markers (UMI) and error correction software reduced sequencing error rates from 0.5% to <0.007%. The detection Limit (LOD) is 0.5%, and when the initial amount of ctDNA is 30ng, the sequencing depth sensitivity of 35000x can reach 99.88%, and the specificity is 99.99%.
TABLE 5 sequencing information
High throughput sequencing analysis method see DRAGEN TruSight shown in fig. 1 TM Oncology 500ctDNA analysis procedure. The genes detected are shown in Table 6:
TABLE 6 high throughput sequencing assay Gene list
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Example 6
Based on the above mentioned preparation and test methods, the specific experimental procedure of example 6 is as follows:
(1) Mnase enzyme digestion
Reference is made to the specific operation of example 3.
(2) Fragmented DNA mix
In this example, the MRD ctDNA quality control product with the gene mutation frequency AF of 0.5% was prepared by nucleosome digestion (Mnase) and purification of the obtained raw material fragment DNA according to the mixture ratio Table 7. Of the 37 gene mutation sites, the codes of the raw materials of DNA-1 to DNA-37 are the DNA obtained by the enzyme digestion and purification of the cells corresponding to the target gene mutation site, the information of the corresponding cell raw materials is shown in Table 7, and the DNA-38 is the DNA obtained by the enzyme digestion and purification of GM12878 cells.
TABLE 7 DNA raw material proportioning table
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(3) Fragmented DNA dilution
In this example, the gene mutation frequency AF 0.05%, and the 0.005% MRD ctDNA quality control was prepared by diluting AF0.5% quality control 1 and 2 times with a 10-fold gradient, respectively. The negative quality control is prepared by regulating the concentration of GM12878 cell DNA fragments purified by enzyme digestion (without doping target gene mutant DNA). Sites indicating dilution accuracy are EGFR L861Q and TPM3-NTRK1. And carrying out digital PCR detection on the two screened sites by using AF 0.05% and AF 0.005% positive quality control products and negative quality control products, wherein the number of multiple holes is 16, and comprehensively analyzing the data of 16 holes. In addition, each of the gradient positive and negative quality controls was subjected to concentration and fragment size detection via Agilent TapeStation 4150 and a spectrophotometer.
(4) Quality control NGS sequencing verification
In this example, sequencing verification was performed on MRD ctDNA negative quality control and AF0.5% positive quality control by TruSight Oncology ctDNA panel.
(5) Results summary and analysis
A. The main peak position of the quality control product DNA is 144-176 bp (shown in figure 2), and the main peak position is the same as the fragment distribution characteristics of clinical ctDNA, which shows that the male and female quality control of the MRD ctDNA prepared by the method can simulate the ctDNA, and overcomes the defect that the current MRD quality control product prepared by an exogenous introduction mode cannot simulate the ctDNA. The detection results of the quality control product Agilent TapeStation 4150 prepared in the embodiment are shown in fig. 3 to 6.
B. The spectrophotometric concentration measurement results of the quality control product prepared in this example are shown in table 8. The result is that the range of 17.ltoreq.X.ltoreq.23 ng/. Mu.L is satisfied. The concentration setting range can facilitate clinical examination of 15-30 ng for single use and ctDNA examination.
C. The digital PCR results of the quality control prepared in this example are shown in table 9. As a result, the result range of MRD ctDNA AF was 0.2% -2.7%, and the number of gene mutation sites in the AF range of 0.1% -1.0% was 82% of the total number; the detection data of the EGFR L861Q and the TPM3-NTRK1 in the AF0.5%,0.05% and 0.005% quality control are calculated, and the calculation result shows that the ultra-low frequency quality control product can be obtained by a gradient dilution mode; the measured AF of the negative quality control product is less than 0.005 percent. The result shows that the number of gene mutations of the MRD ctDNA quality control product accurately quantified by digital PCR and conforming to clinical detection sites is up to 37, and the defect of few detection sites of the MRD ctDNA quality control product in the current market can be overcome, so that different requirements of stages from platform development verification to clinical detection quality control and the like of the MRD can be met.
D. The results of the negative quality control and AF0.5% positive quality control prepared in this example, verified by TruSight Oncology ctDNA panel, for 37 sites are summarized in Table 9. The detection result of the NGS mutation frequency of 90% of the gene mutation sites is identical to the digital PCR result, and is in the same order of magnitude. The results further indicate the accuracy of the quality control. In addition, more than 600 mutation sites of the gene associated with tumor and having mutation frequency in the range of 0 < X < 2 are summarized in Table 10.
TABLE 8 MRD ctDNA quality control concentration
| Quality control product | Negative quality control product | AF0.5% quality control product | AF 0.05% quality control product | AF 0.005% quality control material |
| Concentration (ng/. Mu.L) | 19.8 | 20.9 | 20.5 | 21.3 |
The target concentrations were 20 ng/. Mu.L. In practice it is possible to allow fluctuations within the range without affecting the use. The range is 17 ng/. Mu.L to 23 ng/. Mu.L.
TABLE 9 MRD ctDNA quality control 37 Hot mutation digital PCR and NGS detection results
TABLE 10TSO500 sequencing mutation site detection information
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The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (11)
1. The quality control product of the micro residual focus ctDNA is characterized by comprising fragmented DNA obtained by enzyme digestion and purification of 38 cells, wherein each fragmented DNA contains gene mutation shown in the following table, and the mass parts of each fragmented DNA are shown in the following table:
2. The ctDNA quality control product for minimal residual disease according to claim 1, wherein the gene mutation sites represented by DNA-1 to DNA-37 are gene mutation sites carried by tumor cells themselves or obtained by gene editing.
3. The micro residual focus ctDNA quality control of claim 2, wherein the cells used for DNA-1-37 include, but are not limited to, MDA-MB-134-VI, SNU-C1, SW1116, SW48, MDA-MB-468, MCF7, KATOIII, SW620, SK-LMS-1, a-253, SH-4, SNU-449, SW1088, SK-OV-3, a549, BXPC-3, NCI-H1975, NCI-N87, ME-180, RKO, HCT116, HEK293T, GM12878, and KBM-7; cells used for DNA-38 include, but are not limited to, GM12878, KBM-7, HEK293A, HEK293T, and HCT116.
4. The quality control product according to claim 1, wherein the size of the main band of the fragmented DNA of the DNA-1 to DNA-38 is 144bp to 176bp.
5. The ctDNA quality control product for micro residual lesions according to any one of claims 1 to 4, wherein the mass fraction of each fragmented DNA is as follows:
6. the micro-residual focus ctDNA quality control of any one of claims 1-5, wherein the quality control comprises a positive quality control with a mutation frequency of 0.5%, a positive quality control with a mutation frequency of 0.05%, a positive quality control with a mutation frequency of 0.005%, and a negative quality control; the negative quality control product is fragmented DNA obtained by enzyme digestion and purification of cells without the gene mutation sites of DNA-1 to DNA-37.
7. The preparation method of the ctDNA quality control product for the tiny residual focus is characterized by comprising the following steps:
s1: selecting cells for gene editing; the method specifically comprises the following steps:
(1) Designing specific guide RNA aiming at a target site of a target gene;
(2) Co-transfecting the expression vector of the guide RNA and the single-stranded DNA into a target cell;
(3) Performing single cell cloning;
(4) After cell clone formation, sanger sequencing is carried out to determine that gene editing is successful;
s2: mnase enzyme cutting;
(1) Performing enzyme digestion on the cell nucleus corpuscles by using Mnase enzyme, and then screening and purifying by using magnetic beads;
(2) Measuring the purity;
OD260/280: x is more than or equal to 1.8 and less than or equal to 2.0, and the qualification is judged;
OD260/230: x is more than or equal to 1.5 and less than or equal to 5.0, and the qualification is judged;
(3) Detecting the distribution of DNA fragments;
the main band size is in the range of 144bp-176bp, and the main band size is judged to be qualified;
s3: mixing and diluting fragmented DNA;
the DNA obtained by the enzyme slicing, sectioning and purifying is mixed according to the mass part of claim 1, and a positive quality control product with the gene mutation frequency of 0.5% of each gene mutation target point is obtained; verifying mutation site variation information through digital PCR;
diluting the positive quality control product with the gene mutation frequency of 0.5% by 10 times to obtain a positive quality control product with the gene mutation frequency of 0.05%, and diluting the positive quality control product with the gene mutation frequency of 0.05% by 10 times to obtain a positive quality control product with the gene mutation frequency of 0.005%; the DNA fragment obtained by the enzyme digestion and purification of the cells of the DNA-38 is a negative quality control product after the concentration adjustment; determining the mutation frequency of the gene by digital PCR, and calculating the ratio of the mutation frequency of the gene;
s4: and (5) NGS sequencing verification.
8. The method according to claim 7, wherein the digital PCR verifies mutation site variation information in step S3 as follows:
9. the method according to claim 7, wherein the multi-gene panel high-depth sequencing is performed in step S4, and mutation frequency analysis is performed on the sequencing data, and the detected genes are shown in the following table:
10. a micro-residual focus ctDNA detection kit comprising the micro-residual focus ctDNA quality control product according to any one of claims 1 to 6, or the micro-residual focus ctDNA quality control product prepared by the method according to any one of claims 7 to 9.
11. Use of a ctDNA quality control product containing the minimal residual disease focus according to any one of claims 1 to 6, or a ctDNA quality control product containing the minimal residual disease focus prepared by the method according to any one of claims 7 to 9, or a ctDNA detection kit containing the minimal residual disease focus according to claim 10, wherein the use is selected from any one or more of the following:
(1) Preparing a micro residual focus ctDNA quality control product;
(2) Preparing an application of a reagent for evaluating a ctDNA detection or platform of a tiny residual focus;
(3) Preparing an application of a reagent for calibrating the ctDNA result of the tiny residual focus;
(4) The application of the reagent for preparing and optimally calibrating the ctDNA detection method or system of the tiny residual focus is provided.
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