Extraction method of biological matrix DNA

Abstract

The invention belongs to the technical field of biology, and particularly relates to a biological matrix DNA extraction method which comprises the following steps: 1) taking a biological matrix, adding a solvent for homogenizing, adding a protease K solution for cracking and centrifuging, and taking a supernatant to obtain a biological sample solution; and 2) taking the biological sample solution, the magnetic beads and the eluent obtained in the step 1), and performing nucleic acid extraction in a full-automatic nucleic acid extraction and purification instrument. The extraction method provided by the invention has high extraction efficiency on tissue samples (heart, liver, lung, kidney, brain, muscle, stomach, testis, duodenum and uterus) and secretions (excrement), and can be used for accurate PCR detection.

Description

A method for extracting DNA from a biological matrix Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for extracting DNA from a biological matrix. Background Technology

[0002] The process of isolating and purifying DNA from biological samples. This process involves a series of steps, including cell lysis, release of nucleic acids, separation of DNA from proteins, lipids, and other cellular components, and purification and concentration of the DNA. The extracted DNA can be used in various molecular biology techniques, such as PCR (polymerase chain reaction), sequencing, cloning, and gene expression analysis.

[0003] DNA extraction methods vary depending on the biological matrix, such as blood, tissue, secretions, plants, or microorganisms, to suit the characteristics of the sample and the required DNA quality. For example, extracting DNA from blood typically involves the lysis of red blood cells and the separation of white blood cells, while extracting DNA from soil samples requires additional steps to remove soil particles. The quality of the extracted DNA is also crucial for the success of subsequent experiments; therefore, optimizing extraction methods to obtain high-purity and high-yield DNA is an important aspect of molecular biology research.

[0004] However, there is currently no in-depth research on DNA extraction methods for different animal tissues and their secretions, in order to improve the quality of DNA extraction. Summary of the Invention

[0005] To address the above problems, this invention provides a method for extracting DNA from a biological matrix, comprising the following steps:

[0006] 1) Take the biological matrix, add solvent to homogenize, then add proteinase K solution to lyse, centrifuge, take the supernatant to obtain the biological sample solution;

[0007] 2) Take the biological sample solution, magnetic beads and elution buffer obtained in step 1) and extract nucleic acid in a fully automated nucleic acid extraction and purification instrument.

[0008] Further, the solvent in step 1) is a lysis buffer; the volume-to-mass ratio of the lysis buffer to the biological matrix is ​​20 μL: 10 mg.

[0009] Furthermore, the biological matrix is ​​animal tissue; the animal tissue is heart, liver, lung, kidney, brain, muscle, stomach, testis, duodenum or uterus; the lysis buffer is the lysis buffer provided with the Chemagic NATissue Kit LH reagent kit, or BufferATL.

[0010] Furthermore, the volume ratio of the homogenized animal tissue solution to the proteinase K solution is 400 μL: 15 μL; the lysis temperature is 10–30 °C, and the time is 1 h.

[0011] Furthermore, the volume ratio of the biological sample solution of the animal tissue, the magnetic beads, and the elution buffer is 415 μL: 50 μL: 200 μL.

[0012] Further, the solvent in step 1) is Lysis Buffer; the volume-to-mass ratio of Lysis Buffer to biological matrix is ​​800 μL: 100 mg.

[0013] Furthermore, the biological substrate is animal secretions; the animal secretions are feces.

[0014] Furthermore, the volume ratio of the homogenized animal secretion solution to the proteinase K solution is 390 μL: 10 μL; the lysis conditions are incubation at 70°C and 500 rpm for 10 min, followed by incubation at 95°C for 5 min.

[0015] Furthermore, the volume ratio of the biological sample solution of the animal secretions, the magnetic beads, and the elution buffer is 400 μL: 50 μL: 100 μL.

[0016] Furthermore, the concentration of the proteinase K solution is 20 mg / mL.

[0017] Further, the fully automated nucleic acid extraction and purification instrument described in step 2) is a Perkin Elmer, Chemagic 360; nucleic acid extraction is performed according to the program [Chemagic DNA360 H96 prefilling.che].

[0018] This invention relates to a method for extracting DNA from biological matrices. Through investigation of extraction methods and recovery rates on various matrices in mice, rats, and rhesus monkeys, it was found that the same extraction method yielded varying DNA quality from different animal parts. Some tissue samples, such as the spleen, had significant magnetic bead residue, resulting in low extraction and recovery rates; some tissue samples, such as blood, showed species-specific differences in extraction quality; and some animal secretions, such as urine, could not yield detectable DNA. Therefore, it is necessary to optimize the DNA extraction conditions according to different animal body parts or their secretions to ensure extraction precision and recovery rate.

[0019] Methodological verification revealed that the extraction method of this invention for spleen, whole blood, and urine presents risks in terms of extraction quality, requiring further research and investigation of novel extraction methods. However, for other tissue samples (heart, liver, lung, kidney, brain, muscle, stomach, testis, duodenum, uterus) and secretions (feces), the extraction method of this invention demonstrates high extraction efficiency and can be used for accurate PCR detection.

[0020] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0021] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0022] Figure 1. Typical standard curves for detecting the target gene concentration of linearized plasmid standards in samples from various genera using real-time quantitative PCR.

[0023] Figure 2. Typical amplification curves for detecting the target gene concentration of linearized plasmid standards in samples from various genera using real-time quantitative PCR. Detailed Implementation

[0024] Example 1: DNA Extraction from Tissue Samples

[0025] (1) Tissue homogenization: Resuscitate solid tissue in an ice bath, weigh the tissue, add 3-5 ceramic beads with a diameter of 3 mm, add the lysis buffer provided in the Chemagic NATissue Kit LH kit at a ratio of 1:20 (m / v), homogenize in a homogenizer for 30 s, pause for 10 s, repeat 3-6 times, centrifuge briefly, collect 400 μL of sample at the bottom of the tube, add 15 μL of 20 mg / mL proteinase K solution, mix well, and incubate at 10-30℃ for 1 h to obtain tissue homogenate;

[0026] (2) Extraction on the instrument: Add 415 μL of the tissue homogenate prepared in step (1) to the wells of the sample plate of the fully automated nucleic acid extraction and purification instrument (Perkin Elmer, Chemagic360), add 50 μL of magnetic beads to the magnetic bead plate, add 200 μL of the DNA extraction kit (Chemagic NATissue Kit LH) elution buffer to the elution plate, and select the program [ChemagicDNA360 H96 prefilling.che] for nucleic acid extraction.

[0027] Example 2 DNA Extraction from Fecal Samples

[0028] (1) Fecal treatment: Weigh the fecal sample in a centrifuge tube, add Lysis Buffer according to the weight at a ratio of 100mg:800μL, vortex to mix, collect 390μL of sample to the bottom of the tube, add 10μL of 20mg / mL proteinase K solution, incubate at 70℃ and 500rpm for 10min, then incubate at 95℃ for 5min, centrifuge at 13000rpm for 5min, take the supernatant to obtain the fecal sample solution.

[0029] (2) Extraction on the instrument: Add 400 μL of the fecal sample solution obtained in step (1) to the sample plate wells of the fully automated nucleic acid extraction and purification instrument (Perkin Elmer, Chemagic360), add 50 μL of magnetic beads to the magnetic bead plate, add 100 μL of the DNA extraction kit (Chemagic NATissue Kit LH) elution buffer to the elution plate, and select the program [Chemagic DNA360H96 prefilling.che] for nucleic acid extraction.

[0030] The following experimental examples further illustrate the beneficial effects of the present invention.

[0031] Experimental Example 1: Efficiency Study of DNA Extraction Method in Biological Matrix of the Present Invention

[0032] 1. Main instruments and equipment

[0033] 1.1 Main instruments, equipment, and instruments

[0034]

[0035]

[0036] 1.2 Main Software / Data Systems Used in the Experiment

[0037] Software / Data System Version Number Purpose SkanIt DDEABIQ5 QuantStudio TM Design & Analysis SE Software v1.6.0 for Real-Time PCR Reaction Data Acquisition and Analysis; Microsoft Office Excel 2010 for Data Entry and Statistical Analysis. surface

[0038] 2. Methods and Materials

[0039] 2.1 Information on Standard Reference Materials

[0040] Name or code: Linear plasmid standard, concentration: 2.00 × 10⁻⁶ 9copies / μL; Source: Suzhou Genewiz Biotechnology Co., Ltd.;

[0041] 2.2 Standard Stock Solution

[0042] Solvent: EASYDilution (forReal Time PCR);

[0043] Preparation method: Take one branch of linear plasmid standard and dilute it to 5.00 × 10⁻⁶ with EASYDilution (for Real Time PCR). 7 Copies / μL are stored as standard stock solutions for future use.

[0044] 2.3 Main Reagents

[0045] Reagent Name Manufacturer Batch Number *bGH-Fwd Sangon Biotech 3200084248 *bGH-Rwd Sangon Biotech 3200084249 *bGH-Probe Sangon Biotech 3200084250 TaqMan TM Fast Advanced master mixThermo Fisher91250154 surface

[0046] Note: *Primers / probes are stored as dry powder. When using, reconstitute them with deionized water to a final concentration of 10 μM.

[0047] 2.4 Blank matrix and blank DNA: The tissues, secretions and whole blood were obtained from the blank matrix library of Chengdu Huaxi Haiqi Pharmaceutical Technology Co., Ltd. (Tianfu New Drug Research Center). They were stored at -66℃ and below for later use. When used, DNA was extracted and the extracted blank DNA was stored at -66℃ and below for later use.

[0048] 3. Test Content

[0049] The extraction methods and recovery rates of various matrices from mice, rats, and rhesus monkeys were investigated.

[0050] Dilute the standard stock solution to a high concentration (2.00 × 10⁻⁶). 5 copies / μL), low (2.00×10 3Two quality control samples (copies / μL) at two concentrations were added to 100 μL each in tissue homogenate, whole blood, urine, and processed fecal supernatant for nucleic acid extraction. Six samples were prepared for each matrix, and nucleic acid extraction was performed in three batches per well, resulting in three independent and effective analytical batches. After extraction, each sample was set up in two replicates for PCR detection. Simultaneously, high- and low-concentration quality control samples from the same source were used as unextracted samples. These unextracted samples were diluted with blank DNA matrix, with two sets prepared for each concentration. The extracted samples were then diluted with deionized water, and both were diluted at least 5-fold before PCR detection. The recovery rate was calculated using the following formula:

[0051] Recovery rate (Recovery%) = [(Nucleic acid concentration of extracted sample) × Elution volume] / [(Nucleic acid concentration of unextracted sample) × Original addition volume] × 100%

[0052] Acceptable recovery criteria: The recovery precision (CV%) of at least 2 / 3 of the samples for each matrix should be within 30.0%.

[0053] 4. Test Methods

[0054] 4.1 DNA Sample Extraction

[0055] 4.1.1 DNA extraction from tissue samples

[0056] (1) Tissue homogenization: Resuscitate solid tissue in an ice bath, weigh the tissue, add 3-5 ceramic beads with a diameter of 3 mm, and homogenize at a ratio of 1:20 (m / v, i.e., tissue weight / lysis buffer volume = 20 mg / 400 μL) (the lysis buffer is the lysis buffer provided in the Chemagic NA Tissue Kit LH kit, or BufferATL provided by QIAGEN; the lysis buffer used in this experiment is the lysis buffer provided in the Chemagic NA Tissue Kit LH kit). If the sample volume is less than 20 mg, add 400 μL of lysis buffer directly. Use a homogenizer (Speed ​​selected 3-4 m / s) to homogenize for 30 s, pause for 10 s, and perform 3-6 cycles in total. Centrifuge briefly and collect the sample at the bottom of the tube.

[0057] (2) Lysis: Add 15 μL of reconstituted proteinase K solution to the homogenized sample tube (reconstitution method: take one vial of proteinase K dry powder, add 2500 μL of sterile deionized water to dissolve and mix well to obtain a proteinase K solution with a concentration of 20 mg / mL), mix well and let stand at 10-30℃ for 1 h for lysis.

[0058] (3) Sample loading: Add samples according to the sample arrangement order of the pre-defined 96-well plate arrangement diagram, add 415 μL of the lysed tissue homogenate, and try to transfer the sample to the bottom of the sample loading well;

[0059] (4) Adding magnetic beads: Add 50 μL of extraction magnetic beads to the corresponding positions of the wells according to the pre-defined 96-well plate arrangement diagram.

[0060] (5) Elution buffer addition: Add 200 μL of elution buffer to the corresponding position of the 96-well plate according to the pre-defined 96-well plate arrangement diagram.

[0061] (6) Extraction on the machine: Place the magnetic rod sleeve in position 1, the magnetic bead plate in position 2, the sample plate in position 3, empty deep-well plates in positions 4-7 respectively, and the elution buffer (the elution buffer that comes with the DNA extraction kit (Chemagic NA Tissue Kit LH)) in position 8. Select the program

Chemagic DNA 360H96prefiling.che

[0062] (7) DNA concentration determination: Add 3 μL of the extracted DNA sample to μDrop Duo Plates and use a multi-functional microplate reader (software: SkanIt Software (Drug Discovery Edition) / v5.0) to determine the DNA concentration by selecting the program

ThermoScientificμDrop Plates

[0063] (8) DNA preservation: DNA is preserved at -66℃ or below for testing.

[0064] 4.1.2 DNA extraction from whole blood and urine samples

[0065] (1) Sample addition: Add samples according to the pre-defined 96-well plate arrangement diagram and sample arrangement order diagram. After the whole blood and urine have returned to room temperature, add 100 μL to the sample plate and then add 10 μL of reconstituted proteinase K.

[0066] (2) Adding magnetic beads: Add 50 μL of extraction magnetic beads to the corresponding positions of the well plate according to the pre-defined 96-well plate arrangement diagram.

[0067] (3) Elution buffer addition: Add 100 μL of elution buffer to the corresponding position of the 96-well plate according to the pre-defined 96-well plate arrangement diagram.

[0068] (4) Extraction on the instrument: Place the magnetic rod sleeve in position 1, the magnetic bead plate in position 2, the sample plate in position 3, empty deep well plates in positions 4-7 respectively, and the elution buffer in position 8. Select the program

Chemagic DNA 360H96 prefiling.che

[0069] (5) DNA concentration determination: Take 3 μL of the extracted DNA sample and add it to μDrop Duo Plates. Use a multi-functional microplate reader (software: SkanIt Software (Drug Discovery Edition) / v5.0) and select the program

ThermoScientificμDrop Plates

[0070] (6) DNA preservation: DNA is preserved at -66°C or below for testing.

[0071] 4.1.3 DNA extraction from fecal samples

[0072] (1) Sample processing: Weigh the collected fecal samples in centrifuge tubes, add Lysis Buffer according to the weight at a ratio of 100mg:800μL, vortex to mix, add 10ul proteinase K, incubate at 70℃ and 500rpm for 10min, then incubate at 95℃ for 5min, and centrifuge at 13000rpm for 5min.

[0073] (2) Sample addition: Add the sample according to the pre-defined 96-well plate arrangement diagram, and aspirate 400 μL of supernatant to transfer the sample to the bottom of the well.

[0074] (3) Adding magnetic beads: Add 50 μL of extraction magnetic beads to the corresponding positions of the wells according to the pre-defined 96-well plate arrangement diagram.

[0075] (4) Elution buffer addition: Add 100 μL of elution buffer to the corresponding position of the 96-well plate according to the pre-defined 96-well plate arrangement diagram.

[0076] (5) Extraction on the instrument: Place the magnetic rod sleeve in position 1, the magnetic bead plate in position 2, the sample plate in position 3, empty deep well plates in positions 4-7 respectively, and the elution buffer in position 8. Select the program

Chemagic DNA 360H96 prefiling.che

[0077] (6) DNA concentration determination: Take 3 μL of the extracted DNA sample and add it to μDrop Duo Plates. Use a multi-functional microplate reader (software: SkanIt Software (Drug Discovery Edition) / v5.0) and select the program

ThermoScientificμDrop Plates

[0078] (7) DNA preservation: DNA is preserved at -66℃ or below for testing.

[0079] The specific arrangement diagram above is as follows:

[0080] 010203040506070809101112ANSC01-01 Blood, Heart, Liver, Lung, Kidney, Brain, Muscle, Stomach, Testis, Duodenum NSC01-02B Uterus, Feces NSC01-03 / / / / / / / / / / C / / / / / / / / / / / / D / / / / / / / / / / / / E / / / / / / / / / / / / F / / / / / / / / / / / / G / / / / / / / / / / / / H / / / / / / / / / / / / / surface

[0081] 4.2 DNA Sample Dilution: The DNA sample to be tested should be diluted at least 5 times after extraction.

[0082] 4.3 Preparation of Standard Curve and Quality Control Samples

[0083] 4.3.1 Preparation of Standard Curve Samples Prepare the standard curve samples according to the table below. First, add a certain volume of standard diluent (blank DNA diluent) to the centrifuge tube. Then, take the corresponding volume of standard stock solution (concentration: 5.00 × 10⁻⁶). 7 Add copies / μL) of the standard curve sample to centrifuge tubes (see Table 1), vortex to mix, and the standard curve sample is valid for use on the same day it is prepared.

[0084] Table 1. Preparation of Standard Curve Samples

[0085] Solution Name | Amount of Solution Taken (μL) | Amount of Diluent Taken (μL) | Concentration Prepared (copies / μL) | STD1 Standard Stock Solution | 5455.00 × 10 6 STD2STD15455.00×10 5 STD3STD25455.00×10 4 STD4STD35455.00×10 3 STD5STD45455.00×10 2 STD6STD55455.00×10 1 STD7STD65455.00 surface

[0086] 4.3.2 Preparation of quality control samples

[0087] Prepare quality control samples according to Table 2. First, add a certain volume of standard diluent (blank DNA diluent) to the centrifuge tube, then add the corresponding volumes of HQC, A, MQC, B, C, and LQC solutions to the centrifuge tube and vortex to mix. The quality control samples are valid for use on the same day they are prepared.

[0088] Table 2 Preparation of quality control samples

[0089] Solution Name | Amount of Solution Taken (μL) | Amount of Diluent Taken (μL) | Concentration Prepared (copies / μL) | Solution Name | Standard Stock Solution | 5455.00×10 6 HQCULOQ40104.00×10 6 AHQC5952.00×10 5 BA5951.00×10 4 MQCB5451.00×10 3 CMQC5451.00×10 2 LQCC5451.00×10 1 surface

[0090] 4.4 Preparation of Negative (Neg) / Blank Control Samples (NTC)

[0091] NTC uses deionized water instead of samples to detect whether the system is contaminated; Neg is derived from mouse, rat, and rhesus monkey blank DNA diluted to monitor nonspecific amplification, in duplicate.

[0092] 4.5 Sample Preparation

[0093] Take tissue sample homogenate, whole blood, urine, and processed fecal supernatant from section "4.1 DNA Sample Extraction" and prepare a quality control sample (concentration: high (2.00 × 10⁻⁶)). 5 copies / μL), low (2.00×10 3 For organ tissue homogenates, mix at a ratio of 100 μL of quality control sample per 300 μL. For urine and whole blood, mix at a ratio of 100 μL of quality control sample per 100 μL.

[0094] Add 400 μL of prepared tissue homogenate to each well of the tissue sample collection, followed by 15 μL of reconstituted proteinase K. Add 200 μL of whole blood and urine samples to the plate, and 400 μL of fecal samples to the plate, followed by 10 μL of reconstituted proteinase K. Add 50 μL of magnetic beads, 200 μL of tissue sample elution buffer, and 100 μL of whole blood, urine, and fecal samples. Perform nucleic acid extraction using an automated nucleic acid extraction and purification instrument with the program

Chemagic DNA 360H96 prefilling.che

[0095] 4.6 Preparation of the reaction system

[0096] Except for the template, the other components are prepared according to Table 3 below to form the qPCR reaction system. The scale is increased proportionally according to the number of reaction wells. After mixing, the reaction premix is ​​added to the corresponding reaction wells according to the pre-defined 96-well plate arrangement diagram, 15 μL / well.

[0097] Table 3. Preparation of the reaction system

[0098] Component reaction volume TaqMan TM Fast Advanced Master Mix 10.0 μL, L WD (10 μM) 0.8 μL, L WD (10 μM) 0.8 μL, Probe (10 μM) 0.4 μL, Deionized Water 3 μL, DNA Template 5 μL, Total Volume 20 μL surface

[0099] Note: The DNA templates are the standard curve sample, quality control sample, test sample, blank genomic DNA, and deionized water, respectively. When the template is deionized water, it serves as the blank control sample (NTC) for each analytical batch. When the template is blank genomic DNA, it serves as the negative control sample (Neg) for each analytical batch.

[0100] 4.7 Sample addition

[0101] Add the corresponding blank control sample (NTC), negative control sample (Neg), standard curve sample, quality control sample, and test sample to the corresponding wells according to the pre-defined 96-well plate arrangement diagram, 5 μL / well, 2 replicates, cover with membrane, and collect the sample to the bottom of the plate by instant centrifugation.

[0102] 4.8 qPCR amplification reaction

[0103] (1) Set up the PCR reaction amplification program: The PCR amplification program is as follows: Select FAM for the experiment, the amplification volume is 20μL, and the hot cap temperature is 105℃.

[0104]

[0105] (2) Set the corresponding layout according to the pre-defined 96-well plate arrangement diagram.

[0106] (3) Run the PCR reaction amplification program.

[0107] 5. Data Processing and Analysis

[0108] 5.1 Data Calculation

[0109] CT value, CT SD, amplification efficiency, and R value of the standard curve samples obtained from PCR analysis software. 2The standard curve equation includes the slope and intercept (y-int), and the concentration fitting formula for the standard curve is: Conc. = 10(CT value - y-int) / Slope. Sample concentration results are retained to 3 significant figures, and relative error (%RE) and precision (%CV) are retained to 1 decimal place.

[0110] The relative error (%RE), standard deviation (SD), and precision (%CV) values ​​used in this report were calculated using Microsoft Excel 2010 software. The calculation formulas are as follows:

[0111]

[0112] Relative error percentage: Relative error (%RE) = (C t -C n ) / C n ×100% (Ct is the measured concentration, Cn is the theoretical concentration);

[0113] Percentage of coefficient of variation:

[0114]

[0115] 5.2 Data Analysis

[0116] Judgment criteria for the test sample: (1) If the concentration of the test sample is within the range of the standard curve, a quantitative result shall be issued; (2) If the concentration of the test sample is lower than LLOQ, the result shall be expressed as BLQ.

[0117] 6. Test Results

[0118] The extraction and detection of samples from three species were investigated: rhesus monkeys (13 samples, including heart, liver, spleen, lung, kidney, brain, muscle, stomach, testis, duodenum, feces, urine, and whole blood); C57BL / 6J mice (11 samples, including heart, liver, spleen, lung, kidney, brain, testis, uterus, feces, urine, and whole blood); and SD rats (13 samples, including heart, liver, spleen, lung, kidney, brain, muscle, stomach, testis, uterus, feces, urine, and whole blood). Six samples were extracted and detected for each sample. A total of 222 extracted samples were tested, and 74 samples without extraction were also tested.

[0119] The experimental samples involved 10 DNA extraction analysis batches and 33 qPCR detection analysis batches, all of which were valid analysis batches. Typical standard curves and typical amplification curves for the target gene concentration of linearized plasmid standards in the samples determined by qPCR are shown in Figures 1-2. Table 4 summarizes the standard curve fitting parameters for the target gene concentration of linearized plasmid standards in samples from various genera; Tables 5-6 show the statistical results of the standard curve data; Tables 7-8 show the statistical results of the %RE data for quality control samples; Table 9 shows the statistical results of the data for negative control samples; Tables 10-12 show the extraction recovery rate and precision results for each matrix sample; and Table 13 shows the extraction recovery rate and precision (%CV) results for the same matrix for various genera.

[0120] Table 4. Summary of standard curve fitting parameters for detecting the gene concentration of linearized plasmid standards in samples from various genera by real-time quantitative PCR.

[0121]

[0122] Table 5. Standard curve results for detecting the gene concentration of linearized plasmid standards in various genera of samples by real-time quantitative PCR ①

[0123]

[0124] Table 6. Standard curve results for detecting the gene concentration of linearized plasmid standards in various genera of samples by real-time quantitative PCR. ②

[0125]

[0126] Note: " / " indicates no data was calculated.

[0127] Table 7. Statistical analysis of quality control sample data for detecting the gene concentration of linearized plasmid standards in various genera by real-time quantitative PCR ①

[0128]

[0129] Table 8. Statistical analysis of quality control sample data for detecting the gene concentration of linearized plasmid standards in various genera by real-time quantitative PCR.

[0130]

[0131] Note: "*" indicates that the sample's SD > 0.5, which does not meet the acceptance criteria; " / " indicates that no calculation data is available.

[0132] Table 9. Statistics of negative control samples for detecting the gene concentration of linearized plasmid standards in various genera by real-time quantitative PCR.

[0133]

[0134] Note: "NaN" indicates that the instrument displays an "Undetermined" value.

[0135] Table 10. Statistics on extraction recovery rate and precision of target gene concentration of linearized plasmid standard in rhesus monkey samples by real-time fluorescence quantitative PCR.

[0136]

[0137] Note: " / " indicates no data; "*" indicates that the sample values ​​differ significantly and are not included in the calculation.

[0138] Table 11. Statistics on the extraction recovery rate and precision of linearized plasmid standard gene concentration in C57BL / 6J mouse samples by real-time quantitative PCR.

[0139] Notes: " / " indicates no data; "*" indicates large sample value differences and is not included in the calculation; "#" indicates no value was detected in urine samples. Table 12: Statistics on extraction recovery rate and precision of target gene concentration of linearized plasmid standard in SD rat samples by real-time fluorescence quantitative PCR.

[0140]

[0141] Note: " / " indicates no data; "*" indicates that the sample values ​​vary greatly and are not included in the calculation; "#" indicates that no value was detected in the urine sample.

[0142] Table 13 Precision statistics of extraction and recovery rates of linearized plasmid standard gene concentrations in various genera of samples detected by real-time quantitative PCR.

[0143]

[0144] 7. Quality control sample evaluation

[0145] All analytical batches were accompanied by two sets of quality control samples at three levels (low, medium, and high concentrations). At least 67% of the quality control samples and at least 50% of the samples at each concentration in each analytical batch met the accuracy requirements within ±50%. Analytical batches that did not meet the acceptance criteria were rejected. Results showed that the accuracy of the low, medium, and high concentration quality control samples for all analytical batches was within the range of -36.2% to 48.0%, meeting the acceptance criteria. Specific results are shown in Tables 7 and 8.

[0146] 8 Conclusions

[0147] The average recovery rates of rhesus monkey matrix samples ranged from 20.9% to 67.3%, with the lowest recovery rate for whole blood samples (20.9%) and the highest recovery rate for lung samples (67.3%). The precision of the recovery rates ranged from 3.0% to 30.0%.

[0148] The recovery rates of C57BL / 6J mouse matrix samples ranged from 19.2% to 81.4%, with the lowest recovery rate in spleen samples (19.2%) and the highest recovery rate in fecal samples (81.4%). The precision of the recovery rates ranged from 4.7% to 28.0%.

[0149] The recovery rates of the matrix samples from SD rats ranged from 34.3% to 71.3%, with the lowest recovery rate for heart samples (20.9%) and the highest recovery rate for fecal samples (71.3%). The precision of the recovery rates ranged from 3.5% to 29.9%.

[0150] The precision (CV%) of the recovery rate of the same matrix for different species ranged from 10.4% to 53.4%. However, magnetic bead residue after spleen DNA extraction led to significant differences in extraction, with a recovery precision of 48.6%. Whole blood showed significant differences among different species, which may be due to matrix effects. The recovery rate of urine was low for various species, and further improvements in extraction methods are needed.

[0151] The results show that for matrices with severe magnetic bead residue (spleen), matrices with low extraction recovery (less than 30%), and matrices with species differences in extraction quality (whole blood), the loading amount should be strictly limited or the pretreatment conditions should be optimized to ensure extraction precision and recovery rate during sample extraction.

[0152] Because the extraction method of this invention for spleen, whole blood, and urine carries risks to extraction quality, further research and investigation of new extraction methods are needed. For other tissue samples (heart, liver, lung, kidney, brain, muscle, stomach, testis, duodenum, uterus) and secretions (feces), the extraction method of this invention has high extraction efficiency and can be used for accurate PCR detection.

Claims

1. A method for extracting DNA from a biological matrix, characterized in that: The process includes the following steps: 1) Take the biological matrix, add solvent to homogenize, then add proteinase K solution to lyse, centrifuge, take the supernatant to obtain the biological sample solution; 2) Take the biological sample solution, magnetic beads and elution buffer obtained in step 1) and extract nucleic acid in a fully automated nucleic acid extraction and purification instrument.

2. The extraction method according to claim 1, characterized in that: Step 1) The solvent is a lysis buffer; the volume-to-mass ratio of the lysis buffer to the biological matrix is ​​20 μL: 10 mg.

3. The extraction method according to claim 2, characterized in that: The biological matrix is ​​animal tissue; the animal tissue is heart, liver, lung, kidney, brain, muscle, stomach, testis, duodenum or uterus; the lysis buffer is the lysis buffer provided with the Chemagic NATissue Kit LH reagent kit, or Buffer ATL.

4. The extraction method according to claim 3, characterized in that: The volume ratio of the homogenized animal tissue solution to the proteinase K solution was 400 μL: 15 μL; the concentration of the proteinase K solution was 20 mg / mL; the lysis temperature was 10–30 °C, and the time was 1 h.

5. The extraction method according to claim 4, characterized in that: The volume ratio of the biological sample solution, magnetic beads, and elution buffer for the animal tissue was 415 μL: 50 μL: 200 μL.

6. The extraction method according to claim 1, characterized in that: Step 1) The solvent is Lysis Buffer; the volume-to-mass ratio of Lysis Buffer to biological matrix is ​​800 μL: 100 mg.

7. The extraction method according to claim 6, characterized in that: The biological substrate is animal secretions; the animal secretions are feces.

8. The extraction method according to claim 7, characterized in that: The volume ratio of the homogenized animal secretion solution to the proteinase K solution was 390 μL: 10 μL; the concentration of the proteinase K solution was 20 mg / mL; the lysis conditions were incubation at 70°C and 500 rpm for 10 min, followed by incubation at 95°C for 5 min.

9. The extraction method according to claim 6, characterized in that: The volume ratio of the biological sample solution of the animal secretions, magnetic beads, and elution buffer was 400 μL: 50 μL: 100 μL.

10. The extraction method according to claim 1, characterized in that: Step 2) The fully automated nucleic acid extraction and purification instrument used is a Perkin Elmer Chemagic 360; nucleic acid extraction is performed according to the program 【Chemagic DNA 360H96 prefilling.che】.

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