CN113667751A - Preparation method and application of calibrator and reference for BCR-ABL gene fusion detection - Google Patents
Preparation method and application of calibrator and reference for BCR-ABL gene fusion detection Download PDFInfo
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Abstract
The invention provides a preparation method and application of a calibrator and a reference substance for BCR-ABL gene fusion detection, wherein the method comprises the following steps: respectively preparing ABL wild type armor RNA and BCR-ABL fusion type armor RNA; dissolving the ABL wild-type armor RNA and the BCR-ABL fusion-type armor RNA respectively by using a protective diluent; detecting the concentration of the ABL wild-type armor RNA and the BCR-ABL fused armor RNA by digital PCR; carrying out gradient dilution on the ABL wild type armor RNA and the BCR-ABL fusion type armor RNA by using a protection diluent to obtain a calibrator; and mixing the ABL wild type armor RNA calibrator and the BCR-ABL fusion type armor RNA calibrator in proportion to obtain a reference product. The prepared calibrator and reference product have good traceability with WHO primary standard product and accurate result.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of a calibrator and a reference substance for BCR-ABL gene fusion detection.
Background
Chronic Myelogenous Leukemia (CML) is a disease characterized by malignant proliferation of middle-late juvenile cells, and BCR-ABL fusion genes (i.e., ABL genes on chromosome 9 translocate to BCR genes on chromosome 22 to form fusion genes) can be detected in about 90-95% of CML patients, and can be used for typing diagnosis, efficacy evaluation, and detection of minimal residual disease and reliable index of prognosis evaluation of CML.
Currently, the detection methods commonly used in clinic include conventional chromosome analysis, Fluorescence In Situ Hybridization (FISH), and fluorescent quantitative PCR. The NCCN guidelines recommend that CML patients should undergo BCR-ABL fluorescent quantitative PCR every three months after reaching CCyR (complete cytological remission) to monitor whether residual leukemic cells are ascending, descending, or in a stable state.
At present, BCR-ABL fluorescent quantitative PCR detection IS internationally standardized, and in consideration of differences of different conditions (such as different reference genes, PCR primers, an extraction method, a fluorescent quantitative PCR system, a reverse transcription system and the like) of an experiment, detection results of laboratories need to be compared with WHO primary standard products, and a conversion Coefficient (CF) IS established, so that the quantitative results of the BCR-ABL are converted into an international standard value (IS).
However, the WHO primary standard product has a long shelf life and a lot of procedures, and the RNA quantity obtained after extraction is very small, so that the demands of research, development, verification and quality inspection of enterprises can not be met. In addition, the prior art cannot trace the source with a WHO primary reference product, and the quantitative result is lack of reliability; or even if the source can be traced, the difference with the theoretical value is larger; or large batch-to-batch differences exist between different reference preparation batches, and good consistency cannot be maintained.
Therefore, it is a problem to be solved how to provide an easily available calibrator and reference material that can be used for reagent development, verification and quality inspection.
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention provides a preparation method and application of a calibrator and a reference product for BCR-ABL gene fusion detection, the prepared calibrator and reference product have good amplification efficiency, small batch difference, capability of tracing sources with WHO primary standard products well, a correction coefficient very close to 1.0 (namely, a detection value is equal to a theoretical value), and extremely high application value.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for preparing a calibrator and a reference for BCR-ABL gene fusion detection, the method comprising:
respectively preparing ABL wild type armor RNA and BCR-ABL fusion type armor RNA;
dissolving the ABL wild-type armor RNA and the BCR-ABL fusion-type armor RNA respectively by using a protective diluent;
detecting the concentration of the ABL wild-type armor RNA and the BCR-ABL fused armor RNA by digital PCR;
carrying out gradient dilution on the ABL wild type armor RNA and the BCR-ABL fusion type armor RNA by using a protection diluent to obtain a calibrator; and mixing the ABL wild type armor RNA calibrator and the BCR-ABL fusion type armor RNA calibrator in proportion to obtain a reference product.
In the invention, armored RNA is selected for preparing a calibrator and a reference substance, and can be consistent with a WHO primary standard substance on a nucleic acid level; the addition of the protective diluent can improve and stabilize the amplification efficiency of the reaction and reduce or eliminate the difference between batches; the quantitative detection of the armored RNA is more accurate by selecting the digital PCR for detection; the prepared calibration product and reference product can be well traced to the source with WHO primary standard product, and the correction coefficient is very close to 1.0.
Preferably, the nucleotide sequence encoding the ABL wild-type armored RNA is shown in SEQ ID No. 1.
SEQ ID No.1：
caaaccaaaaatggccaaggctgggtcccaagcaactacatcacgccagtcaacagtctggagaaacactcctggtaccatgggcctgtgtcccgcaatgccgctgagtatctgctgagcagcgggatcaatggcagcttcttggtgcgtgagagtgagagcagtcctggccagaggtccatctcgctgagatacgaagggagggtgtaccattacaggatcaacactgcttctgatggcaagctctacgtctcctccgagagccgcttcaacaccctggccgagttggttcatcatcattcaacggtggccgacgggctcatcaccacgctccattatccagccccaaagcgcaacaagcccactgtctatggtgtgtcccctaactacgacaagt。
Preferably, the nucleotide sequence encoding the BCR-ABL fusion type armor RNA is shown as SEQ ID No. 2.
SEQ ID No.2：
tacacgttcctgatctcctctgactatgagcgtgcagagtggagggagaacatccgggagcagcagaagaagtgtttcagaagcttctccctgacatccgtggagctgcagatgctgaccaactcgtgtgtgaaactccagactgtccacagcattccgctgaccatcaataaggaagatgatgagtctccggggctctatgggtttctgaatgtcatcgtccactcagccactggatttaagcagagttcaaaagcccttcagcggccagtagcatctgactttgagcctcagggtctgagtgaagccgctcgttggaactccaaggaaaaccttctcgctggacccagtgaaaatgaccccaaccttttcgttgcactgtatgattttgtggccagtggagataacactctaagcataactaaaggtgaaaagctccgggtcttaggctataatcacaatggggaatggtgtgaagcc。
Preferably, before the dissolving, the method further comprises a step of performing water bath cracking on the ABL wild-type armored RNA and the BCR-ABL fusion-type armored RNA at 85-95 ℃ for 4-8 min, wherein the temperature may be, for example, 85 ℃, 86 ℃, 87 ℃, 88 ℃, 89 ℃, 90 ℃, 91 ℃, 92 ℃, 93 ℃, 94 ℃ or 95 ℃, and the time may be, for example, 4min, 4.5min, 5min, 5.5min, 6min, 6.5min, 7min, 7.5min or 8min, and other specific points in the numerical range may be selected, and thus, the description is omitted.
Preferably, the protecting diluent comprises a Tris-EDTA buffer, a protecting nucleic acid and a protecting agent.
Preferably, the pH of the Tris-EDTA buffer is 7.0-8.5, for example, 7.0, 7.5, 8.0, or 8.5, and other specific values within the range may be selected, which is not repeated herein.
Preferably, the protective nucleic acid comprises carrier rna (carrier rna) and/or salmon sperm DNA.
Preferably, the final concentration of the protected nucleic acid is 10-60 ng/. mu.L, for example, 10 ng/. mu.L, 15 ng/. mu.L, 20 ng/. mu.L, 25 ng/. mu.L, 30 ng/. mu.L, 35 ng/. mu.L, 40 ng/. mu.L, 45 ng/. mu.L, 50 ng/. mu.L, 55 ng/. mu.L or 60 ng/. mu.L, and the like, and other specific points within the numerical range can be selected, which is not repeated herein.
Preferably, the protectant comprises Triton X-100 and/or Tween 20.
Preferably, the final concentration of the Triton X-100 is 0.005% to 0.05%, for example, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, or 0.05%, and other specific values within the numerical range can be selected, which is not described herein again.
Preferably, the final concentration of Tween20 is 0.05% to 0.5%, for example, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%, etc., and other specific values in the value range can be selected, which is not described herein again.
According to the invention, by adding the protective nucleic acid and the protective agent into the buffer solution, the stability of the target sequence can be enhanced, the target sequence can be effectively detected, the batch difference is reduced, and the detection result is more accurate.
Preferably, the reaction system of the digital PCR comprises a primer pair, a fluorescent probe and a premix.
Preferably, the final concentration of the primer pair in the reaction system is 600nM to 1200nM, for example, 600nM, 650nM, 700nM, 750nM, 800nM, 850nM, 900nM, 950nM, 1000nM, 1050nM, 1100nM, 1150nM, or 1200nM, etc., and the final concentration of the fluorescent probe in the reaction system is 80nM to 400nM, for example, 80nM, 100nM, 150nM, 200nM, 250nM, 300nM, 350nM, or 400nM, etc., and other specific points in the value range can be selected, which is not repeated herein.
Preferably, the sequence of a primer pair for amplifying the ABL wild-type armored RNA is shown as SEQ ID No. 3-4, and the sequence of a fluorescent probe for detecting the ABL wild-type armored RNA is shown as SEQ ID No. 5.
Preferably, the sequence of a primer pair for amplifying the BCR-ABL fused armored RNA is shown as SEQ ID No. 6-7, and the sequence of a fluorescent probe for detecting the BCR-ABL fused armored RNA is shown as SEQ ID No. 8.
SEQ ID No.3：CTACGTCTCCTCCGAGAGCCGCTTCAACA；
SEQ ID No.4：CCCGTCGGCCACCGTTGAATGATGATG；
SEQ ID No.5：CCCTGGCCGAGTTGGTT；
SEQ ID No.6：ACGAGCGGCTTCACTCAGACCCTGAGGCTC；
SEQ ID No.7：TCCACAGCATTCCGCTGACCATCAATAAGGAAG；
SEQ ID No.8：AAGCCCTTCAGCGGCCAGTAGCATCTGACTTT。
Preferably, the fluorescent probe is labeled with a fluorescence generating group at the 5 'end and a fluorescence quenching group at the 3' end.
Preferably, the fluorogenic group comprises any one of FAM, HEX, VIC, TAMRA, ROX, Cy3, or Cy 5.
Preferably, the fluorescence quenching group comprises any one of MGB, BHQ1, BHQ2 or BHQ 3.
Preferably, the premix comprises a buffer, a reverse transcriptase, a DNA polymerase and dNTPs.
Preferably, the reaction procedure of the digital PCR comprises:
reverse transcription:
40-60 ℃, 13-16 min, for example, the temperature can be 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃, 46 ℃, 47 ℃, 48 ℃, 49 ℃, 50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃ or 60 ℃, and the time can be 13min, 13.5min, 14min, 14.5min, 15min, 15.5min or 16min, and other specific point values in the numerical range can be selected, and are not described in detail herein.
Pre-denaturation:
94-98 deg.C, 8-12 min, such as 94 deg.C, 94.5 deg.C, 95 deg.C, 95.5 deg.C, 96 deg.C, 96.5 deg.C, 97 deg.C, 97.5 deg.C or 98 deg.C, and the time can be, for example, 8min, 8.5min, 9min, 9.5min, 10min, 10.5min, 11min, 11.5min or 12min, and other specific values in the value range can be selected, and are not described in detail herein.
And (3) circulating amplification:
94-98 ℃ for 10-30 s, the temperature can be 94 ℃, 94.5 ℃, 95 ℃, 95.5 ℃, 96 ℃, 96.5 ℃, 97 ℃, 97.5 ℃ or 98 ℃ for example, the time can be 10s, 11s, 12s, 13s, 14s, 15s, 16s, 17s, 18s, 19s, 20s, 21s, 22s, 23s, 24s, 25s, 26s, 27s, 28s, 29s or 30s for example, and other specific point values in the numerical range can be selected, which is not described in detail herein.
50-60 ℃ for 20-60 s, the temperature can be 50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃ or 60 ℃ for example, the time can be 20s, 25s, 30s, 35s, 40s, 45s, 50s, 55s or 60s for example, and other specific values in the numerical range can be selected, which is not described in detail herein.
The cycle is carried out 40 to 50 times, and may be, for example, 40 times, 41 times, 42 times, 43 times, 44 times, 45 times, 46 times, 47 times, 48 times, 49 times or 50 times.
Preferably, after the gradient dilution, the final concentration of the ABL wild-type armor RNA calibrator and the final concentration of the BCR-ABL fusion-type armor RNA calibrator are (1-5) multiplied by 10 in sequence5Copies/. mu.L, for example, may be 1X 105Copy/. mu.L, 2X 105Copy/. mu.L, 3X 105Copy/. mu.L, 4X 105Copies/. mu.L or 5X 105Copy/. mu.L, etc., and other specific point values in the numerical range can be selected, which are not described in detail herein;
(1～5)×104copies/. mu.L, for example, may be 1X 104Copy/. mu.L, 2X 104Copy/. mu.L, 3X 104Copy/. mu.L, 4X 104Copies/. mu.L or 5X 104Copy/. mu.L, etc., and other specific point values in the numerical range can be selected, which are not described in detail herein;
(1～5)×103copies/. mu.L, for example, may be 1X 103Copy/. mu.L, 2X 103Copy/. mu.L, 3X 103Copy/. mu.L, 4X 103Copies/. mu.L or 5X 103Copy/. mu.L, etc., and other specific point values in the numerical range can be selected, which are not described in detail herein;
(1～5)×102copies/. mu.L, for example, may be 1X 102Copy/. mu.L, 2X 102Copy/. mu.L, 3X 102Copy/. mu.L, 4X 102Copies/. mu.L or 5X 102Copy/. mu.L, etc., and other specific point values in the numerical range can be selected, which are not described in detail herein;
(1～5)×101copies/. mu.L, for example, may be 1X 101Copy/. mu.L, 2X 101Copy/. mu.L, 3X 101Copy/. mu.L, 4X 101Copies/. mu.L or 5X 101Copy/. mu.L, etc., and other specific point values in the numerical range can be selected, which are not described in detail herein;
1-5 copies/. mu.L, for example, 1 copy/. mu.L, 2 copies/. mu.L, 3 copies/. mu.L, 4 copies/. mu.L, or 5 copies/. mu.L, etc., and other specific values within the numerical range can be selected, which is not described herein again.
Preferably, in the reference product, the concentration ratio of the ABL wild type armor RNA calibrator to the BCR-ABL fusion type armor RNA calibrator is 10:1, 100:1, 1000:1 and 10000:1 in sequence.
As a preferred technical scheme, the preparation method of the calibrator and the reference for BCR-ABL gene fusion detection comprises the following steps:
(1) preparation of armored RNA:
respectively preparing ABL wild type armor RNA and BCR-ABL fusion type armor RNA, wherein the nucleotide sequence for coding the ABL wild type armor RNA is shown as SEQ ID No.1, and the nucleotide sequence for coding the BCR-ABL fusion type armor RNA is shown as SEQ ID No. 2;
(2) dissolving:
cracking the ABL wild armor RNA and the BCR-ABL fusion type armor RNA in water bath at 85-95 ℃ for 4-8 min, and then respectively dissolving by using protection diluent;
wherein the protective diluent comprises Tris-EDTA buffer solution with the pH value of 7-8.5, carrier RNA and/or salmon sperm DNA with the final concentration of 10-60 ng/mu L, and 0.005-0.05% of Triton X-100 and/or 0.05-0.5% of Tween 20;
(3) and (3) concentration detection:
the concentrations of the ABL wild-type armor RNA and BCR-ABL fused armor RNA were detected by digital PCR:
the reaction system of the digital PCR comprises a primer pair with the final concentration of 600-1200 nM, a fluorescent probe with the final concentration of 80-400 nM and a premixed solution;
the sequence of a primer pair for amplifying the ABL wild-type armored RNA is shown as SEQ ID No. 3-4, and the sequence of a fluorescent probe for detecting the ABL wild-type armored RNA is shown as SEQ ID No. 5;
the sequence of a primer pair for amplifying the BCR-ABL fused armored RNA is shown as SEQ ID No. 6-7, and the sequence of a fluorescent probe for detecting the BCR-ABL fused armored RNA is shown as SEQ ID No. 8;
the premixed liquid comprises buffer solution, reverse transcriptase, DNA polymerase and dNTPs;
the reaction procedure of the digital PCR comprises:
reverse transcription: at 40-60 ℃ for 13-16 min;
pre-denaturation: 94-98 ℃ for 8-12 min;
and (3) circulating amplification: 94-98 ℃ for 10-30 s; 50-60 ℃ for 20-60 s; circulating for 40-50 times;
(4) preparation of calibrators and reference samples:
using protection diluent to carry out gradient dilution on the ABL wild armor RNA and the BCR-ABL fusion type armor RNA until the final concentration is (1-5) multiplied by 10 in sequence5Copy/. mu.L, (1-5). times.104Copy/. mu.L, (1-5). times.103Copy/. mu.L, (1-5). times.102Copy/. mu.L, (1-5). times.101Copying/mu L and 1-5 copies/mu L to obtain a calibrator;
mixing the ABL wild type armor RNA calibrator and the BCR-ABL fusion type armor RNA calibrator in a concentration ratio of 10:1, 100:1, 1000:1 and 10000:1 in sequence to obtain a reference product.
In a second aspect, the invention provides a calibrator and a reference for BCR-ABL gene fusion detection, wherein the calibrator and the reference are prepared by the preparation method of the calibrator and the reference for BCR-ABL gene fusion detection described in the first aspect.
In the invention, the calibrator and the reference prepared by the method have good traceability with WHO primary standard, are easier to prepare and obtain, can be applied to relevant detection, and have practical application value.
In a third aspect, the present invention provides a method for detecting the accuracy of the calibrator and the reference for BCR-ABL gene fusion detection described in the second aspect, comprising:
amplifying the calibration product, the reference product and the WHO primary standard product, constructing a standard curve of the cycle number and the copy number of the calibration product, then constructing a linear relation curve of an actual detection value and a theoretical value of the WHO primary standard product, then constructing a linear relation curve of an actual detection value and a theoretical value of the reference product, and judging the accuracy according to curve parameters.
According to the method, the accuracy of the calibration product can be judged according to the standard curve of the calibration product and the curve parameter of the linear relation curve of the actual detection value and the theoretical value of the WHO primary standard product. If R of the standard curve2Close to 1, the amplification efficiency is close to 100%; and the linear relation is similar to y ═ x, R2And if the accuracy is close to 1, the calibration product has good accuracy and can be used for relevant detection of products.
According to the method and the device, the accuracy of the calibration product can be judged according to the curve parameter of the linear relation curve of the actual detection value and the theoretical value of the reference product. If the linear relationship is similar to y ═ x, R2And if the result is close to 1, the accuracy of the reference product is good, and the reference product can be used for relevant detection of products.
In the invention, the actual detection value is a logarithmic value of BCR-ABL/ABL percentage, and the theoretical value is a logarithmic value of BCR-ABL/ABL percentage.
Preferably, the reaction system for amplification comprises a primer pair, a fluorescent probe, a PCR buffer, a reverse transcription buffer, a reverse transcriptase, a DNA polymerase, an RNase inhibitor and dNTPs.
Preferably, the final concentration of the primer pair is 200nM to 1000nM, such as 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, or 1000nM, and the final concentration of the fluorescent probe is 100nM to 500nM, such as 100nM, 150nM, 200nM, 250nM, 300nM, 350nM, 400nM, 450nM, or 500nM, and other specific point values in the value range can be selected, which is not described herein again.
Preferably, the final concentration of the reverse transcriptase is 0.1-3U/. mu.L, for example, 0.1U/. mu.L, 0.5U/. mu.L, 1U/. mu.L, 1.5U/. mu.L, 2U/. mu.L, 2.5U/. mu.L or 3U/. mu.L, and the like, and other specific values in the numerical range can be selected, which is not repeated herein.
Preferably, the final concentration of the DNA polymerase is 0.05-0.2U/. mu.L, for example, 0.05U/. mu.L, 0.1U/. mu.L, 0.15U/. mu.L, or 0.2U/. mu.L, etc., and other specific values within the value range can be selected, which is not described herein again.
Preferably, the final concentration of the RNase inhibitor is 0.1-1U/. mu.L, for example, 0.1U/. mu.L, 0.2U/. mu.L, 0.3U/. mu.L, 0.4U/. mu.L, 0.5U/. mu.L, 0.6U/. mu.L, 0.7U/. mu.L, 0.8U/. mu.L, 0.9U/. mu.L, or 1U/. mu.L, etc., and other specific values in the numerical range can be selected, which is not repeated herein.
Preferably, the final concentration of the dNTPs is 100-500. mu.M, for example, 100. mu.M, 150. mu.M, 200. mu.M, 250. mu.M, 300. mu.M, 350. mu.M, 400. mu.M, 450. mu.M or 500. mu.M, and the like, and other specific values within the numerical range can be selected, and are not repeated herein.
Preferably, the reaction procedure for the amplification is the same as that for digital PCR.
As a preferred technical scheme, the method for detecting the accuracy of the calibrator and the reference substance for BCR-ABL gene fusion detection comprises the following steps:
(1) amplifying a calibrator, a reference and a WHO primary standard, wherein an amplified reaction system comprises a primer pair with a final concentration of 200-1000 nM, a fluorescent probe with a final concentration of 100-500 nM, a PCR buffer solution, a reverse transcription buffer solution, reverse transcriptase with a final concentration of 0.1-3U/muL, DNA polymerase with a final concentration of 0.05-0.2U/muL, RNase inhibitor with a final concentration of 0.1-1U/muL and dNTPs with a final concentration of 100-500 muM, and the amplified reaction procedure is the same as that of digital PCR;
(2) and (3) establishing a standard curve of the cycle number and the copy number of the calibration product, then establishing a linear relation curve of an actual detection value and a theoretical value of the WHO primary standard product, then establishing a linear relation curve of an actual detection value and a theoretical value of the reference product, and judging the accuracy according to curve parameters.
In a fourth aspect, the invention provides a preparation method of a calibrator and a reference for BCR-ABL gene fusion detection in the first aspect and/or an application of the method for detecting the accuracy of the calibrator and the reference for BCR-ABL gene fusion detection in preparation of the calibrator and the reference for BCR-ABL gene fusion detection in the third aspect.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention selects armored RNA to prepare the calibrator and the reference substance, which can be consistent with WHO primary standard substance (RNA) at the nucleic acid level; by adding the protective diluent, the amplification efficiency in the amplification reaction process can be improved and stabilized, the parallelism and the repeatability of the experiment are better, the difference between batches is reduced, and the result is more accurate;
(2) the method has the advantages that quantification is carried out through digital PCR in the preparation process, the quantification of the original copy number of the armored RNA is more accurate, the prepared calibrator and reference product can be well traced with WHO primary standard, the correction coefficient is very close to 1.0, the method is easy to obtain and simple to operate, conditions are provided for relevant detection reactions, and the method has extremely high application value.
Drawings
FIG. 1A is a standard curve for an ABL wild-type armored RNA calibrator;
FIG. 1B is a standard curve of a BCR-ABL fused armored RNA calibrator;
FIG. 2 is a linear relationship curve of an actual detection value and a theoretical value of a WHO primary standard product;
FIG. 3 is a linear relationship curve of the actual detection value and the theoretical value of the reference product;
fig. 4 is a schematic diagram of a reference product tracing process.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Materials:
the premix was purchased from biotechnology limited, Xinyi, Beijing;
PCR buffer, reverse transcription buffer, reverse transcriptase, DNA polymerase, RNase inhibitor and dNTPs were purchased from Fenpeng Bio Inc.;
WHO primary standards (09/138) were purchased from national biologicals institute of Biotechnology (NIBSC), and obtained after extraction from Qiagen RNeasy Mini Kit.
Example 1
This example provides a calibrator and a reference for BCR-ABL gene fusion assay, which are prepared by the following steps:
(1) preparation of armored RNA:
armored RNA is consigned to Xiamen Shangshi technology GmbH for synthesis. Respectively preparing ABL wild type armor RNA and BCR-ABL fusion type armor RNA, wherein the nucleotide sequence for coding the ABL wild type armor RNA is shown as SEQ ID No.1, and the nucleotide sequence for coding the BCR-ABL fusion type armor RNA is shown as SEQ ID No. 2.
SEQ ID No.1：
caaaccaaaaatggccaaggctgggtcccaagcaactacatcacgccagtcaacagtctggagaaacactcctggtaccatgggcctgtgtcccgcaatgccgctgagtatctgctgagcagcgggatcaatggcagcttcttggtgcgtgagagtgagagcagtcctggccagaggtccatctcgctgagatacgaagggagggtgtaccattacaggatcaacactgcttctgatggcaagctctacgtctcctccgagagccgcttcaacaccctggccgagttggttcatcatcattcaacggtggccgacgggctcatcaccacgctccattatccagccccaaagcgcaacaagcccactgtctatggtgtgtcccctaactacgacaagt。
SEQ ID No.2：
tacacgttcctgatctcctctgactatgagcgtgcagagtggagggagaacatccgggagcagcagaagaagtgtttcagaagcttctccctgacatccgtggagctgcagatgctgaccaactcgtgtgtgaaactccagactgtccacagcattccgctgaccatcaataaggaagatgatgagtctccggggctctatgggtttctgaatgtcatcgtccactcagccactggatttaagcagagttcaaaagcccttcagcggccagtagcatctgactttgagcctcagggtctgagtgaagccgctcgttggaactccaaggaaaaccttctcgctggacccagtgaaaatgaccccaaccttttcgttgcactgtatgattttgtggccagtggagataacactctaagcataactaaaggtgaaaagctccgggtcttaggctataatcacaatggggaatggtgtgaagcc。
(2) Dissolving:
the ABL wild armor RNA and the BCR-ABL fusion type armor RNA are cracked in a water bath at 90 ℃ for 5min, and then are respectively dissolved by using protective diluent, so that the concentration is 10 times of concentration gradient in sequence until the theoretical concentration is 2 multiplied by 103Copy/. mu.L;
wherein the protective diluent comprises Tris-EDTA buffer solution with pH of 8.0, carrier RNA with final concentration of 20 ng/mu L and 0.01% Triton X-100.
(3) And (3) concentration detection:
the concentrations of the ABL wild-type armor RNA and BCR-ABL fused armor RNA were detected by digital PCR:
the reaction system of the digital PCR comprises a primer pair with the final concentration of 800nM, a fluorescent probe with the final concentration of 250nM and a premixed solution;
the sequence of a primer pair for amplifying the ABL wild type armored RNA is shown as SEQ ID No. 3-4, the sequence of a fluorescent probe for detecting the ABL wild type armored RNA is shown as SEQ ID No.5, a fluorescence generating group of the fluorescent probe is FAM, and a fluorescence quenching group is MGB;
the sequence of a primer pair for amplifying the BCR-ABL fused armored RNA is shown as SEQ ID No. 6-7, the sequence of a fluorescent probe for detecting the BCR-ABL fused armored RNA is shown as SEQ ID No.8, a fluorescence generating group of the fluorescent probe is FAM, and a fluorescence quenching group is BHQ 1;
SEQ ID No.3：CTACGTCTCCTCCGAGAGCCGCTTCAACA；
SEQ ID No.4：CCCGTCGGCCACCGTTGAATGATGATG；
SEQ ID No.5：CCCTGGCCGAGTTGGTT；
SEQ ID No.6：ACGAGCGGCTTCACTCAGACCCTGAGGCTC；
SEQ ID No.7：TCCACAGCATTCCGCTGACCATCAATAAGGAAG；
SEQ ID No.8：AAGCCCTTCAGCGGCCAGTAGCATCTGACTTT。
the premixed liquid comprises buffer solution, reverse transcriptase, DNA polymerase and dNTPs;
the reaction system is as follows:
the reaction procedure of the digital PCR comprises:
reverse transcription: 15min at 55 ℃;
pre-denaturation: at 95 ℃ for 10 min;
and (3) circulating amplification: 30s at 94 ℃; 60s at 55 ℃; circulating for 40 times;
cooling an instrument: 12 ℃ for 5 min.
After amplification was complete, the quantitative results were analyzed using a chip analyzer to obtain the actual concentration of both armored RNA (copies/. mu.L). And calculating the actual concentration of the original armor RNA solution according to the actual concentration and the gradient dissolution times.
(4) Preparation of calibrators and reference samples:
using protection diluent to perform gradient dilution on the ABL wild-type armor RNA and the BCR-ABL fusion type armor RNA until the final concentration is sequentially 2 multiplied by 105Copy/. mu.L, 2X 104Copy/. mu.L, 2X 103Copy/. mu.L, 2X 102Copy/. mu.L, 2X 101Copy/. mu.L and 2 copies/. mu.L to obtain a calibrator.
Mixing the ABL wild type armor RNA calibrator and the BCR-ABL fusion type armor RNA calibrator in a concentration ratio of 10:1, 100:1, 1000:1 and 10000:1 in sequence to obtain a reference product.
Mixing, and collecting 20 μ L of the mixture at a concentration of 2 × 105Copy/. mu.L ABL wild-type armored RNA calibrator at a concentration of 2X 10, respectively, and 20. mu.L4Copy/. mu.L, 2X 103Copy/. mu.L, 2X 102Copy/. mu.L, 2X 101And mixing the copied/mu L BCR-ABL fused armored RNA calibrator, and supplementing 160 mu L of protection diluent to obtain the reference substance.
The invention uses armored RNA to prepare the calibrator and the reference substance, which can be consistent with WHO primary standard; the amplification efficiency can be stabilized by adding the protective diluent, and the difference between batches is reduced; and the digital PCR is used for quantification, so that the calculation result is more accurate, and the source tracing with the WHO primary standard product can be better realized.
Example 2
This example tests the accuracy of the calibrator and reference for BCR-ABL gene fusion testing prepared in example 1, and includes the following steps:
(1) and (3) amplifying the calibrator, the reference and the WHO primary standard, wherein the amplified reaction system comprises a primer pair with a final concentration of 600nM, a fluorescent probe with a final concentration of 200nM, a PCR buffer solution, a reverse transcription buffer solution, a reverse transcriptase with a concentration of 0.4U/. mu.L, a DNA polymerase with a concentration of 0.075U/. mu.L, an RNase inhibitor with a concentration of 0.125U/. mu.L and dNTPs with a concentration of 250. mu.M.
The reaction system is as follows:
the reaction procedure was the same as in example 1.
(2) And (3) establishing a standard curve of the cycle number and the copy number of the calibration product, then establishing a linear relation curve of an actual detection value and a theoretical value of the WHO primary standard product, then establishing a linear relation curve of an actual detection value and a theoretical value of the reference product, and judging the accuracy according to curve parameters.
The standard curve of the ABL wild-type armor RNA calibrator is shown in FIG. 1A, and the standard curve of the BCR-ABL fusion-type armor RNA calibrator is shown in FIG. 1B. As can be seen from the figure, the linear relationship of the standard curve is good (R)21.000 and 0.999 respectively), the amplification efficiency is higher (the Eff% is 97.636 and 101.84 respectively), and the method can be used for accurate quantification.
The linear relationship between the actual detection value (logarithm of BCR-ABL/ABL percentage) and the theoretical value (logarithm of BCR-ABL/ABL percentage) of the WHO primary standard is shown in FIG. 2, and it can be seen that R is2Linear relationship 0.995, slope 1.0776 (approximately 1), intercept 0.0213 (approximately 0), linear relationshipGood, linear equations are approximated by y ═ x, Correction Factor (CF) ═ 0.9521, and 1-bit fractional number left is equal to 1.0. (the correction factor is the inverse of the inverse logarithm of the intercept).
The linear relationship between the actual measured value (logarithm of BCR-ABL/ABL percentage) and the theoretical value (logarithm of BCR-ABL/ABL percentage) of the reference is shown in FIG. 3, and it can be seen from the graph that R is2At 0.995, the slope is 1.0767 (close to 1), the intercept is 0.0391 (close to 0), the linearity is good, the linear formula is approximated by y x, and the quantitative result is as expected.
The results show that the correction coefficient of the prepared calibrator under a matched detection system is very close to 1.0; the prepared reference product is basically consistent with a theoretical value, can be traced to a WHO primary standard product, can be directly used for product verification or quality inspection, and can also be used for tracing a subsequent preparation batch reference product, and a flow chart is shown in FIG. 4.
In conclusion, the calibrator and the reference prepared by the invention can be consistent with the WHO primary standard product in the aspect of nucleic acid, and the result is more accurate; through digital PCR quantification in the preparation process, the original copy number of the armored RNA is quantified more accurately; the armored RNA is diluted by using the protective diluent, so that the amplification efficiency of the reaction can be stabilized and improved, the difference between batches is reduced, and the repeatability of the experiment is better; the method can be well traced with WHO primary standard products, the correction coefficient is very close to 1.0, the preparation method is simple, the use is convenient, and the method is easier to obtain.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Sequence listing
<110> Mejie transformation medical research (Suzhou) Co., Ltd
<120> preparation method and application of calibrator and reference substance for BCR-ABL gene fusion detection
<130> 2021
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 397
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<213> Artificial sequence
<400> 1
caaaccaaaa atggccaagg ctgggtccca agcaactaca tcacgccagt caacagtctg 60
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agcgggatca atggcagctt cttggtgcgt gagagtgaga gcagtcctgg ccagaggtcc 180
atctcgctga gatacgaagg gagggtgtac cattacagga tcaacactgc ttctgatggc 240
aagctctacg tctcctccga gagccgcttc aacaccctgg ccgagttggt tcatcatcat 300
tcaacggtgg ccgacgggct catcaccacg ctccattatc cagccccaaa gcgcaacaag 360
cccactgtct atggtgtgtc ccctaactac gacaagt 397
<210> 2
<211> 480
<212> DNA
<213> Artificial sequence
<400> 2
tacacgttcc tgatctcctc tgactatgag cgtgcagagt ggagggagaa catccgggag 60
cagcagaaga agtgtttcag aagcttctcc ctgacatccg tggagctgca gatgctgacc 120
aactcgtgtg tgaaactcca gactgtccac agcattccgc tgaccatcaa taaggaagat 180
gatgagtctc cggggctcta tgggtttctg aatgtcatcg tccactcagc cactggattt 240
aagcagagtt caaaagccct tcagcggcca gtagcatctg actttgagcc tcagggtctg 300
agtgaagccg ctcgttggaa ctccaaggaa aaccttctcg ctggacccag tgaaaatgac 360
cccaaccttt tcgttgcact gtatgatttt gtggccagtg gagataacac tctaagcata 420
actaaaggtg aaaagctccg ggtcttaggc tataatcaca atggggaatg gtgtgaagcc 480
<210> 3
<211> 29
<212> DNA
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<400> 3
ctacgtctcc tccgagagcc gcttcaaca 29
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<400> 4
cccgtcggcc accgttgaat gatgatg 27
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ccctggccga gttggtt 17
<210> 6
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acgagcggct tcactcagac cctgaggctc 30
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tccacagcat tccgctgacc atcaataagg aag 33
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Claims (10)
1. A preparation method of a calibrator and a reference substance for BCR-ABL gene fusion detection is characterized by comprising the following steps:
respectively preparing ABL wild type armor RNA and BCR-ABL fusion type armor RNA;
dissolving the ABL wild-type armor RNA and the BCR-ABL fusion-type armor RNA respectively by using a protective diluent;
detecting the concentration of the ABL wild-type armor RNA and the BCR-ABL fused armor RNA by digital PCR;
carrying out gradient dilution on the ABL wild type armor RNA and the BCR-ABL fusion type armor RNA by using a protection diluent to obtain a calibrator; and mixing the ABL wild type armor RNA calibrator and the BCR-ABL fusion type armor RNA calibrator in proportion to obtain a reference product.
2. The method for preparing a calibrator and a reference for BCR-ABL gene fusion detection according to claim 1, wherein the nucleotide sequence encoding the ABL wild-type armor RNA is represented by SEQ ID No. 1;
preferably, the nucleotide sequence for coding the BCR-ABL fusion type armor RNA is shown as SEQ ID No. 2;
preferably, the method also comprises the step of cracking the ABL wild-type armor RNA and the BCR-ABL fusion-type armor RNA in water bath at 85-95 ℃ for 4-8 min before dissolving;
preferably, the protection diluent comprises a Tris-EDTA buffer, a protection nucleic acid and a protective agent;
preferably, the pH value of the Tris-EDTA buffer solution is 7.0-8.5;
preferably, the protective nucleic acid comprises vector RNA and/or salmon sperm DNA;
preferably, the final concentration of the protective nucleic acid is 10-60 ng/. mu.L;
preferably, the protectant comprises Triton X-100 and/or Tween 20;
preferably, the final concentration of the Triton X-100 is 0.005% -0.05%;
preferably, the final concentration of the Tween20 is 0.05-0.5%.
3. The method for preparing a calibrator and a reference for BCR-ABL gene fusion detection according to claim 1 or 2, wherein the reaction system of the digital PCR comprises a primer pair, a fluorescent probe and a premix;
preferably, the final concentration of the primer pair in the reaction system is 600-1200 nM, and the final concentration of the fluorescent probe in the reaction system is 80-400 nM;
preferably, the sequence of a primer pair for amplifying the ABL wild-type armored RNA is shown as SEQ ID No. 3-4, and the sequence of a fluorescent probe for detecting the ABL wild-type armored RNA is shown as SEQ ID No. 5;
preferably, the sequence of a primer pair for amplifying the BCR-ABL fused armor RNA is shown as SEQ ID No. 6-7, and the sequence of a fluorescent probe for detecting the BCR-ABL fused armor RNA is shown as SEQ ID No. 8;
preferably, the 5 'end of the fluorescent probe is marked with a fluorescence generating group, and the 3' end of the fluorescent probe is marked with a fluorescence quenching group;
preferably, the fluorogenic group comprises any one of FAM, HEX, VIC, TAMRA, ROX, Cy3, or Cy 5;
preferably, the fluorescence quenching group comprises any one of MGB, BHQ1, BHQ2 or BHQ 3;
preferably, the premix comprises a buffer, a reverse transcriptase, a DNA polymerase and dNTPs;
preferably, the reaction procedure of the digital PCR comprises:
reverse transcription: at 40-60 ℃ for 13-16 min;
pre-denaturation: 94-98 ℃ for 8-12 min;
and (3) circulating amplification: 94-98 ℃ for 10-30 s; 50-60 ℃ for 20-60 s; and circulating for 40-50 times.
4. The method for preparing a calibrator and a reference for BCR-ABL gene fusion assay according to any one of claims 1 to 3, wherein the final concentrations of the ABL wild-type armored RNA calibrator and the BCR-ABL fusion armored RNA calibrator after the gradient dilution are (1-5) x 105Copy/. mu.L, (1-5). times.104Copy/. mu.L, (1-5). times.103Copy/. mu.L, (1-5). times.102Copy/. mu.L, (1-5). times.101Copy/. mu.L and 1-5 copies/. mu.L;
preferably, in the reference product, the concentration ratio of the ABL wild type armor RNA calibrator to the BCR-ABL fusion type armor RNA calibrator is 10:1, 100:1, 1000:1 and 10000:1 in sequence.
5. The method for preparing a calibrator and a reference for BCR-ABL gene fusion detection according to any one of claims 1 to 4, wherein the method comprises the following steps:
(1) preparation of armored RNA:
respectively preparing ABL wild type armor RNA and BCR-ABL fusion type armor RNA, wherein the nucleotide sequence for coding the ABL wild type armor RNA is shown as SEQ ID No.1, and the nucleotide sequence for coding the BCR-ABL fusion type armor RNA is shown as SEQ ID No. 2;
(2) dissolving:
cracking the ABL wild armor RNA and the BCR-ABL fusion type armor RNA in water bath at 85-95 ℃ for 4-8 min, and then respectively dissolving by using protection diluent;
wherein the protective diluent comprises Tris-EDTA buffer solution with the pH value of 7-8.5, carrier RNA and/or salmon sperm DNA with the final concentration of 10-60 ng/mu L, and 0.005-0.05% of Triton X-100 and/or 0.05-0.5% of Tween 20;
(3) and (3) concentration detection:
the concentrations of the ABL wild-type armor RNA and BCR-ABL fused armor RNA were detected by digital PCR:
the reaction system of the digital PCR comprises a primer pair with the final concentration of 600-1200 nM, a fluorescent probe with the final concentration of 80-400 nM and a premixed solution;
the sequence of a primer pair for amplifying the ABL wild-type armored RNA is shown as SEQ ID No. 3-4, and the sequence of a fluorescent probe for detecting the ABL wild-type armored RNA is shown as SEQ ID No. 5;
the sequence of a primer pair for amplifying the BCR-ABL fused armored RNA is shown as SEQ ID No. 6-7, and the sequence of a fluorescent probe for detecting the BCR-ABL fused armored RNA is shown as SEQ ID No. 8;
the premixed liquid comprises buffer solution, reverse transcriptase, DNA polymerase and dNTPs;
the reaction procedure of the digital PCR comprises:
reverse transcription: at 40-60 ℃ for 13-16 min;
pre-denaturation: 94-98 ℃ for 8-12 min;
and (3) circulating amplification: 94-98 ℃ for 10-30 s; 50-60 ℃ for 20-60 s; circulating for 40-50 times;
(4) preparation of calibrators and reference samples:
using protection diluent to carry out gradient dilution on the ABL wild armor RNA and the BCR-ABL fusion type armor RNA until the final concentration is (1-5) multiplied by 10 in sequence5Copy/. mu.L, (1-5). times.104Copy/. mu.L, (1-5). times.103Copy/. mu.L, (1-5). times.102Copy/. mu.L, (1-5). times.101Copying/mu L and 1-5 copies/mu L to obtain a calibrator;
mixing the ABL wild type armor RNA calibrator and the BCR-ABL fusion type armor RNA calibrator in a concentration ratio of 10:1, 100:1, 1000:1 and 10000:1 in sequence to obtain a reference product.
6. A calibrator and a reference for BCR-ABL gene fusion detection, wherein the calibrator and the reference are prepared by the preparation method of the calibrator and the reference for BCR-ABL gene fusion detection according to any one of claims 1 to 5.
7. A method of testing the accuracy of a calibrator and a reference for the BCR-ABL gene fusion test of claim 6, comprising:
amplifying the calibration product, the reference product and the WHO primary standard product, constructing a standard curve of the cycle number and the copy number of the calibration product, then constructing a linear relation curve of an actual detection value and a theoretical value of the WHO primary standard product, then constructing a linear relation curve of an actual detection value and a theoretical value of the reference product, and judging the accuracy according to curve parameters.
8. The method of claim 7, wherein the reaction system for amplification comprises primer pairs, fluorescent probes, PCR buffer, reverse transcription buffer, reverse transcriptase, DNA polymerase, rnase inhibitors, and dNTPs;
preferably, the final concentration of the primer pair is 200-1000 nM, and the final concentration of the fluorescent probe is 100-500 nM;
preferably, the final concentration of the reverse transcriptase is 0.1-3U/mu L;
preferably, the final concentration of the DNA polymerase is 0.05-0.2U/muL;
preferably, the final concentration of the RNase inhibitor is 0.1-1U/. mu.L;
preferably, the final concentration of the dNTPs is 100-500 mu M;
preferably, the reaction procedure for the amplification is the same as that for digital PCR.
9. The method of claim 7 or 8, wherein the method comprises:
(1) amplifying a calibrator, a reference and a WHO primary standard, wherein an amplified reaction system comprises a primer pair with a final concentration of 200-1000 nM, a fluorescent probe with a final concentration of 100-500 nM, a PCR buffer solution, a reverse transcription buffer solution, reverse transcriptase with a final concentration of 0.1-3U/muL, DNA polymerase with a final concentration of 0.05-0.2U/muL, RNase inhibitor with a final concentration of 0.1-1U/muL and dNTPs with a final concentration of 100-500 muM, and the amplified reaction procedure is the same as that of digital PCR;
(2) and (3) establishing a standard curve of the cycle number and the copy number of the calibration product, then establishing a linear relation curve of an actual detection value and a theoretical value of the WHO primary standard product, then establishing a linear relation curve of an actual detection value and a theoretical value of the reference product, and judging the accuracy according to curve parameters.
10. The use of the method for preparing the calibrator and the reference for BCR-ABL gene fusion detection according to any one of claims 1 to 5 and/or the method for detecting the accuracy of the calibrator and the reference for BCR-ABL gene fusion detection according to any one of claims 7 to 9 in the preparation of the calibrator and the reference for BCR-ABL gene fusion detection.
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