A fluorescent PCR method, kit and reagent

Abstract

The present application relates to a fluorescent PCR method, kit and corresponding detection reagent using white blood cells in blood as templates, which comprises resuspending collected white blood cells in a buffer, and then using the white blood cells as templates for fluorescent PCR. The method solves the problems of long time consumption, high cost and low efficiency caused by nucleic acid extraction from blood when performing fluorescent quantitative PCR amplification on whole blood.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and medicine, specifically to a fluorescent PCR method using crude blood leukocyte extract as a template, as well as a kit and detection reagents. Background Technology

[0002] With the continuous development of PCR technology, it has been widely used in both biological and medical fields. Meanwhile, with the application of PCR technology, quantitative real-time PCR (qPCR) technology has gained widespread popularity. Whether it's PCR or qPCR, the application of these technologies is inseparable from the amplification template, namely nucleic acid. Therefore, nucleic acid extraction or purification is a key point in PCR technology. Currently, commonly used nucleic acid extraction or purification techniques mainly fall into three categories: (1) traditional phenol-chloroform extraction, which takes a long time, about 3-4 hours, and the purified nucleic acid purity is also low; (2) silica gel column extraction, which takes less time than traditional phenol-chloroform extraction, about 1-2 hours, and the purified nucleic acid purity is higher; (3) magnetic bead extraction, which takes less time than traditional phenol-chloroform extraction, about 1-2 hours, and the purified nucleic acid purity is higher. Nevertheless, although silica gel column and magnetic bead methods have improved in terms of time consumption, compared to the subsequent PCR time (about 1 hour), they increase the overall nucleic acid detection time to about twice the time of simple PCR. Therefore, both biological experiments and clinical testing urgently require a simple and efficient method.

[0003] Therefore, some researchers have proposed a direct whole blood PCR method, which involves performing PCR directly on whole blood or its crude extract using Taq polymerase and PCR buffer, which have strong interference resistance. While this method has proven effective in qualitative PCR, significantly shortening nucleic acid detection time, it is not well-suited for quantitative real-time PCR (qPCR). The reason is that qPCR requires the acquisition of fluorescence signals during the PCR process, and heme in blood, due to the absorption of light by its porphyrin ring at 415-430 nm, severely interferes with this process, leading to qPCR failure.

[0004] The presence of heme limits the application of direct quantitative PCR in whole blood. We know that heme is mainly found in red blood cells, while nucleic acids in human blood are mainly found in white blood cells. Therefore, to achieve direct quantitative PCR in whole blood, it is necessary to separate red blood cells from white blood cells. Several studies have reported methods for separating white blood cells, such as Sun Lili and Zhao Yujie. Development of a novel whole white blood cell separation solution [J]. (China Medical Guide, 2008, 5(16):23-25.), but the effect of using white blood cells obtained by this method directly for quantitative PCR is not ideal. Currently, commercially available reagents for separating red blood cells and white blood cells are not only expensive but also relatively complex to operate. Therefore, there is an urgent need for a rapid, effective, and low-cost method to reduce heme in whole blood, thereby supporting direct quantitative PCR in whole blood. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a fluorescent PCR method using leukocytes in blood as a template, comprising obtaining leukocytes from a test sample, performing fluorescent PCR using the leukocytes as a template, wherein the leukocytes used as the template are resuspended in buffer A, and the formulation of buffer A includes:

[0006] Tris-HCl: 5-8 mmol / L

[0007] EDTA: 0.5-1.5 mmol / L

[0008] NP-40: 0.5-1%

[0009] BSA: 0.1-0.5 mg / ml

[0010] Furthermore, the method for preparing leukocytes as templates includes the following steps:

[0011] (1) Add a red blood cell rupture reagent to a whole blood sample to rupture the red blood cells and release hemoglobin;

[0012] (2) Centrifuge the treatment solution obtained in step (1) to precipitate white blood cells and remove the supernatant;

[0013] (3) Resuspend the white blood cells in buffer solution A.

[0014] Further, add 30-100 μl of buffer A solution to resuspend the white blood cells.

[0015] The red blood cell lysis reagent is selected from TE or purified water. The DNA polymerase is selected from rTaq polymerase.

[0016] More specific fluorescent PCR methods, including

[0017] (1) Add 1 ml of TE solution or purified water to 20-100 μl of whole blood, vortex for 30 seconds to cause red blood cells to rupture and release hemoglobin;

[0018] (2) Centrifuge at 12000×g for 1 min to allow white blood cells to settle, and remove the supernatant;

[0019] (3) Resuspend the white blood cells in 30-100 μl of buffer A solution;

[0020] (4) Prepare the fluorescent PCR system;

[0021] (5) Place the prepared reaction system in a real-time PCR instrument for reaction, and analyze the results after the reaction is completed.

[0022] Furthermore, the fluorescent PCR system includes:

[0023] 10×PCR buffer: 2 μl

[0024] dNTP (10mM): 0.2-1μl

[0025] MgCl2 (25mM): 0-5μl

[0026] DNA polymerase: 1-3 U

[0027] Upstream primer (10 μM): 0.2-1 μl

[0028] Downstream primer (10 μM): 0.2-1 μl

[0029] Probe 1 (10μm): 0.2-1.2μl

[0030] Probe 2 (10μm): 0.2-1.2μl

[0031] Template: 1-5μl

[0032] Purified water: Add to 20 μl.

[0033] This invention also provides a fluorescent PCR kit using leukocytes in blood as a template, comprising leukocyte resuspension buffer A, wherein the formulation of buffer A includes:

[0034] Tris-HCl: 5-8 mmol / L

[0035] EDTA: 0.5-1.5 mmol / L

[0036] NP-40: 0.5-1%

[0037] BSA: 0.1-0.5 mg / ml.

[0038] The kit also includes a red blood cell lysis reagent, which is selected from TE or purified water.

[0039] Furthermore, the kit also includes a DNA polymerase selected from rTaq polymerase.

[0040] The present invention also provides a buffer solution for eliminating the inhibitory effect of residual heme on fluorescent PCR, wherein the buffer solution formulation comprises: Tris-HCl, EDTA, NP-40 and BSA.

[0041] More specifically, the buffer formulation includes:

[0042] Tris-HCl: 5-8 mmol / L

[0043] EDTA: 0.5-1.5 mmol / L

[0044] NP-40: 0.5-1%

[0045] BSA: 0.1-0.5 mg / ml.

[0046] The advantages of this invention are: It provides a rapid, efficient, and low-cost method for direct fluorescent PCR of blood, solving the problems of long processing times, high costs, and low efficiency associated with existing techniques for quantitative PCR amplification of whole blood, which require nucleic acid extraction. This invention also addresses the issue of significant heme-related interference when using leukocytes as a template for fluorescent PCR detection. Furthermore, the method for obtaining leukocytes is simple, shortening the entire fluorescent PCR detection cycle and improving detection efficiency. Attached Figure Description

[0047] Figure 1 Example 1: Amplification results of ARB1 wild-type sample.

[0048] Figure 2 Example 1: Amplification results of ADRB1 mutant sample.

[0049] Figure 3 Example 1: Amplification results of ADRB1 heterozygous samples.

[0050] Figure 4 Example 2: Amplification results of ADRB1 wild-type samples using the method of the present invention.

[0051] Figure 5 Example 2: Amplification results of the ADRB1 mutant sample using the method of the present invention.

[0052] Figure 6 Example 2: Amplification results of the ADRB1 heterozygous sample using the method of the present invention.

[0053] Figure 7 Example 2: Detection results of ADRB1 wild-type samples using Sanger sequencing.

[0054] Figure 8 Example 2: Detection results of ADRB1 mutant samples using Sanger sequencing.

[0055] Figure 9 Example 2: Detection results of ARB1 heterozygous samples using Sanger sequencing.

[0056] Figure 10 Example 2: Amplification results of ALDH2 wild-type samples using the method of the present invention.

[0057] Figure 11 Example 2: Amplification results of the ALDH2 mutant sample using the method of the present invention.

[0058] Figure 12 Example 2: Amplification results of the method of the present invention on ALDH2 heterozygous samples.

[0059] Figure 13 Example 2: Detection results of ALDH2 wild-type samples using Sanger sequencing.

[0060] Figure 14 Example 2: Detection results of ALDH2 mutant samples using Sanger sequencing.

[0061] Figure 15 Example 2: Detection results of ALDH2 heterozygous samples using Sanger sequencing.

[0062] Figure 16 Example 3: Amplification results of hemoglobin as an interfering agent.

[0063] Figure 17 Example 3: Amplification results of bilirubin as an interfering agent.

[0064] Figure 18 Example 3: Amplification results with cholesterol as an interfering agent.

[0065] Figure 19 Example 3: Amplification results using triglycerides as an interfering agent.

[0066] Figure 20 Amplification results of sample 1 in Example 4.

[0067] Figure 21 Amplification results of sample 2 in Example 4.

[0068] Figure 22 Amplification results of sample 3 in Example 4.

[0069] Figure 23 Amplification results of sample 4 in Example 4.

[0070] Figure 24 Amplification results of sample 5 in Example 4.

[0071] Figure 25 Amplification results of sample 6 in Example 4.

[0072] Figure 26 The amplification results of sample number 13 in Example 2 of Example 5.

[0073] Figure 27 The amplification results of sample number 14 in Example 2 of Example 5.

[0074] Figure 28 The amplification results of sample number 24 in Example 2 of Example 5. Detailed Implementation

[0075] Example 1: Fluorescent PCR experiments were conducted using samples obtained by different extraction methods as templates.

[0076] For fluorescent PCR detection of blood samples, the method of this invention only requires simple pretreatment of the blood sample to isolate leukocytes, which can then be used as templates for fluorescent PCR. Traditional methods first extract DNA from the sample, and then use the extracted DNA as a template for fluorescent PCR detection. There are three main types of traditional sample extraction methods: organic reagent extraction, silica gel column extraction, and nanomagnetic microsphere extraction. This embodiment uses leukocytes extracted by the method of this invention and DNA extracted by traditional methods as templates, and conducts a comparative experiment using fluorescent PCR, as detailed below:

[0077] I. Experimental Methods

[0078] (I) Sample processing

[0079] 1. The method of this invention for extracting leukocytes

[0080] (1) Take 80 μl of EDTA-anticoagulated whole blood into a 1.5 ml centrifuge tube, add 1 ml of purified water, and vortex for 30 s.

[0081] (2) Centrifuge at 12000×g for 1 min to allow white blood cells to settle and remove the supernatant.

[0082] (3) Resuspend the white blood cells in 50 μl of buffer A solution. The formulation of buffer A is as follows:

[0083] Tris-HCl: 5.5 mmol / L

[0084] EDTA: 0.5 mmol / L

[0085] NP-40: 0.5%

[0086] BSA: 0.1 mg / ml.

[0087] 2. DNA extraction using organic reagent extraction method

[0088] (1) Take 3 ml of EDTA-anticoagulated whole blood into a 50 ml centrifuge tube, add 30 ml of purified water, shake for 20 s, let stand for 10 min, and then centrifuge at 3800 rpm for 20 min at 4℃. Carefully remove the supernatant.

[0089] (2) Add 5 ml of TES to the precipitate and mix by inverting.

[0090] (3) Add 350 μl of 10% SDS and 700 ng of proteinase K, and lyse overnight at 37°C.

[0091] (4) After the centrifuge tube has cooled to room temperature, add 5 ml of Tris-saturated phenol, mix by inversion, then add 5 ml of chloroform-isoamyl alcohol (24:1), mix by inversion, and centrifuge at 2500 rpm for 15 min at 4°C.

[0092] (5) Take all the supernatant into another centrifuge tube, add 2.5 times the volume of ice-cold anhydrous ethanol, invert repeatedly, centrifuge at 12000 rpm for 10 min, and collect the supernatant.

[0093] (6) Wash twice with 75% ethanol, air dry, and then add 200 μl of TB solution to dissolve the precipitate.

[0094] 3. DNA extraction using silica gel column extraction method

[0095] The product manufactured by Hangzhou Baimai Biotechnology Co., Ltd. is called " The "Rapid Blood Genomic DNA Small-Scale Preparation Kit" has the following specific steps:

[0096] (1) Add 500 μl of buffer solution X1 to a 1.5 ml centrifuge tube.

[0097] (2) Add 200 μl of anticoagulated whole blood to buffer X1, and pipette back and forth several times to completely dissolve any blood remaining on the pipette tip. Tighten the cap of the centrifuge tube and vortex for 10 seconds.

[0098] (3) Add 100 μl buffer X2 and vortex for 10 s.

[0099] (4) Centrifuge at 12,000×g for 10 min.

[0100] (5) Place the preparation tube into a 2ml centrifuge tube, add the filtrate from step 4 into the preparation tube, and centrifuge at 12,000×g for 1min.

[0101] (6) Discard the filtrate, place the preparation tube back into the original 2ml centrifuge tube, add 700μl Wash 1B, and incubate at room temperature for 2min. Centrifuge at 12,000×g for 30s.

[0102] (7) Discard the filtrate, put the preparation tube back into the original 2ml centrifuge tube, add 800μl of Wash 2A with anhydrous ethanol, and centrifuge at 12,000×g for 1min.

[0103] (8) Place the preparation tube back into the original 2ml centrifuge tube, add 500μl Wash 2A to the preparation tube, and centrifuge at 12,000×g for 1min.

[0104] (9) Discard the filtrate, put the preparation tube back into the original 2ml centrifuge tube, and centrifuge at 12,000×g for 1min.

[0105] (10) Place the preparation tube into another clean 1.5 ml centrifuge tube (provided in the kit), add 100 μl of Eluent C to the center of the membrane in the preparation tube, and let stand at room temperature for 1 min. Elute the genomic DNA by centrifuging at 12,000 × g for 1 min.

[0106] 4. DNA extraction using nanomagnetic microspheres

[0107] MagPure, produced by Hangzhou Baimai Biotechnology Co., Ltd. TM - Magnetic Bead Blood Genome Small-Scale Extraction Kit, the specific steps are as follows:

[0108] (1) Add 500 μl of buffer solution X1 to a 1.5 ml centrifuge tube.

[0109] (2) Add 200 μl of anticoagulated whole blood to buffer X1 and vortex vigorously for 2 min.

[0110] (3) Add 100 μl of buffer MX2 and vortex vigorously for 2 min. Centrifuge at 120,00Xg for 5 min. Carefully aspirate the supernatant from the centrifugation into a clean 2 ml centrifuge tube.

[0111] (4) Add 300 μl Beads A and 550 μl isopropanol to the supernatant and vortex for 10 min to make the liquid completely vortexed.

[0112] (5) Place the centrifuge tube on the magnetic rack and wait for the supernatant to clear. Carefully aspirate the supernatant, being careful not to remove the magnetic beads.

[0113] (6) Remove the magnetic rack, add 500 μl Wash 1B, and vortex for 1 min to make all the liquid in the tube vortex.

[0114] (7) Place the centrifuge tube on the magnetic rack and wait for the supernatant to clear. Carefully aspirate the supernatant, being careful not to remove the magnetic beads.

[0115] (8) Repeat steps (6)-(7).

[0116] (9) Remove the magnetic rack, add 500 μl Wash 2A, and vortex for 1 min to make all the liquid in the tube vortex.

[0117] (10) Place the centrifuge tube on the magnetic rack and wait for the supernatant to clear. Carefully aspirate the supernatant, being careful not to remove the magnetic beads.

[0118] (11) Repeat steps (9)-(10).

[0119] (12) Remove the magnetic rack and place the centrifuge tubes in a 65℃ oven to dry for 10-15 minutes.

[0120] (13) Add 100 μl of Eluent B and vortex for 5 min to resuspend the magnetic beads.

[0121] (14) Place the centrifuge tube on a magnetic rack and let it stand at room temperature for 1 minute or until the supernatant is clear. Carefully aspirate the supernatant along the tube wall opposite the magnetic beads to the test plate and store it. The supernatant contains the purified blood genomic DNA.

[0122] (II) Fluorescent PCR Method

[0123] Fluorescent PCR was used for SNP genotyping at the c.1165 site of the ADRB1 gene. The primer and probe sequences required for fluorescent PCR are as follows:

[0124] ADRB1 upstream primer: 5'-GGCCTTCAACCCCATCATCTA-3'

[0125] ADRB1 downstream primer: 5'-CCGGTCTCCGTGGGTCGCGT-3'

[0126] ADRB1 probe 1: 5'-FAM-CCTTCCAGGGACTGCTCT-MGBNFQ-3'

[0127] ADRB1 probe 2: 5'-VIC-CCTTCCAGCGACTGCTCT-MGBNFQ-3'

[0128] 1. Prepare the fluorescent PCR system (dual probe) as follows:

[0129] Table 1 Fluorescent PCR System

[0130] 10×PCR buffer 2μl dNTPs (10mM) 0.5μl <![CDATA[MgCl2(25mM)]]> 2μl DNA polymerase 1U Upstream primer (10 μm) 1μl Downstream primer (10 μm) 1μl Probe 1 (10μm) 0.8μl Probe 2 (10μm) 0.8μl template* See the table below for details. Purified water Add to a total volume of 20 μl

[0131] *Different methods require different template volumes due to variations in the initial sample and final product volumes. To minimize errors in result comparison caused by differences in the amount of template added to the PCR system, sample addition was performed according to Table 2. The template volume was calculated as follows: Using this invention as a reference, an 80 μL initial sample was resuspended in 50 μL, resulting in a sample concentration of 80 / 50. The template volume added to the PCR system was 2 μL, indicating that the added template volume was 80 / 50 * 2 = 3.2 μL. All methods used a template volume of 3.2 μL. Taking the organic reagent extraction method as an example, with a sample concentration of 3000 / 200, the template volume added to the PCR system should be 3.2 / (3000 / 200) = 0.213333 μL. And so on.

[0132] Table 2 Sample Dosage

[0133]

[0134] 2. Place the prepared reaction system in a real-time PCR instrument for reaction, and analyze the results after the reaction is complete.

[0135] Fluorescent PCR amplification procedure:

[0136]

[0137] II. Comparative Experiment

[0138] Following the experimental method described above, fluorescent PCR amplification was performed on three samples of different genotypes: Sample 1 (ADRB1 wild-type), Sample 2 (ADRB1 mutant), and Sample 3 (ADRB1 heterozygous). The following indicators were compared:

[0139] (I) Comparison of amplification performance

[0140] The amplification signals from the four methods described above were analyzed, and the results are shown in the appendix. Figure 1 (ADRB1 wild-type sample), attached Figure 2 (ADRB1 mutant sample), attached Figure 3 (ADRB1 heterozygous sample) is shown. As can be seen from the figure, the method of the present invention has the same effect as DNA extraction (silica gel column extraction method) and DNA extraction (nanomagnetic microsphere extraction method), and is significantly better than DNA extraction (organic reagent extraction method).

[0141] Table 3 Comparison of Ct values ​​amplified by different methods

[0142]

[0143] The Ct values ​​of the four fluorescent PCR methods described above were analyzed and compared, as detailed in Table 3. Generally, a smaller Ct value indicates a better amplification effect. The results in this table show that, regardless of whether the sample is wild-type, mutant, or heterozygous, the method of this invention has the same effect as DNA extraction (silica gel column extraction) and DNA extraction (nanomagnetic microsphere extraction), and is significantly superior to DNA extraction (organic reagent extraction).

[0144] (II) Comparison of operation time

[0145] In the application of molecular biology detection methods, especially in clinical testing, timeliness is an important evaluation factor. Therefore, the operation time of the method of this invention was compared with several other methods, and the results are shown in Table 4.

[0146] Table 4 Comparison of operation time for different methods

[0147] Leukocyte extraction (method of this invention) 5min 55min 60min DNA extraction (organic reagent extraction method) 540min 55min 595min DNA extraction (silica gel column extraction method) 30min 55min 85min DNA extraction (nanomagnetic microsphere extraction method) 50min 55min 105min

[0148] Example 2: Accuracy of Genotyping Using Templates Extracted by the Method of the Present Invention

[0149] The method of this invention was used to detect different gene loci (ADRB1 and ALDH2 genes) in samples, and the results were compared with the Sanger sequencing method, the "gold standard" for genotyping, to analyze its accuracy. The primer and probe sequences for ADRB1 and ALDH2 are as follows:

[0150] artificial synthesis

[0151] ADRB1 upstream primer: 5'-GGCCTTCAACCCCATCATCTA-3'

[0152] ADRB1 downstream primer: 5'-CCGGTCTCCGTGGGTCGCGT-3'

[0153] ADRB1 probe 1: 5'-FAM-CCTTCCAGGGACTGCTCT-MGBNFQ-3'

[0154] ADRB1 probe 2: 5'-VIC-CCTTCCAGCGACTGCTCT-MGBNFQ-3'

[0155] ALDH2 upstream primer: 5'-GGCTACAAGATGTCGGGGAG-3'

[0156] ALDH2 downstream primer: 5'-AGACCCTCAAGCCCCAACA-3'

[0157] ALDH2 probe 1: 5'-FAM-CATACACTGAAGTGAAAA-MGBNFQ-3'

[0158] ALDH2 probe 2: 5'-VIC-GCATACACTAAAGTGAAA-MGBNFQ-3'

[0159] I. Experimental Methods

[0160] (1) Take 80 μl of EDTA-anticoagulated whole blood into a 1.5 ml centrifuge tube, add 1 ml of TE solution, and vortex for 30 s.

[0161] (2) Centrifuge at 12000×g for 1 min to allow white blood cells to settle and remove the supernatant.

[0162] (3) Resuspend the white blood cells in 50 μl of buffer A solution. The formulation of buffer A is as follows:

[0163] Tris-HCl: 6.5 mmol / L

[0164] EDTA: 1.1 mmol / L

[0165] NP-40: 0.85%

[0166] BSA: 0.3 mg / ml.

[0167] (4) Prepare the fluorescent PCR system (dual probe) as follows:

[0168] Table 5 Fluorescent PCR System

[0169]

[0170]

[0171] II. Experimental Results

[0172] After analysis, the results of the ADRB1 gene obtained using the method of this invention and the Sanger sequencing method were interpreted, and the specific statistics are shown in Table 6. Examples of amplification maps for three different genotypes using the method of this invention are detailed in the appendix. Figures 4-6 (One sample from each genotype was selected as an example of an amplification map, in which...) Figure 4 This is the amplification pattern for sample number 1. Figure 5 This is the amplification pattern for sample number 3. Figure 6 (The amplification map of sample number 4) is an example of sequencing maps for three different genotypes using the Sanger sequencing method (one sample from each genotype is selected as the sequencing map example, where...). Figure 7 This is the sequencing map of sample number 1. Figure 8 This is the sequencing map of sample number 3. Figure 9 See attached sequencing map for sample number 4. Figures 7-9 .

[0173] Table 6. Accuracy of ADRB1 genotyping

[0174]

[0175] After analysis, the results of the ALDH2 gene sequencing using the method of this invention and the Sanger sequencing method were interpreted, and the specific statistics are shown in Table 7. Examples of amplification maps for three different genotypes using the method of this invention are detailed in the appendix. Figures 10-12 (One sample from each genotype was selected as an example of an amplification map, in which...) Figure 10 This is the amplification pattern for sample number 6. Figure 11 This is the amplification pattern for sample number 3. Figure 12 (This is the amplification map of sample number 1). See the appendix for examples of sequencing maps for three different genotypes using the Sanger sequencing method. Figures 13-15 (One sample from each genotype was selected as an example of the sequencing profile, in which...) Figure 13 This is the sequencing map of sample number 6. Figure 14 This is the sequencing map of sample number 3. Figure 15 (The sequencing map of sample number 1).

[0176] Table 7. Accuracy of ALDH2 Genotyping

[0177]

[0178] The results above show that, with each gene tested in 30 samples, the detection results of the method of this invention achieved a 100% concordance rate with the results of Sanger sequencing. This demonstrates that the method of this invention has high accuracy.

[0179] Example 3: Anti-interference capability of the method of the present invention

[0180] Traditional methods, due to DNA extraction, can minimize the impact of interfering substances on fluorescent PCR. However, the present method, which directly uses pretreated blood products for fluorescent PCR, raises the question of whether it has the same anti-interference ability as fluorescent PCR using traditional DNA extraction methods. Therefore, the influence of anti-interference substances in blood is investigated.

[0181] Select the sample verified by sequencing in Example 2, and perform the following experiments on the same sample: (1) extract leukocytes using the method of the present invention (wherein the formulation of buffer A used is: Tris-HCl: 8 mmol / L, EDTA: 1.5 mmol / L, NP-40: 1%, BSA: 0.5 mg / ml); (2) extract DNA using the conventional silica column method, and use fluorescent PCR to detect and classify samples with different concentrations of interfering substances.

[0182] Table 8 Fluorescent PCR System

[0183] 10×PCR buffer 2μl dNTPs (10mM) 1μl DNA polymerase 2U Upstream primer (10 μm) 0.6μl Downstream primer (10 μm) 0.6μl Probe 1 (10μm) 1.2μl Probe 2 (10μm) 1.2μl template 2μl Purified water Add to a total volume of 20 μl

[0184] The results showed that, among randomly selected different concentrations of interfering agents, the amplification effects were consistent when using leukocytes extracted by the method of this invention and DNA extracted by the traditional silica column method as templates for fluorescent PCR detection (the amplification results of hemoglobin as an interfering agent are attached). Figure 16 The amplification results for bilirubin as an interfering agent are shown in the appendix. Figure 17 The amplification results for cholesterol as an interfering agent are shown in the appendix. Figure 18 The amplification results for triglycerides as interfering agents are shown in the appendix. Figure 19 The Ct values ​​shown in Table 9 are basically consistent.

[0185] Table 9. Anti-interference capability against different interfering objects

[0186]

[0187] The amplification site in this embodiment is ALDH2, and its primer and probe sequences are as follows:

[0188] ALDH2 upstream primer: 5'-GGCTACAAGATGTCGGGGAG-3'

[0189] ALDH2 downstream primer: 5'-AGACCCTCAAGCCCCAACA-3'

[0190] ALDH2 probe 1: 5'-FAM-CATACACTGAAGTGAAAA-MGBNFQ-3'

[0191] ALDH2 probe 2: 5'-VIC-GCATACACTAAAGTGAAA-MGBNFQ-3'

[0192] Example 4: Resistance to heme interference by the method of the present invention in fluorescent PCR

[0193] In the PCR process using blood, the key lies in reducing the interference of heme in red blood cells, especially in fluorescent PCR, where the acquisition of fluorescence signals is crucial for the success of the PCR. Therefore, evaluating the anti-heme interference capability of the method of this invention in fluorescent PCR is of great significance for the application of this invention in fluorescent PCR.

[0194] To evaluate the effectiveness and practicality of the method of this invention, it was compared with methods reported in the literature, other prior art, and commercially available leukocyte separation solutions. The comparison was performed using amplification of the ALDH2 gene locus; specific primer and probe information is as follows:

[0195] ALDH2 upstream primer: 5'-GGCTACAAGATGTCGGGGAG-3'

[0196] ALDH2 downstream primer: 5'-AGACCCTCAAGCCCCAACA-3'

[0197] ALDH2 probe 1: 5'-FAM-CATACACTGAAGTGAAAA-MGBNFQ-3'

[0198] ALDH2 probe 2: 5'-VIC-GCATACACTAAAGTGAAA-MGBNFQ-3'

[0199] I. Experimental Methods

[0200] (I) Sample processing

[0201] 1. The method of the present invention

[0202] (1) Take 80 μl of EDTA-anticoagulated whole blood into a 1.5 ml centrifuge tube, add 1 ml of purified water, and vortex for 30 s.

[0203] (2) Centrifuge at 12000×g for 1 min to allow white blood cells to settle and remove the supernatant.

[0204] (3) Resuspend the white blood cells in 50 μl of buffer A solution. The formulation of buffer A is as follows:

[0205] Tris-HCl: 7 mmol / L

[0206] EDTA: 1.2 mmol / L

[0207] NP-40: 0.3%

[0208] BSA: 0.2 mg / ml.

[0209] 2. Ficoll whole leukocyte separation solution method

[0210] One of the methods reported in the literature (Sun Lili, Zhao Yujie. Development of a novel whole leukocyte separation solution [J]. China Medical Herald, 2008, 5(16):23-25.) was adopted, namely the Ficoll whole leukocyte separation solution method, the specific contents of which are as follows:

[0211] (1) Prepare Ficoll whole leukocyte separation medium as follows:

[0212] Sucrose: 9%

[0213] Diazometrine: 15.2%

[0214] Sodium chloride: 3 mg / ml

[0215] (2) Take 1 ml of EDTA-anticoagulated whole blood, add an equal amount of EDTA-PBS (0.2 mol / L, pH 7.4) to dilute, and slowly add it to the surface of 2 ml of the above Dextran whole leukocyte separation solution along the wall of the centrifuge tube.

[0216] (3) 657×g, centrifuged for 20min.

[0217] (4) The milky white, cloud-like layer between the separated liquids is the white blood cell layer.

[0218] (5) Wash twice with EDTA-PBS and resuspend the white blood cells in 625 μl TE solution.

[0219] 3. Anti-interference liquid using existing technology

[0220] The anti-interference solution contains: 10 mM Tris-HCl, 0.2 mM Na2EDTA, 0.01% (v / v) Triton X-100, 0.06% (v / v) allyl isothiocyanate, pH 8.6. The allyl isothiocyanate content is 0.04%-0.06%, preferably 0.06%.

[0221] The specific sample processing methods are as follows:

[0222] (1) Take 80 μl of EDTA-anticoagulated whole blood, add 2 ml of double-distilled water to rupture red blood cells, centrifuge at 3500 rpm for 5 min, and discard the supernatant.

[0223] (1) Add 50 μl of anti-interference solution (the composition of the anti-interference solution is: 10 mM Tris-HCl, 0.2 mM Na2EDTA, 0.01% (V / V) Triton X-100, 0.06% (V / V) allyl isothiocyanate, pH 8.6) and mix for 2 min.

[0224] 4. Commercially available leukocyte separation solution

[0225] The blood was processed using Solarbio's Human Peripheral Blood Leukocyte Separation Kit (trade name: Human Peripheral Blood Leukocyte Separation Kit, catalog number P8670), as follows:

[0226] (1) Take 1 ml of EDTA anticoagulated whole blood, mix it with whole blood and tissue diluent at a ratio of 1:1, and carefully add it to the surface of the separation solution.

[0227] (2) Centrifuge at 500×g for 25 min and separate into layers.

[0228] (3) Discard the first plasma layer, collect the second white blood cell layer, the third separation fluid layer and the fourth red blood cell layer, put them into a test tube containing 10 ml of cell washing solution and mix thoroughly.

[0229] (4) Centrifuge at 500×g for 30 min and discard the supernatant.

[0230] (5) Add 10 times the volume of red blood cell lysis buffer to lyse the red blood cells, gently pipette to mix, and lyse for 1-2 min. Centrifuge at 500×g for 5 min and discard the supernatant.

[0231] (6) Add 1 ml PBS, wash the white blood cells, centrifuge at 500 × g for 3 min, and discard the supernatant. Wash a total of 3 times.

[0232] (7) Add 625 μl of TE for resuspension.

[0233] (II) Fluorescent PCR Detection

[0234] (1) Prepare the following fluorescent PCR system:

[0235] 10×PCR buffer 2μl dNTPs (10mM) 1μl DNA polymerase 3U Upstream primer (10 μm) 0.4μl Downstream primer (10 μm) 0.4μl Probe 1 (10μm) 0.5μl Probe 2 (10μm) 0.5μl template 2μl Purified water Add to a total volume of 20 μl

[0236] (2) Add 2 μl of leukocyte suspension obtained by different sample processing methods.

[0237] (3) Place the prepared reaction system in a real-time PCR instrument for reaction, and analyze the results after the reaction is completed.

[0238] II. Result Comparison

[0239] The interference resistance of the four methods mentioned above to heme in fluorescent PCR was analyzed. Generally speaking, in equal amounts of amplification template, the stronger the resistance to heme interference, the smaller the Ct value, and the stronger the fluorescent amplification signal.

[0240] Six samples were selected from those verified by sequencing in Example 2. Samples 1 to 6 were samples numbered 4, 6, 11, 12, 19, and 27 in Example 2, respectively. The Ct values ​​of the samples were compared using four different fluorescent PCR methods. See the attached diagram for detailed amplification signal images. Figure 20-25 The results show that the method of this invention has the same effect as commercially available leukocyte separation reagents. Compared with the methods described in the above-mentioned literature and other existing technologies, it has significant advantages.

[0241] Table 10 Anti-heme interference ability of different methods

[0242]

[0243] Example 5: Anti-heme interference ability of buffer A

[0244] To demonstrate that the main component of the method for resisting heme interference in this invention is buffer A, the effects of different leukocyte resuspensions were compared. Three samples were selected from Example 2 after sequencing verification, of which samples 7# to 9# were samples 13, 14, and 24 in Example 2, respectively. They were processed and subjected to fluorescent PCR detection according to the following methods.

[0245] (I) Sample processing

[0246] (1) Take 80 μl of EDTA-anticoagulated whole blood into a 1.5 ml centrifuge tube, add 1 ml of purified water, and vortex for 30 s.

[0247] (2) Centrifuge at 12000×g for 1 min to allow white blood cells to settle and remove the supernatant.

[0248] (2) Add 50 μl of leukocyte resuspension solution. Details are as follows:

[0249] 1 Buffer solution A (in this invention) 2 TE 3 Purified water

[0250] The formulation of buffer A is as follows:

[0251] Tris-HCl: 5.5 mmol / L

[0252] EDTA: 0.65 mmol / L

[0253] NP-40: 0.75%

[0254] BSA: 0.25 mg / ml.

[0255] (II) Fluorescent PCR Detection

[0256] The ALDH2 gene was detected using the following primer and probe sequences:

[0257] ALDH2 upstream primer: 5'-GGCTACAAGATGTCGGGGAG-3'

[0258] ALDH2 downstream primer: 5'-AGACCCTCAAGCCCCAACA-3'

[0259] ALDH2 probe 1: 5'-FAM-CATACACTGAAGTGAAAA-MGBNFQ-3'

[0260] ALDH2 probe 2: 5'-VIC-GCATACACTAAAGTGAAA-MGBNFQ-3'

[0261] ① Prepare the following fluorescent PCR system:

[0262]

[0263]

[0264] (III) Comparison of Results

[0265] Generally, in equal amounts of amplification template, the stronger the resistance to heme interference, the smaller the Ct value. Therefore, the Ct values ​​of the two leukocyte resuspensions for fluorescent PCR were compared. The results are shown in the table below.

[0266]

[0267] According to experimental results, the buffer A of the present invention can, to a certain extent, resist the inhibitory effect of residual heme in white blood cells on fluorescent PCR.

[0268]

[0269]

Claims

1. A fluorescent PCR method using leukocytes in blood as a template, comprising obtaining leukocytes from a test sample and performing fluorescent PCR using the leukocytes as a template, characterized in that, Leukocytes, serving as templates, are resuspended in buffer A, which is composed of Tris-HCl: 5-8 mmol / L, EDTA: 0.5-1.5 mmol / L, NP-40: 0.5-1%, and BSA: 0.1-0.5 mg / ml.

2. The fluorescent PCR method according to claim 1, characterized in that, The method for preparing leukocytes as templates includes the following steps: (1) Add a red blood cell rupture reagent to a whole blood sample to rupture red blood cells and release hemoglobin; (2) Centrifuge the treatment solution obtained in step (1) to precipitate white blood cells and remove the supernatant; (3) Resuspend the white blood cells in buffer solution A.

3. The fluorescent PCR method according to any one of claims 1 to 2, characterized in that, Resuspend the white blood cells in 30-100 µl of buffer A solution.

4. The fluorescent PCR method according to claim 2, characterized in that, The red blood cell lysis reagent is selected from TE or purified water.

5. The fluorescent PCR method according to claim 1, characterized in that, (1) Add 1 ml of TE solution or purified water to 20-100 µl of whole blood, vortex for 30 seconds to cause red blood cells to rupture and release hemoglobin; (2) Centrifuge at 12000×g for 1 min to allow white blood cells to settle, and remove the supernatant; (3) Resuspend the white blood cells in 30-100 µl of buffer A solution; (4) Prepare the fluorescent PCR system; (5) Place the prepared reaction system in a real-time PCR instrument for reaction, and analyze the results after the reaction is completed.

6. The fluorescent PCR method according to claim 5, characterized in that, The fluorescent PCR system is as follows: 10×PCR buffer: 2 µl dNTP (10mM): 0.2-1 µl MgCl2 (25mM): 0-5 µl DNA polymerase: 1-3 U Upstream primer (10 μM): 0.2–1 µl Downstream primer (10 μM): 0.2–1 µl Probe 1 (10µm): 0.2-1.2 µl Probe 2 (10µm): 0.2-1.2 µl Template: 1-5 µl Purified water: Add to 20µl.

7. The fluorescent PCR method according to claim 6, characterized in that, The DNA polymerase is selected from rTaq polymerase.

8. The fluorescent PCR method according to claim 1, characterized in that, Fluorescent PCR amplification program 。 9. A fluorescent PCR kit using leukocytes in blood as a template, comprising leukocyte resuspension buffer A, wherein the buffer A is formulated with Tris-HCl: 5-8 mmol / L, EDTA: 0.5-1.5 mmol / L, NP-40: 0.5-1%, and BSA: 0.1-0.5 mg / ml.

10. The reagent kit according to claim 9, characterized in that, It also includes a red blood cell lysis reagent, which is selected from TE or purified water.

11. The reagent kit according to claim 9, characterized in that, It also includes a DNA polymerase selected from rTaq enzymes.

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