CN113789389A - Human leukemia BCR-ABL fusion mutation one-step detection kit - Google Patents
Human leukemia BCR-ABL fusion mutation one-step detection kit Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, in particular to a human leukemia BCR-ABL fusion mutation one-step detection kit based on digital PCR technology one-step detection; comprises a premix solution for micro-drop digital PCR; BCR-ABL P210 mutation detection mixed liquor comprises a BCR-ABL P210 mRNA primer and probe combination and an internal standard gene GUSB mRNA primer and probe combination; positive control, negative control and blank control; according to the invention, on a MicroDrop-100 digital PCR platform, RNA extracted from peripheral blood or bone marrow samples is directly used as a template through a one-step method, the RNA is reversely transcribed into cDNA without additional experiments, the RNA is not required to be concentrated, the target gene copy number in the RNA samples is detected to obtain an absolute quantitative result, a standard curve is not required to be established, and the single-hole detection sensitivity can reach 0.001%, so that the method has the advantages of simple and efficient detection process, absolute quantification, high specificity, high accuracy, high sensitivity and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a human leukemia BCR-ABL fusion mutation one-step detection kit based on digital PCR technology one-step detection.
Background
Philadelphia chromosome (Ph) is a minute chromosome resulting from a balanced translocation between human chromosome 9 and chromosome 22 that was first discovered in 1960. This translocation results in the parallel translocation of the long-arm ABL gene (position q34) located on human chromosome 9 with the long-arm BCR gene (position q11) on chromosome 22 to produce a novel fusion gene BCR-ABL; there are three variants of the BCR-ABL fusion gene: the P190 type, the P210 type and the P230 type, wherein the P210 type (also called B3a2/B2a 2) (or called "e 14a2/e13a 2") is the most common, and accounts for more than 90% of Chronic myelogenous leukemia patients (CML) and 1/3 Ph positive adult precursor B-cell lymphocytic leukemia (BALL) patients. The gene can transcribe an abnormal mRNA of 8.5kb, and is finally translated into a protein P210 with the size of 210kD, wherein the P210 has stronger tyrosine protein kinase activity, and can activate protooncogenes and certain cell factors through various signal transduction pathways to cause the malignant transformation of cells.
The expression level of the BCR-ABL fusion gene can be used as a diagnosis basis for the classification of leukemia patients, a targeted medication guide, and a reliable index for the detection and prognosis evaluation of minimal residual disease; monitoring BCR-ABL% mutation levels in the blood of patients has become an international standard for patient management.
At present, the detection methods commonly used for the BCR-ABL fusion gene comprise cell conventional chromosome detection, Fluorescence In Situ Hybridization (FISH), immunophenotyping, polymerase chain reaction and the like. The conventional chromosome detection is a basic clinical auxiliary diagnosis CML method, and the FISH is the progress of the conventional chromosome detection, takes bone marrow or peripheral blood as a research object, can detect metaphase and interphase nuclear cells, and can synchronously detect and analyze occult types and variability. The sensitivity is high, the specificity is good, but when a common bi-color single fusion probe is used for detecting bcr/abl fusion genes, the false positive rate of 4% -10% can be caused by random distribution or optical overlapping of bcr and abl gene signals. An excessively high false positive rate not only affects the sensitivity and specificity, but also affects the interpretation of the detection result of the patient to a certain extent. The Flow Cytometry (FCM) can be used for quickly analyzing a large number of cells, and is accurate and convenient. However, the FCM method requires special instruments, has high operation technical requirements, is expensive, and has poor result repeatability. The polymerase chain reaction detection method mainly comprises a direct sequencing method after reverse transcription, common PCR after reverse transcription, fluorescent quantitative PCR and the like. The common PCR or direct sequencing method after reverse transcription is the most direct BCR-ABL fusion gene detection method, but the detection sensitivity is low, and the method is commonly used for patients in initial diagnosis. The qPCR technology is low in detection cost, but the qPCR technology depends on a standard reference substance in quantitative detection, is easily influenced by primer amplification efficiency, so that the detection sensitivity and accuracy are not high enough, and the effective monitoring on the minimal residual disease cannot be realized. In conclusion, the detection accuracy and sensitivity of the existing BCR-ABL fusion gene or Ph positive chromosome detection method are not high.
The accuracy of the detection report results directly influences the diagnosis of clinical diseases and the judgment of treatment effects, so that the development of more sensitive and accurate quantitative methods and reagents for the expression level of the BCR-ABL fusion gene mRNA in blood and the establishment of standardized detection and result judgment methods are urgently needed to improve the reliability of detection clinical application. Digital PCR (digital PCR) is a technology that has emerged in recent years, and its principle is to distribute a standard PCR reaction into a large number of tiny reactors, each of which may or may not contain one or more copies of a target molecule (DNA template), to achieve "single-molecule template PCR amplification", and after PCR amplification, to perform fluorescence intensity detection by a biochip analyzer. The intensity of the fluorescence signal of the positive droplets comprising the nucleic acid molecule is increased compared to the negative droplets, and the absolute copy number of the target nucleic acid molecule is calculated by the proportion of positive droplets according to the principle of poisson distribution. The whole process does not depend on a standard curve and a reference sample, and the sample is directly detected to obtain the copy number of the target sequence. Digital PCR is particularly useful in applications that do not resolve well depending on Ct values: copy number variation, mutation detection, gene relative expression research, second-generation sequencing result verification, miRNA expression analysis, single-cell gene expression analysis and the like. Therefore, the method is also suitable for effective monitoring for detecting the BCR-ABL fusion mutation minimal residual disease.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a detection kit for detecting human leukemia BCR-ABL fusion mutation based on a digital PCR (polymerase chain reaction) one-step method, and aims to improve the accuracy and sensitivity of BCR-ABL fusion gene detection.
In order to achieve the purpose, the purpose of the invention is realized by the following technical scheme:
the invention provides a human leukemia BCR-ABL fusion mutation one-step detection kit, which is characterized by comprising the following components in parts by weight:
a premix for droplet-type digital PCR;
BCR-ABL P210 mutation detection mixed liquor comprises a BCR-ABL P210 mRNA primer and probe combination and an internal standard gene GUSB mRNA primer and probe combination;
positive control, negative control and blank control.
Preferably, the sequence of the BCR-ABL P210 mRNA primer and probe combination is as follows:
BCR-ABL-P210 F1a: 5' -CCGCTGACCATCAAYAAG-3’；
BCR-ABL-P210 R5: 5' -CCAACGAGCGGCTTCA-3’；
BCR-ABL-P210 P5:5' -FAM-CCAGTAGCATCTGACTTTGAG-MGB-3'。
preferably, the sequences of the primer and probe combination of the internal standard gene GUSB mRNA are as follows:
GUSB-F1：5'-ACGCAGAAAATACGTGGT-3'
GUSB-R1:5'-CCGAGTGAAGATCCCCTTTTTA-3'
GUSB-P1：5'-VIC-GATTTCATGACTGAACAGTCA-MGB-3'。
preferably, in the mRNA primer and probe combination of BCR-ABL-P210, the working concentration of the primer is 500-1000nM, wherein the preferred concentration is 900nM, and the working concentration of the probe is 100-300nM, wherein the preferred concentration is 250 nM.
Preferably, in the primer and probe combination of the internal standard gene GUSB mRNA, the working concentration of the primer is 500-1000nM, wherein the preferred concentration is 900nM, and the working concentration of the probe is 100-300nM, wherein the preferred concentration is 250 nM.
Preferably, the premix solution for the micro-drop digital PCR comprises buffer solution, dNTPs, Taq enzyme and reverse transcriptase.
Preferably, the BCR-ABL P210 mRNA primer and probe combination and the primer and probe combination of the internal standard gene GUSB mRNA are prepared into 10X BCR-ABL P210 mutation detection mixed liquor.
Preferably, the positive control is prepared by RNA extracted from a K562 cell line with mutation of a BCR-ABL fusion gene.
Preferably, the negative control is prepared from RNA extracted from a mutation-negative HL60 cell line.
Preferably, the blank control is obtained by water split charging without ribozyme and is used for monitoring whether the detection reagent is polluted or not.
The invention relates to an application method for detecting by using a human leukemia BCR-ABL fusion mutation one-step detection kit, which comprises the following steps:
(1) firstly, extracting RNA of a sample, and preparing an extraction kit;
(2) preparing a PCR reaction system: taking a premix for micro-drop digital PCR, a BCR-ABL P210 mutation detection mixture, an RNA sample and ribozyme-free water for each sample detection; and simultaneously preparing a positive control, a negative control and a blank control for detection: respectively replacing RNA samples in the PCR reaction system to obtain; after each tube of PCR reaction system is prepared, carrying out rotary oscillation for several seconds, and carrying out centrifugal mixing uniformly;
(3) droplet preparation, PCR amplification: carrying out droplet preparation on the PCR reaction system in the step (2) by adopting a sample preparation instrument chip of a MicroDrop-100 digital PCR platform, and amplifying the obtained droplets by using a PCR (polymerase chain reaction) amplification instrument;
(4) droplet fluorescence detection: and (3) carrying out fluorescence detection on the microdroplets after the PCR amplification in the step (3) by adopting a biochip analyzer of a MicroDrop-100 digital PCR platform, judging whether the sample contains the fusion gene according to the type of a fluorescence signal, and analyzing to obtain the copy number mutation ratio.
Preferably, in the method of application,
in the extraction kit in the step (1), after RNA is extracted, the concentration is detected by a NanoDrop micro spectrophotometer, and the RNA concentration is not lower than 100 ng/mu L;
the sample in the step (2) is RNA extracted from whole blood or marrow fluid;
the adding amount of the RNA sample in the PCR reaction system of the step (2) is not less than 500 ng;
the PCR reaction system of the step (2) is 20 mu L: wherein, the premix 5 mu L, BCR-ABL P210 mutation detection mixture for the micro-drop digital PCR is 12.5 mu L of a2 mu L, RNA sample, and nuclease-free water is supplemented to the total volume of 20 mu L;
the PCR amplification procedure of the step (3) is as follows: reacting at 50 ℃ for 30 min; reacting at 95 ℃ for 10 min; reaction at 95 ℃ for 30sec, at 62 ℃ for 40sec, 45 cycles; reacting at 98 ℃ for 10 min; terminating at 16 ℃; setting the temperature rise and fall speed of 1 ℃/sec in each step;
in the step (4), the channel 1 fluorescent droplet for droplet fluorescence detection is a BCR-ABL P210 signal droplet, the channel 2 fluorescent droplet is an internal reference gene GUSB signal droplet, and the copy number obtained by detecting the two channel signal droplets can be used for calculating the mutation ratio level of BCR-ABL P210/GUSB%.
Preferably, compared with the prior art, the invention has the following beneficial effects:
compared with other existing detection technologies, the technology provided by the invention has the advantages of simpler, more convenient and efficient detection process, high result accuracy, high sensitivity and the like, and specifically comprises the following steps:
(1) the detection process is simpler and more efficient: the method is different from the traditional two-step detection method, the extracted RNA sample is directly added into a PCR detection system, the reverse transcription and the gene amplification are completed in one system by one-time experimental operation, the experiment of reverse transcribing RNA into cDNA in advance is not needed, and the operation steps are simplified;
(2) the detection result has high accuracy: the method is based on a Yongnuo medical MicroDrop-100 micro-drop digital PCR technology platform, an absolute quantitative mode is adopted, a standard curve and a reference sample are not depended on, the copy number of a target sequence is directly detected, and the accuracy is high. The principle is that a standard PCR reaction is distributed into a large number of tiny reactors, each reactor contains or does not contain one or more copies of target molecules (RNA templates), so that 'single-molecule template PCR amplification' is realized, and after the PCR amplification, a biochip reader carries out fluorescence intensity detection on micro-droplets one by one. The intensity of the fluorescence signal of the positive microdroplets comprising the nucleic acid molecules is increased compared to the negative microdroplets. And finally, according to the principle of Poisson distribution, calculating the absolute copy number of the target nucleic acid molecule according to the proportion of the positive micro-droplets. In addition, the GUSB gene is selected as an internal reference gene, so that the generation of false positive in the PCR amplification process can be effectively avoided, and the detection result has strong specificity;
(3) the sensitivity is high: the invention is based on the Yongnuo medical MicroDrop-100 micro-drop digital PCR technical platform, uses the one-step hypersensitive micro-drop digital PCR premix solution for Yongnuo medical production, only needs 1-1.5ml of peripheral blood of a detected person to extract RNA, does not need to additionally perform concentration operation on the extracted RNA sample, can obtain more than 30 ten thousand internal reference gene copy numbers by direct detection, achieves the detection level of 0.001% mutation ratio, realizes the monitoring of MR 5.0, and has the advantage of ultrahigh sensitivity.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention.
FIG. 1 is a two-dimensional graph of the RNA results of the BCR-ABL P210 negative cell line HL60 detection of the present invention;
FIG. 2 is a two-dimensional graph of the RNA results of the test of clinical negative blood samples according to the invention;
FIG. 3 is a two-dimensional graph of the RNA results of the BCR-ABL P210 mutant cell line K562 of the present invention;
FIG. 4 is a two-dimensional graph of the results of the detection of the BCR-ABL fusion mutation international standard ERM-AD623d according to the present invention;
FIG. 5 is a one-dimensional graph of the results of the present invention using 4. mu.L each of 5 tubes of BCR-ABL P210/GUSB% mutation ratio linear sample mixture as a sample;
FIG. 6 is a graph of the linear results of the present invention performed with 5 tubes of BCR-ABL P210/GUSB% mutation ratio linear sample mix;
FIG. 7 is a one-dimensional graph showing the results of the assay of the present invention using 4. mu.L each of the 4-tube RNA BCR-ABL P210/GUSB% mutation ratio mixtures as a sample.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained by a person of ordinary skill in the art without any creative work based on the embodiments of the present invention belong to the protection scope of the present invention.
Example 1
A human leukemia BCR-ABL fusion mutation one-step detection kit comprises:
(1) the premix for the micro-drop digital PCR is a premix for the one-step hypersensitive micro-drop digital PCR: comprises buffer solution, dNTPs, Taq enzyme, reverse transcriptase and the like (product of Guangdong Yongno medical science and technology Limited company, the product number is S0200020401, and the micro drop type digital PCR system is matched for use);
(2) the BCR-ABL P210 mutation detection mixed solution comprises a BCR-ABL P210 mRNA primer and probe combination and an internal standard gene GUSB mRNA primer and probe combination:
the sequence of the BCR-ABL P210 mRNA primer and probe combination is as follows:
BCR-ABL-P210 F1a: 5' -CCGCTGACCATCAAYAAG-3’；
BCR-ABL-P210 R5: 5' -CCAACGAGCGGCTTCA-3’；
BCR-ABL-P210 P5:5' -FAM-CCAGTAGCATCTGACTTTGAG-MGB-3'；
in the mRNA primer and probe combination of the BCR-ABL-P210, the working concentration of the primer is 500-1000nM, preferably 900nM, and the working concentration of the probe is 100-300nM, preferably 250 nM;
the sequences of the primer and probe combination of the internal standard gene GUSB mRNA are as follows:
GUSB-F1：5'-ACGCAGAAAATACGTGGT-3'
GUSB-R1:5'-CCGAGTGAAGATCCCCTTTTTA-3'
GUSB-P1：5'-VIC-GATTTCATGACTGAACAGTCA-MGB-3'；
in the GUSB mRNA primer and probe combination, the working concentration of the primer is 500-1000nM, preferably 900nM, and the working concentration of the probe is 100-300nM, preferably 250 nM;
the BCR-ABL P210 mRNA primer and probe combination and the primer and probe combination of the internal standard gene GUSB mRNA are prepared into 10 multiplied BCR-ABL P210 mutation detection mixed liquor;
(3) positive control: the positive control is prepared by RNA extracted from a K562 cell line with BCR-ABL fusion gene mutation;
(4) negative control: the negative control is prepared by RNA extracted from BCR-ABL fusion gene mutation negative HL60 cell line;
(5) blank control: the blank control is obtained by commercial ribozyme-free water split charging and is used for monitoring the environment or monitoring whether the reagent is polluted by the positive sample.
Secondly, providing a one-step micro-drop digital PCR detection method for human leukemia BCR-ABL fusion mutation, which comprises the following steps:
(1) sample preparation: the type of the applicable sample is whole blood or bone marrow;
(2) sample processing and nucleic acid extraction (sample processing zone)
Purchasing a commercial nucleic acid extraction kit, extracting RNA from a sample according to the instruction of the nucleic acid extraction kit, and measuring the concentration of the extracted RNA solution by using Nano-Drop, wherein the concentration of the RNA is not lower than 100 ng/mu L;
(3) reagent preparation (reagent preparation area)
a. Thawing all components of the kit to room temperature, fully dissolving all the components, then oscillating and mixing uniformly, and centrifuging for a short time;
b. the number of reactions N, N = the number of samples to be tested (N) + the number of control samples (3) +1, and the total volume of the sample reaction system was determined to be 20. mu.L. Taking corresponding sample number of non-ribozyme centrifuge tubes, preparing reaction mixture for the required reaction amount at room temperature according to the following table, sucking and beating a pipette gun for 5-10 times, fully and uniformly mixing, and subpackaging the mixed solution into independent non-ribozyme-polluted centrifuge tubes according to 7 mul/tube after short centrifugation, wherein the following table 1:
TABLE 1
c. Application of sample (sample preparation zone)
Directly using the extracted RNA as a template for detection, adding RNA in an amount not less than 500ng into the split reaction mixed liquid tube in the step, adding the volume of an RNA sample to the total reaction volume of 20 mu L, and supplementing ribozyme-free water to the total reaction volume of 20 mu L if the total reaction volume of the added sample is less than 20 mu L; and tightly covering the pipe cover, shaking for several seconds to mix uniformly, centrifuging for a short time to completely throw the liquid on the pipe wall to the pipe bottom (to avoid generating bubbles), and then preparing micro-droplets.
(4) Preparation of microdroplets
d. And taking 20 mu L of reaction mixed liquor in the reaction tube, and generating micro-droplets, wherein the specific operation steps are carried out according to the instruction of the Yongnuo medical MicroDrop-100 sample preparation instrument.
(5) PCR amplification (amplification detection zone)
Carrying out PCR amplification on the 96-hole PCR reaction plate with the sealed membrane, wherein the amplification procedure is as follows: reacting at 50 ℃ for 30 min; reacting at 95 ℃ for 10 min; reaction at 95 ℃ for 30sec, at 62 ℃ for 40sec, 45 cycles; reacting at 98 ℃ for 10 min; terminating at 16 ℃; the temperature rise and fall speed of 1 ℃/sec is set in each step.
(6) Microdroplet detection and result analysis
After PCR amplification is finished, placing a 96-hole PCR reaction plate in a biochip analyzer of a Yongnuo medical MicroDrop-100 digital PCR platform for detection according to the instruction of a specification to obtain a gene copy number result;
(7) and (3) judging the detection result, specifically comprising the following steps:
h. judging droplet generation effectiveness: the total number of the droplets in each reaction tube is more than or equal to 50000, and if the total number of the droplets is less than 50000, the droplets in the reaction holes are not generated ideally and need to be generated again;
i. the sum of the number of the positive micro-drops falling in the channel 1 and the double-positive area is more than or equal to 3, the positive sample is judged, and the formula is calculated according to the fusion gene proportion: calculating channel 1/(channel 1+ channel 2) to obtain the mutation ratio of the fusion gene;
j. the sum of the number of positive micro drops falling in the channel 1 and the double positive area is less than 3, and the copy number of the internal standard gene is less than 3000, and the nucleic acid is recommended to be re-extracted so as to avoid the detection omission caused by the insufficient addition amount of the nucleic acid;
k. the sum of the number of the positive micro-drops falling in the channel 1 and the double-positive area is less than 3, and the copy number of the internal standard gene is more than or equal to 3000, and the result is judged to be lower than the detection limit.
The detection limit of the kit is related to the total copy number in the RNA sample added to the detection system. When the total copy number of the reference gene and the mutant gene entering a detection system is more than or equal to 500000 copies, the kit can detect 0.001 percent of the mutation level of the BCR-ABL P210 fusion gene.
Example 2: a kit developed by a plasmid sample on a MicroDrop-100 digital PCR platform is used for carrying out a sensitivity and minimum mutation ratio detection limit test experiment, and the method comprises the following specific steps:
(1) a commercially artificially synthesized BCR-ABL P210 positive plasmid (concentration x 10)5cp/. mu.L) with internal standard GUSB plasmid (concentration x 10)5cp/mu L) as mother liquor, and preparing the mother liquor by dilution according to the theoretical mutation proportion gradient to prepare 5 tubes of mixed liquor with the mutation ratio of 10 percent, 1 percent, 0.1 percent, 0.01 percent and 0.001 percent of BCR-ABL P210/GUSB percent;
(2) taking 4 mu L of the 5-tube BCR-ABL P210/GUSB% mutation ratio mixed solution as samples, and detecting according to the detection step method of the kit;
(3) the results of the detection are shown in the one-dimensional graph of fig. 5 and table 2:
TABLE 2
| Sample name | Total number of droplets | Channel 1P 210 | Channel | 2 reference gene copy number | Total number of copies | Measured ratio of |
Measured LOG10 | Theoretical LOG10 |
| BCR-ABLP210/GUSB-0.001% | 70041 | 2.1 | 293432.0 | 293433.9 | 0.0007% | -5.2 | -5.0 | |
| BCR-ABLP210/GUSB-0.01% | 74324 | 16.9 | 309447.8 | 309464.6 | 0.005% | -4.3 | -4.0 | |
| BCR-ABLP210/GUSB-0.1% | 72550 | 178.5 | 297905.6 | 298084.1 | 0.060% | -3.2 | -3.0 | |
| BCR-ABLP210/GUSB-1% | 70907 | 2184.0 | 286452.6 | 288636.7 | 0.762% | -2.1 | -2.0 | |
| BBCR-ABLP210/GUSB-10% | 74171 | 30039.8 | 322743.2 | 352783.0 | 9.308% | -1 | -1.0 |
(4) The lowest mutation ratio sample BCR-ABL P210/GUSB-0.001% is repeated 20 times to obtain the results shown in the following table 3, and the results are all at the mutation ratio level of 0.001% and the positive detection rate is 100%:
TABLE 3
(5) According to the results described in example 2, the detection result of the kit of the invention is consistent with the theoretical value, which shows that the kit of the invention has high sensitivity and accuracy. The present invention detects a mutation level of 0.001%.
Example 3: accuracy test experiment of kit developed on MicroDrop-100 digital PCR platform is carried out by using European Standard office BCR-ABL pDNA calibrator ERM-AD623 plasmid sample
(1) The detection sample is BCR-ABL pDNA calibrator ERM-AD623, the operation steps are carried out according to a kit method, and the obtained results are shown in the following table 4:
TABLE 4
(2) According to the results shown in the table, the kit provided by the invention is used for detecting the international standard substance, the result shows that the R2 is more than 0.99 and is consistent with the theoretical value, the detection result is high in accuracy, and the copy number is within the range of the theoretical value.
Example 4: the kit developed on a MicroDrop-100 digital PCR platform is subjected to linear test by using a cell line RNA sample, and the specific steps are as follows:
(1) after the concentration of RNA extracted from BCR-ABL P210 mutant positive K562 cells and negative HL60 samples is detected by Nano-Drop, the RNA is respectively diluted by ribozyme-free water to be about 125 ng/muL, HL60 RNA (125 ng/muL) is used for preparing a mutation ratio dilution mother solution, the K562 RNA (125 ng/muL) is diluted in a gradient manner according to the volume ratio, and mixed RNA samples with the concentration of about 1%, 0.1%, 0.01% and 0.001% are prepared:
(2) taking 4 mu L of the 4-tube RNA BCR-ABL P210/GUSB% mutation ratio mixed solution respectively, and detecting according to the detection step method of the kit;
(3) the results of the measurements are shown in the following table 5 and the one-dimensional graph of fig. 7:
TABLE 5
(4) According to the results described in example 4, the detection result of the kit of the invention conforms to the theoretical value, which indicates that the kit of the invention has high sensitivity and accuracy, and the mutation level of the invention can be detected to be 0.001%.
Example 5: the kit developed on a MicroDrop-100 digital PCR platform is subjected to specificity test by using a clinical negative whole blood RNA sample, and the specific steps are as follows:
(1) RNA was extracted from 16 clinically negative peripheral whole blood samples: according to the instruction of purchasing a commercial total RNA extraction kit, 250 mu L of whole blood samples are respectively taken for RNA extraction in 8 cases, 1.2ml of whole blood samples are taken for RNA extraction in 8 cases, and the volumes of extracted and eluted RNA are all 30 mu L;
(2) performing nucleic acid concentration determination on the RNA solution extracted in the step (1) by using Nano-Drop, wherein the concentration result is shown in the following table 6;
(3) taking 2 mu L of the RNA sample extracted from 250 mu L of whole blood in the step (1), and detecting according to the detection step method of the kit;
(4) taking 12.5 mu L of RNA sample extracted from 1.2ml of whole blood in the step (1), and detecting according to the detection step method of the kit;
(5) the results of the tests are shown in tables 6 and 7 below:
TABLE 6
TABLE 7
(6) According to the results described in example 5, the kit of the invention detects clinically negative peripheral blood sample RNA with the ratio of less than 0.001%, and the samples are all negative; in addition, the invention can detect the background with the total copy number of more than 30 ten thousand only by less than 1.5mL of peripheral blood sample, thereby reaching the monitoring level of M5.0.
Example 6: clinical positive samples are used for carrying out verification test on kit developed on MicroDrop-100 digital PCR platform
(1) The remaining 3 positive mutant RNA samples were tested at the hospital hematological laboratory: 1/3/8 sample known mutation ratio;
(2) the kit of the invention is used for carrying out the multi-well detection of each sample, and the result is shown in the following table 8;
(3) according to the results described in example 6, the kit of the present invention detects that the clinically positive mutant RNA sample has a known ratio to the clinically positive mutant RNA sample.
TABLE 8
The above description is only a preferred embodiment of the present invention, and is not intended to limit the technical scope of the present invention, and those skilled in the art may make modifications and variations within the spirit of the present invention, and all modifications, equivalents and modifications of the above embodiments according to the technical spirit of the present invention are within the scope of the present invention.
Sequence listing
SEQUENCE LISTING
<110> Guangdong Yongnuo medical science and technology Co., Ltd
<120> human leukemia BCR-ABL fusion mutation one-step detection kit
<160>6
<210>1
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
CCGCTGACCATCAAYAAG 18
<210>2
<211>16
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
CCAACGAGCGGCTTCA 16
<210>3
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
CCAGTAGCATCTGACTTTGAG 21
<210>4
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
ACGCAGAAAATACGTGGT 18
<210>5
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
CCGAGTGAAGATCCCCTTTTTA 22
<210>6
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
GATTTCATGACTGAACAGTCA 21
Claims (6)
1. The human leukemia BCR-ABL fusion mutation one-step detection kit is characterized by comprising:
a premix for droplet-type digital PCR;
BCR-ABL P210 mutation detection mixed liquor comprises a BCR-ABL P210 mRNA primer and probe combination and an internal standard gene GUSB mRNA primer and probe combination; wherein:
the sequence of the BCR-ABL P210 mRNA primer and probe combination is as follows:
BCR-ABL-P210 F1a: 5' -CCGCTGACCATCAAYAAG-3’；
BCR-ABL-P210 R5: 5' -CCAACGAGCGGCTTCA-3’；
BCR-ABL-P210 P5:5' -FAM-CCAGTAGCATCTGACTTTGAG-MGB-3'；
the sequences of the primer and probe combination of the internal standard gene GUSB mRNA are as follows:
GUSB-F1：5'-ACGCAGAAAATACGTGGT-3'
GUSB-R1:5'-CCGAGTGAAGATCCCCTTTTTA-3'
GUSB-P1：5'-VIC-GATTTCATGACTGAACAGTCA-MGB-3'；
positive control, negative control and blank control.
2. The human leukemia BCR-ABL fusion mutation one-step detection kit as claimed in claim 1, wherein in the combination of the mRNA primer and the probe of BCR-ABL-P210, the working concentration of the primer is 500-1000nM, and the working concentration of the probe is 100-300 nM; in the primer and probe combination of the internal standard gene GUSB mRNA, the working concentration of the primer is 500-1000nM, and the working concentration of the probe is 100-300 nM.
3. The human leukemia BCR-ABL fusion mutation one-step detection kit as claimed in claim 2, wherein the pre-mixed solution for the micro-droplet digital PCR comprises buffer solution, dNTPs, Taq enzyme and reverse transcriptase.
4. The human leukemia BCR-ABL fusion mutation one-step detection kit as claimed in claim 3, wherein the BCR-ABL P210 mRNA primer and probe combination, the internal standard gene GUSB mRNA primer and probe combination are prepared into 10 x BCR-ABL P210 mutation detection mixed solution.
5. The human leukemia BCR-ABL fusion mutation one-step detection kit as claimed in claim 4, wherein the positive control is prepared from RNA extracted from K562 cell line with BCR-ABL fusion gene mutation; the negative control is prepared by RNA extracted from a mutation negative HL60 cell line.
6. The human leukemia BCR-ABL fusion mutation one-step detection kit as claimed in claim 5, wherein the blank control is obtained by ribozyme-free water split charging, and is used for monitoring whether the detection reagent is polluted or not.
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Citations (4)
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|---|---|---|---|---|
| CN106701957A (en) * | 2017-01-12 | 2017-05-24 | 广州蓝吉生物技术有限公司 | Kit for detecting fusion gene BCR-ABL (P210) mRNA expression |
| WO2019033065A1 (en) * | 2017-08-11 | 2019-02-14 | Atila Biosystems, Inc. | Digital amplification with primers of limited nucleotide composition |
| CN109554474A (en) * | 2018-12-29 | 2019-04-02 | 中山大学达安基因股份有限公司 | A kind of method and kit of BCR-ABL fusion quantitative detection |
| CN112301129A (en) * | 2020-11-05 | 2021-02-02 | 深圳荻硕贝肯精准医学有限公司 | BCR-ABL fusion gene detection kit and BCR-ABL fusion gene detection method |
-
2021
- 2021-11-18 CN CN202111366474.4A patent/CN113789389A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701957A (en) * | 2017-01-12 | 2017-05-24 | 广州蓝吉生物技术有限公司 | Kit for detecting fusion gene BCR-ABL (P210) mRNA expression |
| WO2019033065A1 (en) * | 2017-08-11 | 2019-02-14 | Atila Biosystems, Inc. | Digital amplification with primers of limited nucleotide composition |
| CN109554474A (en) * | 2018-12-29 | 2019-04-02 | 中山大学达安基因股份有限公司 | A kind of method and kit of BCR-ABL fusion quantitative detection |
| CN112301129A (en) * | 2020-11-05 | 2021-02-02 | 深圳荻硕贝肯精准医学有限公司 | BCR-ABL fusion gene detection kit and BCR-ABL fusion gene detection method |
Non-Patent Citations (2)
| Title |
|---|
| JACQUELINE MAIER等: "Optimized Digital Droplet PCR for BCR-ABL", 《J MOL DIAGN》 * |
| 王文军等: "数字PCR较荧光定量PCR可更敏感监测慢性粒细胞白血病患者BCR/ABL (P210)微小残留病及疾病进展", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
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