CN112662746A - Quality control product for genotyping detection and preparation method thereof - Google Patents
Quality control product for genotyping detection and preparation method thereof Download PDFInfo
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Abstract
The invention provides a quality control product and a preparation method thereof, wherein the quality control product is a crude blood extract and comprises leucocytes and buffer solution, the leucocytes are from a sample with a target gene as a heterozygote, and a Ct value obtained by fluorescence PCR measurement is within a preset Ct value range. The quality control product and the preparation method thereof can be used for effective quality control of blood direct-amplification genotyping products, and the precision and the stability of the quality control product meet the industrial requirements of the quality control product.
Description
Technical Field
The invention belongs to the technical field of biological products, and particularly relates to a preparation method of a quality control product for genotyping detection.
Background
With the continuous development and promotion of precise medical treatment, genotyping detection and products thereof have been approved by clinical diagnosis, and more genotyping products are gradually released on the market. With the continuous deepening of such products, the control of the detection method is very important, and therefore, the preparation and application of quality control products are gradually concerned and paid attention. At present, for a gene typing detection product, two detection targets are mainly provided, namely, genomic DNA and crude extracts of blood or blood. The invention mainly aims at a method or a product which takes blood or crude blood extract as a detection target. For such products, the quality control products adopted in the past are mainly artificially constructed plasmids, but the main problems of adopting the constructed plasmids as the quality control products are that the medical test mainly takes human blood or crude blood extracts as detection targets, the plasmids as substitutes have incomparability, and especially unpredictable interference factors exist in the blood or crude blood extracts, which are not possessed by the plasmids.
Therefore, it is very important to find a quality control product with higher comparability and a preparation method thereof aiming at the detection method or product taking blood or crude blood extract as a detection target. In order to maximize the comparability of quality control products, we often use substances consistent with the detection target, i.e., crude blood or blood extracts in the art. Because the blood has instability in the processes of preservation, use and the like, the invention takes the crude extract of the blood as a quality control product and explains the performance of the quality control product and the preparation of the quality control product.
Disclosure of Invention
The invention aims to solve the technical problem of providing a quality control product which can be suitable for effectively controlling the quality of a blood direct-amplification genotyping product and a preparation method thereof.
The quality control product suitable for the genotyping product comprises white blood cells isolated from a sample in which the target gene is heterozygous.
Further, the Ct value of the quality control product is determined by using fluorescence PCR, and the Ct value is within the range of the preset Ct value for quality control product verification.
Further, the buffer solution A is also included, and the formula of the buffer solution A comprises:
Tris-HCl:5-8mmol/L
EDTA:0.5-1.5mmol/L
NP-40: 0.5-1% (volume mass percentage)
BSA：0.1-0.5mg/ml。
Further, the set method for verifying the Ct value range of the quality control product comprises the following steps: sequencing a clinical blood sample to screen out a sample with a target gene detection site as a heterozygote; screening a heterozygote sample with the leukocyte concentration within a specified range, processing the heterozygote sample to obtain a leukocyte solution, evaluating the Ct value of the leukocyte solution by using fluorescence PCR, and determining the preset Ct value range for quality control product verification.
More specifically, the preset Ct value range is 22-28.
Specifically, the white blood cells are obtained by the following method:
(1) adding purified water or TE solution into the whole blood, and vortexing;
(2) and (4) centrifuging to settle the white blood cells, and removing supernatant to obtain the white blood cells.
The invention also provides a preparation method of the quality control product suitable for the blood direct amplification genotyping product, which comprises the confirmation of the screening condition of the quality control product and the preparation of the quality control product, wherein the confirmation of the screening condition of the quality control product comprises the following steps: firstly, designing and synthesizing a sequencing primer aiming at a target detection site; then, sequencing a clinical blood sample to screen out a sample with a target detection site as a heterozygote; finally, screening a heterozygote sample with the leukocyte concentration within a specified range, processing the heterozygote sample to obtain a leukocyte solution, evaluating the Ct value of the leukocyte solution by using fluorescence PCR, and determining the preset Ct value range of quality control product verification;
preparing a quality control product: sequencing by using the sequencing primer, screening a heterozygous sub-sample with the leukocyte concentration within a specified range, then processing the heterozygous sub-sample to obtain a leukocyte solution, and determining the Ct value of the leukocyte solution by using fluorescence PCR; if the Ct value is within the preset quality control product verification Ct value range, the cell-coated solution is used as the quality control product for subsequent application;
the heterozygous daughter sample processing method comprises the following steps:
(1) adding a red blood cell disruption reagent to the whole blood sample to disrupt red blood cells and release hemoglobin;
(2) centrifuging the treatment solution obtained in step (1) to precipitate leukocytes, and removing the supernatant;
(3) add buffer a solution to resuspend the leukocytes.
Further, the specified range refers to the range of leukocyte concentration in the blood sample of 4 × 109-10×109And (2) per liter.
Further, the processing method of the sample for fluorescence PCR comprises the following steps: adding purified water with 5-10 times of volume into EDTA (ethylene diamine tetraacetic acid) anticoagulated whole blood, and whirling for 30 s; ② 12000 Xg centrifugation for 1min to make the leucocyte settle and remove the supernatant; and adding a buffer solution A with the same volume as the whole blood for resuspension.
Further, the formula of the buffer solution A comprises: 5-8mmol/L Tris-HCl, 0.5-1.5mmol/L EDTA, 0.5-1% NP-40, 0.1-0.5mg/ml BSA.
Furthermore, the template used for sequencing is selected from genomic DNA or blood.
The blood direct amplification is short for blood direct amplification.
The quality control product and the preparation method thereof can be used for effective quality control of blood direct-amplification genotyping products, and the precision and the stability of the quality control product meet the industrial requirements of the quality control product.
Drawings
FIG. 1 results of stability test at 37 ℃ in example 3.
FIG. 2 the results of the stability test at 2-8 ℃ in example 3.
FIG. 3 stability test results at-20 ℃ in example 3.
Detailed Description
The present invention is described in further detail by taking the typing detection of SLCO1B1(388A > G) site mutation as an example.
Taking a buffer solution A as a quality control product storage solution, wherein the formula of the buffer solution A comprises:
Tris-HCl:5-8mmol/L
EDTA:0.5-1.5mmol/L
NP-40: 0.5-1% (volume mass percentage)
BSA：0.1-0.5mg/ml
Example 1 preparation method of quality control Material
The quality control preparation is carried out by taking the typing for detecting SLCO1B1(388A > G) site mutation as the aim, and comprises the following steps:
(1) sequencing primers were designed and synthesized for the SLCO1B1(388A > G) site, with the following primer sequences:
SLCO1B1(388A > G) upstream sequencing primer: 5'-GTGTTGTTAATGGGCGAACTGT-3'
SLCO1B1(388A > G) downstream sequencing primer: 5'-AATGGTGCAAATAAAGGGGAAT-3'
(2) The sequencing primer is used for carrying out blood direct amplification, and after an amplification product is obtained, the amplification product is sent to a sequencing company for Sanger sequencing so as to screen heterozygotes. The specific blood direct expansion system and procedure were as follows:
an amplification system:
| components | Dosage of | Final concentration | |
| 2× | 10μl | 1× | |
| Upstream sequencing primer (10. mu.M) | 0.8μl | 0.4μM | |
| Downstream sequencing primer (10. mu.M) | 0.8μl | 0.4μM | |
| Whole blood | 2μl | / | |
| Purified water | Up to 20μl | / |
And (3) amplification procedure:
(3) in the above-selected heterozygote, a leukocyte concentration of 4X 10 was further selected9-10×109The white blood cell concentration of each sample, 126 cases in total, was 4X 10 in clinically normal humans9-10×109Per liter, i.e., selected white blood cells are within the normal range and their SLCO1B1 (388A)>G) The gene locus is a sample of heterozygotes. And (3) carrying out sample processing on the screened 126 samples meeting the requirements, wherein the processing method comprises the following steps:
add 1ml of purified water or TE solution to 100. mu.l of EDTA-anticoagulated whole blood and vortex for 30 s.
② centrifugation at 12000 Xg for 1min to allow the white blood cells to settle and remove the supernatant.
③ adding 100 mul of buffer solution A for heavy suspension.
Wherein the formula of the buffer solution A comprises:
Tris-HCl:6.5mmol/L
EDTA:1.1mmol/L
NP-40:0.8％
BSA：0.3mg/ml
(4) fluorescence PCR amplification (SLCO1B1(388A > G) primer and probe sequences as described below) yielded Ct values for each sample. The fluorescent PCR amplification system and procedure were as follows:
the sequence of the upstream primer is as follows: 5'-TAAACAAGTGGATAAGGTCGATGTTG-3'
The sequence of the downstream primer is as follows: 5'-AGATAATGGTGCAAATAAAGGGGAAT-3'
Wild-type probe sequence: 5 '-FAM-TCCCCTATTCCACGAAGCA-MGBNFQ-3'
Mutant probe sequence:
5’-VIC-AATGTTTAAAATGAAACACTCTCTTATC-MGBNFQ-3’
an amplification system:
| name of material | Addition amount (μ l) | Final concentration |
| 2 × Premix buffer (dNTP free) | 10 | 1× |
| dNTP Mix(10mM) | 0.4 | 200μM |
| ROX reference dye (0.5. mu.M) | 0.8 | 0.02μM |
| Formamide DMF | 0.15 | / |
| Upstream primer (10. mu.M) | 0.8 | 0.4μM |
| Downstream primer (10. mu.M) | 1.6 | 0.8μM |
| Wild type probe (10. mu.M) | 0.4 | 0.2μM |
| Mutant probe (10. mu.M) | 0.9 | 0.45μM |
| Enzyme(6U/μl) | 0.4 | 1× |
| Form panel | 2 | / |
| Purified water | Up to 20 | / |
Fluorescent PCR amplification procedure:
(5) the Ct values were analyzed by SPSS software, as shown in Table 1.
TABLE 1
| N | Minimum value | Maximum value | Mean value | Standard deviation of | |
| Leukocyte concentration (. times.109/L) | 126 | 4.05 | 10.00 | 6.6997 | 1.65566 |
| Ct(FAM) | 126 | 22.62 | 27.53 | 25.1644 | 1.12487 |
| Ct(VIC) | 126 | 22.64 | 27.93 | 25.5420 | 1.19340 |
| Effective N (List State) | 126 |
According to the table above, the Ct value range is determined to be 22-28.
(6) Screening for leukocytes at a concentration of 4X 109-10×109And (3) carrying out heterozygote sequencing verification on the heterozygote sample of each L in the same steps (1) and (2).
(7) And (4) processing the sample screened in the step (6), wherein the processing method is as follows:
add 1ml purified water to 100. mu.l EDTA-anticoagulated whole blood and vortex for 30 s.
② centrifugation at 12000 Xg for 1min to allow the white blood cells to settle and remove the supernatant.
③ adding 100 mul of buffer solution A for heavy suspension.
Wherein the formula of the buffer solution A comprises:
Tris-HCl:5mmol/L
EDTA:0.5mmol/L
NP-40:0.5％
BSA：0.1mg/ml
(8) and (3) performing fluorescence PCR amplification to obtain a corresponding Ct value, observing whether the Ct value range is between 22 and 28, if so, taking the tube product as a quality control product, and if not, discarding the tube product.
The above steps (1) to (5) can be considered as the quality control screening condition confirmation step. The steps (6) to (8) may be regarded as quality control preparation steps according to quality control screening conditions.
The sample sequencing method in the quality control screening condition confirmation step and the quality control preparation step may be performed after amplification using genomic DNA as a template, or may be performed after amplification using blood as a template. In this example, the sequencing after amplification was performed using blood as a template.
Example 2 precision measurement of quality control Material
1. Experimental methods
The same lot of quality control samples prepared in example 1 were each continuously measured 10 times with reference to the Standard in Industrial Standard (YY/T1182-2010) for nucleic acid amplification assay, and the average value of the measurement results was calculated
And standard deviation S1Calculating the coefficient of variation in the tube according to the following formula
2. The index requirement is as follows: CV is less than or equal to 5 percent
3. The experimental results are as follows: see Table 2 below
TABLE 2
4. Description of the results
The precision of the quality control product in the batch is less than or equal to 5 percent.
Example 3 stability test of quality control Material
1. Experimental methods
The quality control products prepared in example 1 were subjected to stability tests at 37 deg.C, 2-8 deg.C and-20 deg.C, respectively, wherein 37 deg.C is a thermal stability test, 2-8 deg.C is a temporal stability test (to evaluate whether short-term use can be temporally stored at 2-8 deg.C to avoid repeated freeze thawing), and-20 deg.C is an expiration stability test.
The control products thus treated were subjected to fluorescent PCR amplification (SLCO1B1(388A > G) with the following primer and probe sequences) and the Ct values thereof were analyzed. The specific amplification system and procedure were as follows:
the sequence of the upstream primer is as follows: 5'-TAAACAAGTGGATAAGGTCGATGTTG-3'
The sequence of the downstream primer is as follows: 5'-AGATAATGGTGCAAATAAAGGGGAAT-3'
Wild-type probe sequence: 5 '-FAM-TCCCCTATTCCACGAAGCA-MGBNFQ-3'
Mutant probe sequence:
5’-VIC-AATGTTTAAAATGAAACACTCTCTTATC-MGBNFQ-3’
an amplification system:
| name of material | Addition amount (μ l) | Final concentration |
| 2 × Premix buffer (dNTP free) | 10 | 1× |
| dNTP Mix(10mM) | 0.4 | 200μM |
| ROX reference dye (0.5. mu.M) | 0.8 | 0.02μM |
| Formamide DMF | 0.15 | / |
| Upstream primer (10. mu.M) | 0.8 | 0.4μM |
| Downstream primer (10. mu.M) | 1.6 | 0.8μM |
| Wild type probe (10. mu.M) | 0.4 | 0.2μM |
| Mutant probe (10. mu.M) | 0.9 | 0.45μM |
| Enzyme(6U/μl) | 0.4 | 1× |
| Form panel | 2 | / |
| Purified water | Up to 20 | / |
And (3) amplification procedure:
2. results of the experiment
The results of the above three conditions were analyzed and shown in FIGS. 1 to 3. The result shows that the quality control product prepared in the example 1 has better thermal stability for 15 days at the temperature of 37 ℃; under the condition of 2-8 ℃, the stability of the temporary storage is good for 8 weeks; the shelf life stability of the product is good at 24 months under the condition of-20 ℃.
Claims (10)
1. A quality control product suitable for use in a genotyping product, comprising leukocytes isolated from a sample in which the target gene is heterozygous.
2. The quality control product according to claim 1, wherein the Ct value of the quality control product is measured by fluorescence PCR and is within a preset Ct value range for quality control product verification.
3. The quality control product of claim 1, further comprising a buffer a, wherein the formulation of the buffer a comprises:
Tris-HCl:5-8mmol/L
EDTA:0.5-1.5mmol/L
NP-40: 0.5-1% (volume mass percentage)
BSA：0.1-0.5mg/ml。
4. The quality control product of claim 2, wherein the set quality control product verification Ct value range method comprises: sequencing a clinical blood sample to screen out a sample with a target gene detection site as a heterozygote; screening a heterozygote sample with the leukocyte concentration within a specified range, processing the heterozygote sample to obtain a leukocyte solution, evaluating the Ct value of the leukocyte solution by using fluorescence PCR, and determining the preset Ct value range for quality control product verification.
5. The quality control product according to claim 1, wherein the leukocytes are obtained by:
(1) adding purified water or TE solution into the whole blood, and vortexing;
(2) and (4) centrifuging to settle the white blood cells, and removing supernatant to obtain the white blood cells.
6. A preparation method of quality control products suitable for blood direct amplification genotyping products comprises the confirmation of quality control product screening conditions and the preparation of the quality control products, and is characterized in that,
the quality control product screening condition confirming step comprises the following steps: firstly, designing and synthesizing a sequencing primer aiming at a target detection site; then, sequencing a clinical blood sample to screen out a sample with a target detection site as a heterozygote; finally, screening a heterozygote sample with the leukocyte concentration within a specified range, processing the heterozygote sample to obtain a leukocyte solution, evaluating the Ct value of the leukocyte solution by using fluorescence PCR, and determining the preset Ct value range of quality control product verification;
preparing a quality control product: sequencing by using the sequencing primer, screening a heterozygous sub-sample with the leukocyte concentration within a specified range, then processing the heterozygous sub-sample to obtain a leukocyte solution, and determining the Ct value of the leukocyte solution by using fluorescence PCR; if the Ct value is within the preset quality control product verification Ct value range, the cell-coated solution is used as the quality control product for subsequent application;
the heterozygous daughter sample processing method comprises the following steps:
(1) adding a red blood cell disruption reagent to the whole blood sample to disrupt red blood cells and release hemoglobin;
(2) centrifuging the treatment solution obtained in step (1) to precipitate leukocytes, and removing the supernatant;
(3) add buffer a solution to resuspend the leukocytes.
7. The method of claim 6, wherein the specified range is a leukocyte concentration range of 4 x 10 in the blood sample9-10×109And (2) per liter.
8. The method of claim 6, wherein the sample processing method for fluorescence PCR comprises: adding purified water with 5-10 times of volume into EDTA (ethylene diamine tetraacetic acid) anticoagulated whole blood, and whirling for 30 s; ② 12000 Xg centrifugation for 1min to make the leucocyte settle and remove the supernatant; and adding a buffer solution A with the same volume as the whole blood for resuspension.
9. The method of claim 9, wherein the formulation of buffer a comprises: 5-8mmol/L Tris-HCl, 0.5-1.5mmol/L EDTA, 0.5-1% NP-40, 0.1-0.5mg/ml BSA.
10. The method according to claim 6, wherein the template used for the sequencing is selected from the group consisting of genomic DNA or blood.
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