CN101624621B - Kit for quantitatively detecting BCR/ABL mRNA level - Google Patents
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Abstract
The invention discloses a kit for quantitatively detecting a BCR/ABL mRNA level. The kit comprises a standard product which is used for manufacturing a standard curve, an inner reference gene real-time quantitative PCR system and at least one of the following three real-time quantitative PCR systems: an M-type BCR/ABL real-time quantitative PCR system, m-type BCR/ABL real-time quantitative PCR system and a mu-type BCR/ABL real-time quantitative PCR system. The kit can accurately, quickly and quantitatively detect various BCR/ABL mRNA levels, is used for diagnosing chronic myelogenous leukemia and acute lymphoblastic leukemia expressed by BCR/ABL and monitoring minimal residual diseases in a treatment process, and provides an important molecular basis for accurate diagnosis of clinical diseases, determination of a treatment proposal, curative effect evaluation and prognosis.
Description
Technical field
The present invention relates to a kind of test kit of detection by quantitative BCR/ABL mRNA level.
Background technology
The adult's acute lymphoblastic leukemia (ALL) of 95% chronic myelogenous leukemia (CML), 25-30% and the children ALL patient of 2-5% all have distinctive Ph karyomit(e), by t (9; 22) mutual dystopy forms, and molecular level shows as the abl gene formation BCR/ABL fusion gene on No. 22 BCR genes on the karyomit(e) and No. 9 karyomit(e).Breaking point on the abl gene concentrates between the exons 1 b upper reaches, 1b and the 1a or the a2 upper reaches, and ABL fusion part is a2 on the mRNA level.Breaking point on the BCR gene mainly concentrates on following three zones: cross over the 5.8kb zone of exons 1 2-16, i.e. the M-BCR district; 55kb on the 1st intron zone, promptly on m-BCR district and the 19th intron, i.e. μ-BCR district.Because the difference of breaking point causes translating the BCR/ABL fusion rotein of different molecular weight size on the BCR gene, is respectively: P210
BCR/ABLFusion rotein, P10
BCR/ABLFusion rotein and P230
BCR/ABLFusion rotein.CML patient B CR/ABL fusion gene is the M-type basically, is individually μ-type, and the m-type is seldom seen.The BCR/ABL fusion gene of 35% adult and 20% children Ph (+) ALL is the M-type mostly, and all the other are the m-type.Adopt round pcr to detect the BCR/ABL fusion gene and have important use value for the monitoring of minimal residual disease (MRD) in the diagnosis of CML and ALL and therapeutic process.
The real-time quantitative PCR of invention technology (RQ-PCR) had realized the quantitative leap of PCR meaning from qualitative to real in recent years.Compare with regular-PCR, RQ-PCR also has the following advantages except can be quantitatively: (1) through adding probe or through making melting curve in the PCR reaction system, makes that specificity strengthens, sensitivity improves; (2) stopped pipe operation in the amplification procedure, the real-time collecting data have reduced opportunities for contamination, reduce false positive results; (3) need not electrophoresis, shortened the running time; (4) estimate sample quality through quantitative internal control gene, reduced false negative result.RQ-PCR key problem in technology part based on the TaqMan probe is: except the primer of upstream and downstream, also added the TaqMan probe in the PCR reaction system, 5 of this probe ' end and 3 ' end indicates fluorescence report group (like FAM or TET) and fluorescent quenching group (like TAMRA) respectively.Before the PCR reaction, probe is complete, and fluorescence report group fluorescent signal emitted is by the cancellation of fluorescent quenching group; Carrying out along with the PCR reaction; The circumscribed activity of Taq enzyme performance 5 ' → 3 '; Make the fluorescence report group that scales off away from the fluorescent quenching group, produce fluorescent signal, fluorescence signal intensity is directly proportional with the amount of original template DNA; When fluorescence signal intensity during greater than threshold value (10 SD more than the baseline); Can be by fluorescent probe monitoring in real time regularly, the CT value of each sample (in the pcr amplification process, fluorescent signal begins to be got into by background the pairing cycle number of flex point of exponential growth phase) is directly proportional with the logarithmic value of starting template amount; Can utilize typical curve to calculate the amount of goal gene and internal control gene respectively according to the CT value, the two ratio that is divided by be the level relatively of goal gene.
The amount that calculates starting template must be utilized typical curve; The making of typical curve is to adopt the standard substance of the known initial amount of process serial dilution to carry out RQ-PCR; Standard substance can be the cDNA of known quality or the plasmid of known copy number; The former makes fairly simple, but the unknown sample result who obtains is not directly perceived, than indigestion; Although and the plasmid standard preparation is more loaded down with trivial details, utilize it can calculate the copy number of unknown sample, and visual result, be present most widely used standard substance type.
The purpose that internal control gene is selected is in order to eliminate the difference of aspects such as mRNA quality between the different samples, quantity and rt efficient; The selection of internal control gene has following three to require greatly: (1) is equal stably express in all nucleated cells; Have nothing to do with differentiation series and differential period, do not tested the influence of processing; (2) there is not pseudogene; (3) expression level and Degradation Level are consistent with goal gene.
Adopt the clinical value of RQ-PCR technology for detection BCR/ABL mRNA level to be mainly reflected in following three broad aspect: (1) diagnosis: up-to-date WHO Case definition requirement; Must detect Ph karyomit(e) or BCR/ABL mRNA just can be diagnosed as CML, so all should there be the BCR/ABL fusion gene in all CML patient's molecular levels.RQ-PCR technology for detection BCR/ABL mRNA level and chromosome karyotype analysis can complement one another, and overcome influences such as concealment dystopy, improve the correct diagnosis of CML greatly.(2) prognosis: Ph (+) ALL patient's prognosis is very poor, and must carry out HSCT could long-term surviving.Adopt RQ-PCR technology for detection patient B CR/ABL mRNA level can patient's layering be helped the selection of clinical treatment.(3) monitoring of MRD: the treatment to CML and Ph (+) ALL patient has at present had hemopoietic stem cell and ABL tyrosine kinase inhibitor effective ways such as (like imatinib mesylate Glivec); Can make considerable patient obtain genetics and alleviate fully, the monitoring of MRD thereafter can only rely on the most responsive RQ-PCR technology of generally acknowledging at present.The BCR/ABL mRNA level that the RQ-PCR technology for detection goes out not only reflects the intravital white blood disease load of patient, and the decline degree of BCR/ABL mRNA level is relevant with long-term efficacy in the specified time.The MRD level confirm be therapeutic evaluation, regimen select (as improve drug dose, to transplant patient's clinical intervention measures such as donor lymphocyte infusion) important evidence, for the prevention clinical recurrence, improve patient's remission rate, curative ratio and survival rate and have great importance.
Summary of the invention
The test kit that the purpose of this invention is to provide a kind of detection by quantitative BCR/ABL mRNA level.
The test kit of detection by quantitative BCR/ABL mRNA level provided by the present invention comprises at least a of the standard substance, internal control gene real-time quantitative PCR system and the following three kinds of real-time quantitative PCR systems that are used for the production standard curve: M-type BCR/ABL real-time quantitative PCR system, m-type BCR/ABL real-time quantitative PCR system and μ-type BCR/ABL real-time quantitative PCR system;
Said M-type, m-type and μ-type BCR/ABL real-time quantitative PCR system includes upstream primer, downstream primer and TaqMan probe; In the said M-type BCR/ABL real-time quantitative PCR system; The nucleotide sequence of upstream primer is shown in sequence in the sequence table 1; The nucleotide sequence of downstream primer is shown in sequence in the sequence table 2, and the nucleotide sequence of TaqMan probe is shown in sequence in the sequence table 3; In the said m-type BCR/ABL real-time quantitative PCR system, the nucleotide sequence of upstream primer is shown in sequence in the sequence table 4, and the nucleotide sequence of downstream primer is shown in sequence in the sequence table 5, and the nucleotide sequence of TaqMan probe is shown in sequence in the sequence table 6; In said μ-type BCR/ABL real-time quantitative PCR system, the nucleotide sequence of upstream primer is shown in sequence in the sequence table 7, and the nucleotide sequence of downstream primer is shown in sequence in the sequence table 8, and the nucleotide sequence of TaqMan probe is shown in sequence in the sequence table 9.
Said internal control gene is preferably abl gene; With abl gene during as internal control gene; Said internal control gene real-time quantitative PCR system comprises upstream primer, downstream primer and TaqMan probe; The nucleotide sequence of the upstream primer of said internal control gene real-time quantitative PCR system is shown in sequence in the sequence table 10, and the nucleotide sequence of downstream primer is shown in sequence in the sequence table 11, and the nucleotide sequence of TaqMan probe is shown in sequence in the sequence table 12.
The Master Mix that also can comprise fluorescent PCR in above-mentioned internal control gene real-time quantitative PCR system, M-type, m-type and μ-type BCR/ABL real-time quantitative PCR system.
Among the present invention, 3 of the TaqMan probe in above-mentioned M-type BCR/ABL, m-type BCR/ABL, μ-type BCR/ABL and four kinds of real-time quantitative PCR systems of internal control gene ' end is connected with fluorescent quenching group TAMRA, and 5 ' end is connected with fluorescence report group FAM.
Standard substance in the test kit of above-mentioned detection by quantitative BCR/ABL mRNA level are the plasmid standard that contains abl gene, and the nucleotide sequence of said abl gene is shown in sequence in the sequence table 13.
Use for ease, the test kit of above-mentioned detection by quantitative BCR/ABL mRNA level also comprises negative control, M-type, m-type and μ-type BCR/ABL positive control.
The test kit of detection by quantitative BCR/ABL mRNA level of the present invention has following characteristics:
1, broad covered area: this test kit comprises primer and the probe that detects M-type BCR/ABL, m-type BCR/ABL and μ-type BCR/ABL, has covered the possible BCR/ABL fusion gene type of CML and ALL patient basically, has improved positive rate.
2, susceptibility is high: can repeat susceptibility is 5 copies.
3, cost is low: experimental system is merely 10 μ l, and each composition consumption all reduces over half, thereby cost is obviously reduced.In addition, the shared typical curve of testing goal gene and internal control gene, not only easy but also reduce cost.
4, practical: this test kit comprises internal control gene real-time quantitative PCR system and plasmid standard simultaneously, can quantitatively go out simultaneously the copy number of BCR/ABL fusion gene and internal control gene ABL in the sample, finally calculates sample B CR/ABL mRNA level.
5, easy to use: the cDNA that only need add testing sample during use can begin the PCR reaction.
6, the shelf lives is long: can prolonged preservation (＞1.5 years) under-20 ℃ of conditions, and do not influence the detection effect.
Test kit of the present invention can be accurately, fast, the various BCR/ABL mRNA of quantitatively determined level; Be used for chronic myelogenous leukemia and express diagnosis and the monitoring of therapeutic process minimal residual disease of the acute lymphoblastic leukemia of BCR/ABL, for the confirming of the making a definite diagnosis of clinical disease, regimen, therapeutic evaluation and prognosis provide important molecule foundation.
Description of drawings
Fig. 1 is for utilizing internal control gene real-time quantitative PCR system amplification 1 * 10
6-1 * 10
2The typical curve of the ABL plasmid standard preparation of copy
Embodiment
If the pcr amplification efficient of two kinds of genes is consistent, it also is the same utilizing two typical curves of their plasmid standard making so respectively, and promptly the slope of typical curve and vertical intercept and gene type are irrelevant.Therefore the present invention is after the pcr amplification efficient of verifying goal gene BCR/ABL and internal control gene ABL is consistent; Adopt the copy number of simultaneously quantitative goal gene of a typical curve and internal control gene; So not only easy economy; More can eliminate the difference that plasmid quantitatively brings, improve the accuracy of RQ-PCR.
The preparation of the test kit of embodiment 1, detection by quantitative BCR/ABL mRNA level
One, the design of primer and probe in the test kit of detection by quantitative BCR/ABL mRNA level
M-type BCR/ABL real-time quantitative PCR system is formed as follows:
(1) upstream primer (being arranged in BCR exons 1 3): 5 '-CCGCTGACCATCAATAAGGAA-3 ' (sequence table sequence 1), final concentration is 0.3 μ M;
(2) downstream primer (being arranged in the ABL exon 2): 5 '-CTCAGACCCTGAGGCTCAAAGT-3 ' (sequence table sequence 2), final concentration is 0.3 μ M;
(3) TaqMan probe (being arranged in the ABL exon 2): 5 '-AGCCCTTCAGCGGCCAGTAGCATCT-3 ' (sequence table sequence 3), final concentration is 0.2 μ M;
(4) the Master Mix of fluorescent PCR (available from American AB I company).
M-type BCR/ABL real-time quantitative PCR system is formed as follows:
(1) upstream primer (being arranged in the BCR exons 1): 5 '-CTGGCCCAACGATGGCGA-3 ' (sequence table sequence 4), final concentration is 0.3 μ M;
(2) downstream primer (being arranged in the ABL exon 2): 5 '-CACTCAGACCCTGAGGCTCAA-3 ' (sequence table sequence 5), final concentration is 0.3 μ M;
(3) TaqMan probe (being arranged in the ABL exon 2): 5 '-AGCCCTTCAGCGGCCAGTAGCATCT-3 ' (sequence table sequence 6), final concentration is 0.2 μ M;
(4) the Master Mix of fluorescent PCR (available from American AB I company).
μ-type BCR/ABL real-time quantitative PCR system is formed as follows:
(1) upstream primer (being arranged in BCR exons 1 9): 5 '-CGGACATCCAGGCACTGAA-3 ' (sequence table sequence 7), final concentration is 0.3 μ M;
(2) downstream primer (being arranged in the ABL exon 2): 5 '-CTCAGACCCTGAGGCTCAAAGT-3 ' (sequence table sequence 8), final concentration is 0.3 μ M;
(3) TaqMan probe (being arranged in the ABL exon 2): 5 '-AGCCCTTCAGCGGCCAGTAGCATCT-3 ' (sequence table sequence 9), final concentration is 0.2 μ M;
(4) the Master Mix of fluorescent PCR (available from American AB I company).
Internal control gene real-time quantitative PCR system is formed as follows:
(1) upstream primer (being positioned at the ABL exon 2):
5 '-TGGAGATAACACTCTAAGCATAACTAAAGGT-3 ' (sequence 10 in the sequence table), final concentration are 0.3 μ M;
(2) downstream primer (being arranged in the ABL exon 3): 5 '-GATGTAGTTGCTTGGGACCCA 3 ' (sequence table sequence 11), final concentration is 0.3 μ M;
(3) TaqMan probe (being arranged in the ABL exon 3): 5 '-CCATTTTTGGTTTGGGCTTCACACCATT-3 ' (sequence table sequence 8), final concentration is 0.2 μ M;
(4) the Master Mix of fluorescent PCR (available from American AB I company).
3 of TaqMan probe in above-mentioned M-type BCR/ABL, m-type BCR/ABL, μ-type BCR/ABL and four kinds of real-time quantitative PCR systems of internal control gene ' end is connected with fluorescent quenching group TAMRA, and 5 ' end is connected with fluorescence report group FAM.
Two, the preparation of ABL plasmid standard
The ABL plasmid standard comprises 1 * 10
6Copy/μ L, 1 * 10
5Copy/μ L, 1 * 10
4Copy/μ L, 1 * 10
3Copy/μ L and 1 * 10
2The plasmid standard of five concentration of copy/μ L, its concrete preparation method is following:
Establishing criteria article sequence covers and greater than the primer of the principle design pcr amplification ABL plasmid standard of RQ-PCR amplified production, concrete sequence is: upstream primer 5 '-CCTTCAGCGGCCAGTAGC-3 ', downstream primer 5 '-GGACACAGGCCCATGGTAC-3 '.Extract total RNA of the stripped PMNC of normal people; Its reverse transcription is become cDNA and is that template is carried out pcr amplification with this cDNA; Pcr amplification product is carried out agarose gel electrophoresis detection, recovery, purifying; Product behind the purifying is connected on the pGEM-T Easy carrier, and transfection TOP10 competent escherichia coli cell, blue hickie screening positive clone utilized.Extract positive colony and carry out plasmid and carry for a short time and check order, sequencing result shows that the deoxyribonucleotide sequence that is connected to the abl gene on the pGEM-T Easy carrier is shown in sequence in the sequence table 13.Wherein, From 5 ' the 142nd the-the 172nd of end is the upstream primer of the internal control gene real-time quantitative PCR system of above-mentioned steps one; From 5 ' the 210th the-the 237th of end is the TaqMan probe sequence of the internal control gene real-time quantitative PCR system of above-mentioned steps one, is the downstream primer of the internal control gene real-time quantitative PCR system of above-mentioned steps one from 5 ' the 245th the-the 265th of end.
The positive colony that above-mentioned sequencing result is correct carries out carrying in the plasmid, and measures plasmid concentration, calculates plasmid copy number, carries out 10 times of serial dilutions, is distributed into 1 * 10 at last
6Copy/μ L, 1 * 10
5Copy/μ L, 1 * 10
4Copy/μ L, 1 * 10
3Copy/μ L, 1 * 10
2Copy/μ L, 1 * 10
1Copy/μ L, put-80 ℃ frozen subsequent use.
Three, the preparation of negative control plasmid-WT1 and M-type, m-type and μ-type BCR/ABL positive control
1, the preparation of negative control plasmid-WT1
Extract total RNA that 1 example has been diagnosed as acute promyelocytic leukemia patient BMNC; Its reverse transcription is become cDNA; With this cDNA is template, is primer PCR amplification WT1 gene with 5 '-ccacagcacagggtacgaga-3 ' and 5 '-ctcagatgccgaccgtacaa-3 '.Pcr amplification product is carried out agarose gel electrophoresis detect, the product behind recovery, the purifying is connected on the pGEM-T Easy carrier, and transfection TOP10 competent escherichia coli cell, utilizes blue hickie screening positive clone.Extract positive colony and carry out plasmid and carry for a short time and check order, sequencing result shows that the deoxyribonucleotide sequence that is connected to the WT1 gene on the pGEM-T Easy carrier is shown in sequence in the sequence table 14.The correct plasmid of the sequencing result that obtains is negative control plasmid-WT1.
2, the preparation of M-type, m-type and μ-type BCR/ABL positive control
Separating 1 example is the CML patient's of M-type BCR/ABL stripped BMNC from the first visit of the People's Hospital, extracts its total RNA, and its reverse transcription is become cDNA, and this cDNA is M-type BCR/ABL positive control.
Separating 1 example is the ALL patient's of m-type BCR/ABL stripped BMNC from the first visit of the People's Hospital, extracts its total RNA, and its reverse transcription is become cDNA, and this cDNA is m-type BCR/ABL positive control.
Separating 1 example is the CML patient's of μ-type BCR/ABL stripped BMNC from the first visit of the People's Hospital, extracts its total RNA, and its reverse transcription is become cDNA, and this cDNA is μ-type BCR/ABL positive control.
Four, method of use
All testing samples internal control gene ABL that all need increase calculates the copy number of ABL and confirms the quality of testing sample.Testing sample is qualified, and (sample of ABL copy number >=30000 thinks qualified; Otherwise need extract sample again detects again) CML patient; The M-type that at first increases BCR/ABL fusion gene, if testing sample is negative, the μ that increases again-type BCR/ABL fusion gene and m-type BCR/ABL fusion gene.For the ALL patient of testing sample qualified (sample of ABL copy number >=30000 thinks qualified, detects otherwise need extract sample again again), the M-type that increases simultaneously BCR/ABL fusion gene and m-type BCR/ABL fusion gene.Concrete method of use is following:
1, gets stripped patient's marrow to be detected or PMNC, extract its total RNA, and its reverse transcription is become cDNA.
2, the cDNA that in the internal control gene real-time quantitative PCR system of 9 μ L above-mentioned steps one, adds the testing sample of 1 μ L step 1 carries out the real-time quantitative PCR reaction, calculates the quality of copy number and the definite testing sample of ABL then.
Qualified (sample of ABL copy number >=30000 thinks qualified for testing sample; Otherwise need extract sample again detects again) CML patient; The cDNA that in the M-type BCR/ABL of 9 μ L above-mentioned steps one real-time quantitative PCR system, adds the testing sample of 1 μ L step 1 carries out the real-time quantitative PCR reaction; If testing sample is negative, then in the μ-type BCR/ABL real-time quantitative PCR system of 9 μ L above-mentioned steps one and m-type BCR/ABL real-time quantitative PCR system, add the cDNA of the testing sample of 1 μ L step 1 respectively, carry out real-time quantitative PCR and react.
Qualified (sample of ABL copy number >=30000 thinks qualified for testing sample; Otherwise need extract sample again detects again) ALL patient; The cDNA that in the M-type BCR/ABL real-time quantitative PCR system of 9 μ L above-mentioned steps one and m-type BCR/ABL real-time quantitative PCR system, adds the testing sample of 1 μ L step 1 respectively carries out the real-time quantitative PCR reaction.
The condition of above-mentioned real-time quantitative PCR reaction is: 1 circulation of 50 ℃ of 2min earlier; 1 circulation of 95 ℃ of 10min then; 95 ℃ of 15s again, 62 ℃ of 1min, 40 circulations.
3, the making of typical curve: the copy number that in the internal control gene real-time quantitative PCR system of 9 μ L above-mentioned steps one, adds two preparations of 1 μ L above-mentioned steps respectively is 1 * 10
6, 1 * 10
5, 1 * 10
4, 1 * 10
3And 1 * 10
2The ABL plasmid standard of five concentration, 1 circulation of 50 ℃ of 2min earlier; 1 circulation of 95 ℃ of 10min then; 95 ℃ of 15s again, 62 ℃ of 1min, 40 circulations.The production standard curve, wherein 1 * 10
2The ABL plasmid standard of copy is made two pipe parallel pipes simultaneously, ABL plasmid standards of all the other each copy pipe that increases respectively.
Utilize the internal control gene real-time quantitative PCR system amplification 1 * 10 of above-mentioned steps one
6-1 * 10
2The typical curve of the ABL plasmid standard preparation of copy is as shown in Figure 1.
4, every crowd of RQ-PCR reacts also need increase the simultaneously positive and negative control.M-type, m-type and the μ-type BCR/ABL positive control that in M-type, m-type and the μ-type BCR/ABL real-time quantitative PCR system of 9 μ L above-mentioned steps one, adds three preparations of 1 μ L above-mentioned steps respectively; The negative control that in the internal control gene real-time quantitative PCR system of 9 μ L above-mentioned steps one, adds three preparations of 1 μ L above-mentioned steps carries out the real-time quantitative PCR reaction.
5, the BCR/ABL mRNA level of testing sample is calculated according to following formula:
The condition of storage of the test kit of detection by quantitative BCR/ABL mRNA level of the present invention is: 4 ℃ of lucifuges store at least 2 months or-20 ℃ of lucifuges store 1.5 years.
Five, the susceptibility of the test kit of detection by quantitative BCR/ABL mRNA level of the present invention, specificity and stability are measured
1, susceptibility: the repeated susceptibility of internal control gene real-time quantitative PCR system, M-type, m-type and μ-type BCR/ABL real-time quantitative PCR system is 5 copies
Each 5 parts of the ABL plasmid standards of 5 copies that will prepare according to the method for above-mentioned steps two; Utilize the internal control gene real-time quantitative PCR system of above-mentioned steps one to carry out repeated experiments 5 times respectively; In the each experiment of result amplification curve is arranged all, therefore the repeated susceptibility of internal control gene real-time quantitative PCR system of the present invention is 5 copies.
M-type, m-type and the μ-type BCR/ABL positive control of above-mentioned steps three preparations is carried out serial dilution respectively; With the cDNA behind the serial dilution is template; Utilize M-type, m-type and the μ-type BCR/ABL real-time quantitative PCR system of above-mentioned steps one to carry out 5 batches of repetition RQ-PCR reaction respectively; Each ABL plasmid standard that all increases simultaneously that reacts; The production standard curve is equivalent to 5 copies and greater than the M-type after the dilution of 5 copies, m-type and μ-type BCR/ABL positive control amplification curve is arranged all in the each experiment of result, and therefore the repeated susceptibility of M-type of the present invention, m-type and μ-type BCR/ABL real-time quantitative PCR system is 5 copies.
2, specificity
We utilize M-type, m-type and the μ-type BCR/ABL real-time quantitative PCR system of above-mentioned steps one to detect negative control plasmid-WT1 respectively, and amplification curve does not appear in the result, confirms that test kit of the present invention has specificity.
Because human nuclear hematopoietic cell all contains abl gene, utilize the internal control gene real-time quantitative PCR system of above-mentioned steps one to detect the WT1 plasmid that does not contain abl gene, amplification curve does not appear in the result, confirms that the ABL detection has specificity.
3, stability
Sample used in the following stability experiment is the stripped BMNC of voluntary donor all from the People's Hospital.
(1) difference analysis in the daytime
1. extract total RNA of sample G717; Its reverse transcription is become cDNA, and be template, utilize the internal control gene real-time quantitative PCR system of above-mentioned steps one to carry out the RQ-PCR reaction with this cDNA; Carry out 5 batches of experiments altogether; The result is as shown in the table, and data can know that the poor in the daytime variation coefficient (CV) is 1.83% from table.
| Sample number | The CT value | Detection time |
| 1 | 22.44 | 06-6-10AM |
| 2 | 22.91 | 05-6- |
| 3 | 22.84 | 05-7-1PM |
| 4 | 23.35 | 05-7- |
| 5 | 22.3 | 05-7-15AM |
2. extract total RNA of sample H236; Its reverse transcription is become cDNA, and be template, utilize the M-type BCR/ABL real-time quantitative PCR system of above-mentioned steps one to carry out the RQ-PCR reaction with this cDNA; Carry out 5 batches of experiments altogether; The result is as shown in the table, and data can know that the poor in the daytime variation coefficient (CV) is 1.06% from table.
| The sample numbering | The CT value | Detection time |
| 1 | 33.28 | 05-9-16PM |
| 2 | 33.97 | 05-9- |
| 3 | 33.01 | 059-30AM |
| 4 | 33.31 | 05-10- |
| 5 | 33.4 | 05-10-13AM |
3. extract total RNA of sample H852; Its reverse transcription is become cDNA, and be template, utilize the m-type BCR/ABL real-time quantitative PCR system of above-mentioned steps one to carry out the RQ-PCR reaction with this cDNA; Carry out 5 batches of experiments altogether; The result is as shown in the table, and data can know that the poor in the daytime variation coefficient (CV) is 2.88% from table.
| The sample numbering | The CT value | Detection time |
| 1 | 29.53 | 06-3-20PM |
| 2 | 28.97 | 06-3- |
| 3 | 29.3 | 06-4-21AM |
| 4 | 31.03 | 06-9- |
| 5 | 30.47 | 06-9-15AM |
(2) day interior difference analysis
1. extract total RNA of sample H236; Its reverse transcription is become cDNA, and be template, utilize the internal control gene real-time quantitative PCR system of above-mentioned steps one to carry out the RQ-PCR reaction with this cDNA; The same batch of RQ-PCR reaction of having carried out 5 parallel holes; The result is as shown in the table, and data can be known from table, and a day interpolation variation coefficient (CV) is 1.35%.
| Sample number | The CT value | Detection time |
| 1 | 22.02 | 05-9-30AM |
| 2 | 22.2 | 05-9- |
| 3 | 22.21 | 05-9-30AM |
| 4 | 21.83 | 05-9- |
| 5 | 22.64 | 05-9-30AM |
2. extract total RNA of sample H314; Its reverse transcription is become cDNA, and be template, utilize the M-type BCR/ABL real-time quantitative PCR system of above-mentioned steps one to carry out the RQ-PCR reaction with this cDNA; The same batch of RQ-PCR reaction of having carried out 5 parallel holes; The result is as shown in the table, and data can be known from table, and a day interpolation variation coefficient (CV) is 1.64%.
| The sample numbering | The CT value | Detection time |
| 1 | 34.94 | 05-9-30AM |
| 2 | 34.12 | 05-9- |
| 3 | 33.78 | 05-9-30AM |
| 4 | 34.83 | 05-9- |
| 5 | 34.3 | 05-9-30AM |
3. extract total RNA of sample J320; Its reverse transcription is become cDNA, and be template, utilize the m-type BCR/ABL real-time quantitative PCR system of above-mentioned steps one to carry out the RQ-PCR reaction with this cDNA; The same batch of RQ-PCR reaction of having carried out 5 parallel holes; The result is as shown in the table, and data can be known from table, and a day interpolation variation coefficient (CV) is 2.94%.
| The sample numbering | The CT value | Detection time |
| 1 | 31.32 | 06-9-13AM |
| 2 | 31.47 | 06-9- |
| 3 | 32.75 | 06-9-13AM |
| 4 | 30.64 | 06-9- |
| 5 | 30.39 | 06-9-13AM |
4, be the influence of storing temp and the storage time of example detection kit with the ABL plasmid standard of different copy numbers to experimental result
Utilize test kit of the present invention to detect 1 * 10
3And 1 * 10
7The ABL plasmid standard room temperature lucifuge of copy stored for 1 week, 4 ℃ of lucifuges store 3 weeks, 6 weeks, 9 weeks and 12 weeks, the detected result when-20 ℃ of lucifuges store 18 months; Detected result is used the CT value representation; Concrete outcome is shown in following two tables, and data can be found out from table, along with the variation in storing temp and storage time; The no considerable change of CT value shows that test kit is still very stable.
10
3Different storing temps of the ABL plasmid standard of copy and the CT value under the time change
| Numbering | Storing temp (℃) | The CT value | Storage time |
| 1 | Room temperature | 31.39 | 1 week |
| 2 | 4 | 31.3 | 3 |
| 3 | 4 | 30.92 | 6 weeks |
| 4 | 4 | 31.18 | 9 |
| 5 | 4 | 30.34 | 12 weeks |
| 6 | -20 | 31.89 | 18 months |
10
7Different storing temps of the ABL plasmid standard of copy and the CT value under the time change
| Numbering | Storing temp (℃) | The CT value | Storage time |
| 1 | Room temperature | 17.41 | 1 week |
| 2 | 4 | 17.66 | 3 |
| 3 | 4 | 16.87 | 6 weeks |
| 4 | 4 | 16.65 | 9 |
| 5 | 4 | 16.24 | 12 weeks |
| 6 | -20 | 17.82 | 18 months |
Sequence table
<160>14
<210>1
<211>21
<212>DNA
< 213>artificial sequence
<400>1
ccgctgacca?tcaataagga?a 21
<210>2
<211>22
<212>DNA
< 213>artificial sequence
<400>2
ctcagaccct?gaggctcaaa?gt 22
<210>3
<211>25
<212>DNA
< 213>artificial sequence
<400>3
agcccttcag?cggccagtag?catct 25
<210>4
<211>18
<212>DNA
< 213>artificial sequence
<400>4
ctggcccaac?gatggcga 18
<210>5
<211>21
<212>DNA
< 213>artificial sequence
<400>5
cactcagacc?ctgaggctca?a 21
<210>6
<211>25
<212>DNA
< 213>artificial sequence
<400>6
agcccttcag?cggccagtag?catct 25
<210>7
<211>19
<212>DNA
< 213>artificial sequence
<400>7
cggacatcca?ggcactgaa 19
<210>8
<211>22
<212>DNA
< 213>artificial sequence
<400>8
ctcagaccct?gaggctcaaa?gt 22
<210>9
<211>25
<212>DNA
< 213>artificial sequence
<400>9
agcccttcag?cggccagtag?catct 25
<210>10
<211>31
<212>DNA
< 213>artificial sequence
<400>10
tggagataac?actctaagca?taactaaagg?t 31
<210>11
<211>21
<212>DNA
< 213>artificial sequence
<400>11
gatgtagttg?cttgggaccc?a 21
<210>12
<211>28
<212>DNA
< 213>artificial sequence
<400>12
ccatttttgg?tttgggcttc?acaccatt 28
<210>13
<211>316
<212>DNA
< 213>artificial sequence
<400>13
ccttcagcgg?ccagtagcat?ctgactttga?gcctcagggt?ctgagtgaag?ccgctcgttg 60
gaactccaag?gaaaaccttc?tcgctggacc?cagtgaaaat?gaccccaacc?ttttcgttgc 120
actgtatgat?tttgtggcca?gtggagataa?cactctaagc?ataactaaag?gtgaaaagct 180
ccgggtctta?ggctataatc?acaatgggga?atggtgtgaa?gcccaaacca?aaaatggcca 240
aggctgggtc?ccaagcaact?acatcacgcc?agtcaacagt?ctggagaaac?actcctggta 300
ccatgggcct?gtgtcc 316
<210>14
<211>151
<212>DNA
< 213>artificial sequence
<400>14
ccacagcaca?gggtacgaga?gcgataacca?cacaacgccc?atcctctgcg?gagcccaata 60
cagaatacac?acgcacggtg?tcttcagagg?cattcaggat?gtgcgacgtg?tgcctggagt 120
agccccgact?cttgtacggt?cggcatctga?g 151
Claims (6)
1. the test kit of a detection by quantitative BCR/ABL mRNA level comprises the standard substance, internal control gene real-time quantitative PCR system and the following three kinds of real-time quantitative PCR systems that are used for the production standard curve: M-type BCR/ABL real-time quantitative PCR system, m-type BCR/ABL real-time quantitative PCR system and μ-type BCR/ABL real-time quantitative PCR system;
Said M-type, m-type and μ-type BCR/ABL real-time quantitative PCR system includes upstream primer, downstream primer and TaqMan probe; In the said M-type BCR/ABL real-time quantitative PCR system, the nucleotide sequence of upstream primer is shown in sequence in the sequence table 1, and the nucleotide sequence of downstream primer is shown in sequence in the sequence table 2, and the nucleotide sequence of TaqMan probe is shown in sequence in the sequence table 3; In the said m-type BCR/ABL real-time quantitative PCR system, the nucleotide sequence of upstream primer is shown in sequence in the sequence table 4, and the nucleotide sequence of downstream primer is shown in sequence in the sequence table 5, and the nucleotide sequence of TaqMan probe is shown in sequence in the sequence table 6; In said μ-type BCR/ABL real-time quantitative PCR system, the nucleotide sequence of upstream primer is shown in sequence in the sequence table 7, and the nucleotide sequence of downstream primer is shown in sequence in the sequence table 8, and the nucleotide sequence of TaqMan probe is shown in sequence in the sequence table 9; Said internal control gene is an abl gene, and said internal control gene real-time quantitative PCR system comprises upstream primer, downstream primer and TaqMan probe; The nucleotide sequence of the upstream primer of said internal control gene real-time quantitative PCR system is shown in sequence in the sequence table 10, and the nucleotide sequence of downstream primer is shown in sequence in the sequence table 11, and the nucleotide sequence of TaqMan probe is shown in sequence in the sequence table 12; Said standard substance are the plasmid standard that contains abl gene.
2. test kit according to claim 1 is characterized in that: the Master Mix that also comprises fluorescent PCR in said internal control gene real-time quantitative PCR system, M-type, m-type and μ-type BCR/ABL real-time quantitative PCR system.
3. test kit according to claim 2; It is characterized in that: 3 of the TaqMan probe in said M-type, m-type, μ-type BCR/ABL real-time quantitative PCR system and the internal control gene real-time quantitative PCR system ' end is connected with fluorescent quenching group TAMRA, and 5 ' end is connected with fluorescence report group FAM.
4. test kit according to claim 1 is characterized in that: the nucleotide sequence of said abl gene is shown in sequence in the sequence table 13.
5. test kit according to claim 1 is characterized in that: said test kit also comprises M-type, m-type or μ-type BCR/ABL positive control.
6. test kit according to claim 5 is characterized in that: said test kit also comprises negative control.
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| CN101624621B true CN101624621B (en) | 2012-01-18 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102676638B (en) * | 2011-03-08 | 2014-06-04 | 苏州大学附属第一医院 | Method and kit for detecting drug-resistance mutation site of ABL kinase domain of BCR/ABL fusion gene |
| CN103667457B (en) * | 2012-04-25 | 2016-07-06 | 武汉艾迪康医学检验所有限公司 | The primer of detection leukemia BCR/ABL b3a2, b2a2 fusion gene relative expression quantity and method |
| MA35216B1 (en) * | 2012-09-13 | 2014-07-03 | Mascir | Probes and primers for detecting bcr-abl gene in duplex reaction |
| CN103667269B (en) * | 2012-09-18 | 2016-06-08 | 南京世和基因生物技术有限公司 | For the DNA probe storehouse hybridized with BCR or abl gene and the method adopting its enrichment BCR-ABL genetic fragment |
| CN102925556B (en) * | 2012-09-29 | 2014-09-17 | 童永清 | Kit for detecting mRNA expression quantity of m BCR fusion gene |
| MA36330B1 (en) * | 2013-10-10 | 2016-04-29 | Mascir Morrocan Foundation For Advanced Science Innovation & Res | Kit for the diagnosis of chronic myeloide leukemia in multiplex reaction and its applications in the monitoring of treatment and residual disease |
| CN104328213B (en) * | 2014-11-24 | 2016-05-11 | 济南市中心医院 | Primer and the kit of BCR-ABL fusion method for quick |
| CN106399462A (en) * | 2015-07-27 | 2017-02-15 | 上海睿玻生物科技有限公司 | BCR-ABL fusion gene amplification kit and BCR-ABL fusion gene detection kit |
| CN110358825A (en) * | 2018-04-09 | 2019-10-22 | 苏州云泰生物医药科技有限公司 | Detect the kit and its application method of people BCR-ABL fusion |
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| EP1454994A1 (en) * | 2003-03-07 | 2004-09-08 | Université de la Méditerranée | Standardized and optimized real-time quantitative reverse transcriptase polymerase chain reaction method for detection of MRD in leukemia |
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| EP1454994A1 (en) * | 2003-03-07 | 2004-09-08 | Université de la Méditerranée | Standardized and optimized real-time quantitative reverse transcriptase polymerase chain reaction method for detection of MRD in leukemia |
Non-Patent Citations (2)
| Title |
|---|
| E Beillard et al.Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using ‘real-time’ quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR) – a Europe against cancer program.《Leukemia》.2003,2474–2486. * |
| IzumiIshikawa et al.dose modification of imatinib by monitoring the level of bcr-abl transcript in chronic myelogenous leukemia.《Tohoku J. Exp. Med.》.2006,355-363. * |
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