CN102925556B - Kit for detecting mRNA expression quantity of m BCR fusion gene - Google Patents
Kit for detecting mRNA expression quantity of m BCR fusion gene Download PDFInfo
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Abstract
The invention relates to a kit for detecting mRNA expression quantity of an m BCR fusion gene, which belongs to the technical field of biology. The kit comprises a detection primer, a fluorescent probe, a cDNA first strand synthetic agent, an RTFQ PCR (Real-time Fluorescence Quantitative Polymerase Chain Reaction) mixed liquor, a negative control and a positive control, wherein the detection primer and the fluorescent probe comprise m BCR fusion gene primer, a reference gene ABL primer and a Taqman fluorescent probe. The m BCR fusion gene is a molecular market of ALL (Acute Lymphoblastic Leukemia) , the method for quantifying the expression quantity of the mRNA is the most sensitive method for diagnosing the ALL, and the disease prognosis can be judged through the detection for the gene before treatment; and therefore, the RTFQ PCR which has high sensitivity and high specificity is adopted for detecting the level of m BCR mRNA, the specificity and the sensitivity of a detection result are both obviously increased, and a bran-new, fast and convenient gene diagnostic technology is provided for deciding a therapeutic schedule for a clinical ALL patiend and forecasting the prognosis.
Description
Technical field
The present invention relates to the fluorescent quantitative PCR technique of biological technical field, be specifically related to a kind of test kit of the m of detection BCR fusion gene mRNA expression amount.
Background technology
Acute lymphoblastic leukemia (Acute lymphoblastic leukemia, ALL) is the abnormal malignant disease of a kind of lymphocyte series hemopoietic stem cell proliferation.In ALL, Ph karyomit(e) can be detected in 20%～40% adult patients and 2～5% child patients, and this karyomit(e) is to be held after mutual transposition and merged and form by No. 9 karyomit(e) abl genes 5 ' end and No. 22 karyomit(e) BCR genes 3 '.Breaking point in ALL patient B CR gene mainly concentrates on about 30kb place, BCR gene 5 ' end upstream, be on First Intron 3 ' terminal sequence, this region is defined as two fragments---bcr2 and bcr3, be referred to as m BCR fusion gene, amalgamation mode is that e1a2 connects, be transcribed into the mRNA of 7.5kb or 7.0kb, translate into a kind of chimeric protein P190.This albumen has higher protein tyrosine kinase activity, can make to activate many barss pathway after a series of substrate protein generation phosphorylations, causes cell that hyper-proliferative, differentiation and abnormal apoptosis occur the in the situation that of the acellular factor.
ALL patient is at the Acute onset initial stage, and in body, leukemia total cellular score can reach 10
12above, after chemotherapy, most of adult patients can be alleviated completely, but at the alleviation initial stage, all detects less than obvious leukocytic existence in marrow and peripheral blood, and now in body, leukemia total cellular score still has 10
7～10
9individual, we are referred to as MRD(Minimal Residual Desease, MRD).If these MRD can not be completely removed, cause the most at last leukemic recurrence, this is also the root of leukemia relapse.Therefore, MRD level is the key index of judgement result for the treatment of, and the rising of this index can be used as the comprehensive recurrence that indicates in advance malignant hematologic disease.
Past, to ALL patient's the diagnosis morphological criteria that places one's entire reliance upon, but the directly observation of impact to its form of quality due to cell smear and staining technique, during observation, use lens ratio also to have very large impact to the judgement of cellular form, and reviewer's level is limited to experience, can not make the judgement of entirely accurate to normal and abnormal cells, this has inevitably caused, and laboratory is undetected, flase drop, therefore recently develops into gradually and relies on molecular level methodology technology to judge.Although adopt RT-PCR(Real Time PCR, real time fluorescent quantitative poly chain reaction) detecting m BCR fusion gene can assist us to judge sensitiveer, more specifically, but not yet about real time fluorescence quantifying PCR method, detects at present the relevant report of m BCR fusion gene.
Summary of the invention
For solving the problems of the technologies described above, the invention provides a kind of test kit of the m of detection BCR fusion gene mRNA expression amount.Described technical scheme is as follows:
The embodiment of the present invention provides a kind of test kit of the m of detection BCR fusion gene mRNA expression amount, comprise and detecting with primer, fluorescent probe, cDNA the first chain synthetic agent, quantitative fluorescent PCR mixed solution, negative control and positive control, described for detection primer, fluorescent probe comprise m BCR fusion gene primer, reference gene ABL primer and Taqman fluorescent probe, described test kit also comprises the primer of inner positive control sequence, inner positive control sequence and the Taqman fluorescent probe of inner positive control sequence, wherein:
M BCR fusion gene upstream primer sequence is as shown in SEQ ID NO:1 in sequence table;
M BCR fusion gene downstream primer sequence is as shown in SEQ ID NO:2 in sequence table;
M BCR fusion gene Taqman fluorescent probe is as shown in SEQ ID NO:3 in sequence table;
Abl gene upstream primer sequence is as shown in SEQ ID NO:4 in sequence table;
Abl gene downstream primer sequence is as shown in SEQ ID NO:5 in sequence table;
Abl gene Taqman fluorescent probe is as shown in SEQ ID NO:6 in sequence table;
Inner positive control sequence is as shown in SEQ ID NO:7 in sequence table;
The upstream primer of inner positive control sequence is as shown in SEQ ID NO:8 in sequence table;
The downstream primer of inner positive control sequence is as shown in SEQ ID NO:9 in sequence table;
The Taqman fluorescent probe of inner positive control sequence is as shown in SEQ ID NO:10 in sequence table;
Described cDNA the first chain synthetic agent contains MgC1
2, reversed transcriptive enzyme damping fluid, dNTPs, RNA enzyme inhibitors, Oligo (dT)
15, AMV reversed transcriptive enzyme and without RNase deionized water; Described quantitative fluorescent PCR mixed solution contains PCR premix, Mg
2+, dNTPs, dUTP, Taq enzyme, UNG enzyme and without RNase deionized water.
Further, 5 ' end of the Taqman fluorescent probe of described m BCR fusion gene and the Taqman fluorescent probe of abl gene is connected with fluorescence report group FAM, 3 ' end is all connected with fluorescent quenching group TAMRA, 5 ' end of the Taqman fluorescent probe of inner positive control sequence is connected with fluorescence report group TET, and 3 ' end is connected with fluorescent quenching group TAMRA.
Particularly, the nucleotide sequence of described reference gene ABL is as shown in SEQ ID NO:11 in sequence table.
Particularly, described negative control is deionized water; Described positive control is the total RNA sample that contains m BCR fusion gene.
Particularly, described cDNA the first chain synthetic agent is: 25mmol/L MgC1
24 μ L, 10 * reversed transcriptive enzyme damping fluid, 2 μ L, 10mmol/L dNTP2 μ L, RNA enzyme inhibitors 0.5 μ L, Oligo (dT)
150.5 μ g, AMV reversed transcriptive enzyme 1.5U and without RNase deionized water, cumulative volume 11 μ L.
Particularly, the mixed solution of described quantitative fluorescent PCR (the reaction system final concentration of take represents) as: 1 * PCR premix (stoste is 2 * PCR Premix), the Mg of 2.5-4.0mM
2+, the dNTPs of 0.2-0.4mM is, the Taq enzyme of the dUTP of 0.3-0.6mM, 0.2U/ μ L and the UNG enzyme of 0.01-0.05U/ μ L and without RNase deionized water.
Advantage and the effect of test kit of the present invention are as follows:
(1) susceptibility is high: can repeat susceptibility is 0.01%, in 10000 cells, has one containing m BCR fusion gene, just can be detected.
(2) high specificity: use specific probe to identify quantitative molecular, accuracy is high, and meanwhile, target sequence is by primer and the dual control of probe, and specificity is good and false positive is low.
(3) handy and safe: simple to operate, safety, level of automation is high and prevent from polluting.Amplification and detect and can detect in same pipe, does not need to uncap, and is difficult for contaminatedly, and increasing simultaneously and detecting a step completes, and does not need post-processed, need not worry radiocontamination.
(4) complete monitoring: the test kit that the embodiment of the present invention provides has been introduced the inner positive quality control system of controlling, and testing process is carried out to Complete Quality Supervision, effectively avoids false positive or false negative.
(5) quick: the fast and high-throughput of speed, test experience can complete at 3-4 hour.
Test kit of the present invention is detection by quantitative m BCR fusion gene mRNA level fast and accurately, false positive and false-negative generation have effectively been stopped, for the diagnosis of acute lymphoblastic leukemia and the monitoring of therapeutic process minimal residual disease, for the diagnosis of acute lymphoblastic leukemia, formulate treatment plan and therapeutic evaluation and prognosis important detection means is provided.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, below the accompanying drawing of required use during embodiment is described is briefly described, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skills, do not paying under the prerequisite of creative work, can also obtain according to these accompanying drawings other accompanying drawing.
Figure 1A is the fluorescence real-time quantitative PCR reaction system amplification 1.0x10 of the test kit that provides of the embodiment of the present invention 2
6-1.0x10
3the fluorescence curve figure of the positive control sequence standard substance in inside of copy;
Figure 1B is the fluorescence real-time quantitative PCR reaction system amplification 1.0x10 of the test kit that provided by the embodiment of the present invention 2
6-1.0x10
3the canonical plotting that the fluorescence curve figure of the positive control sequence standard substance in inside of copy obtains;
Fig. 2 A is the fluorescence real-time quantitative PCR reaction system amplification 1.0x10 of the test kit that provides of the embodiment of the present invention 2
6-1.0x10
3the fluorescence curve figure of the ABL standard substance of copy;
Fig. 2 B is the fluorescence real-time quantitative PCR reaction system amplification 1.0x10 of the test kit that provided by the embodiment of the present invention 2
6-1.0x10
3the canonical plotting that the ABL standard substance fluorescence curve figure of copy obtains.
Embodiment
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with accompanying drawing, embodiment of the present invention is described further in detail.
The preparation of embodiment 1. test kits of the present invention
1, the design of specific primer and fluorescent probe
According to gene order, (abl gene sequence, BCR gene order derive from American National biotechnology information center nucleic acid database, and wherein abl gene ID is respectively 25, and reference sequences number is NM_005157.4; BCR gene I/D is respectively 613, and reference sequences number is NG_009244.1) design respectively primer and the fluorescent probe special to above-mentioned each gene order.
2, according to each component of the composition reagent preparation box of following test kit
Test kit of the present invention is composed as follows:
1. RNA extracts reagent: Trizol reagent (Invitrogen company, product article No.: 15596-026/100ml), every 1ml myeloid tissue adds 1mlTrizol rapid extraction leukaemic myeloid tissue RNA.
2. cDNA the first chain synthetic agent box (RT-PCR) (Fermentas company, product article No.: K1622): 25mmol/L MgCl
24 μ L, 10 * reversed transcriptive enzyme damping fluid, 2 μ L, 10mmol/L dNTP2 μ L, RNA enzyme inhibitors (RNasin, a kind of acid glycoprotein) 0.5 μ L, Oligo (dT)
150.5 μ g, AMV reversed transcriptive enzyme 1.5U and without RNase deionized water, cumulative volume 11 μ L.
3. primer, probe and standard substance: comprise m BCR fusion gene primer, inner positive control sequence primer, reference gene ABL primer and the Taqman fluorescent probe of answering with primer pair, specific as follows:
M BCR fusion gene upstream primer sequence is: 5 '-CGCAAGACCGGGCAGAT-3 ' (in sequence table, sequence 1);
M BCR fusion gene downstream primer sequence is: 5 '-AACGAGCGGCTTCACTCAGA-3 ' (in sequence table, sequence 2);
M BCR fusion gene Taqman fluorescent probe: sequence 3 in FAM5 '-ACGATGGCGAGGGC-TAMRA3 ' TAMRA(sequence table);
Inner positive control sequence is: 5 '-CGCAAGACCGGGGACAUCUGGCCCAACGAUGCGGAGGG CGCCUUCCAUGGAGACGCAGAAGCCCUUCAGCGGCCAGUAGCAUCUGACUUUGAGC C UCAGGGUGUCAGUGAAGCCGCUCGUU-3 ' (in sequence table, sequence 7);
Inner positive control sequence upstream primer sequence is: 5 '-CGCAAGACCGGGGACAT-3 ' (in sequence table, sequence 8);
Inner positive control sequence downstream primer sequence is: 5 '-AACGAGCGGCTTCACTGACA-3 ' (in sequence table, sequence 9);
Inner positive control sequence Taqman fluorescent probe: sequence 10 in TET5 '-ACGATGCGGAGGGC-3 ' TAMRA(sequence table);
ABL gene sequence is: 5 '- CAGGGTCTGAGTGAAGCCGCTCGTTGGAACTCCAAGGAAAA?CCTTCTCGCTGGACCCAGTGAAAATGACCCCAACCTTTTCGTTGCACTGTATGATTTTGT?GGCCAGTGGAGATAACACTCTAAGCATAACTAAAGGTGAAAAGCTCCGGGTCTTAGGCT?ATAATCACAATGGGGAATGGTGTGAAGCCCAAACCAAAAATGGCCAAGGCTGGGTCCC?AAGCAACTACATCACGCCAGTCAACAGTCTGGAGAAACACTCCTGGTACCATGGGCCTG?TGTCCCGCAATGCCGCTGAGTATCTGCTGAGCAGCGGGATCAATGGCAGCTTCTTGGTG?CGTGAGAGTGAGAGCAGTCCTGGC - 3 'sequence (sequence in the table 11);
Abl gene upstream primer sequence is: 5 '-CCGGGTCTTAGGCTATAATCACA-3 ' (in sequence table, sequence 4);
Abl gene downstream primer sequence is: 5 '-GCCTTGGCCATTTTTGGTT-3 ' (in sequence table, sequence 5);
Abl gene Taqman fluorescent probe: sequence 6 in FAM5 '-TGGTGTGAAGCCC-3 ' TAMRA(sequence table);
Wherein, inner positive control sequence is artificial synthesized sequence, comprises the artificial composition sequence of the known m BCR fusion gene sequence of a part and a part; Inner positive control sequence and abl gene sequence are used separately as standard substance.
Above-mentioned primer sequence, control sequence, gene order and Taqman fluorescent probe sequence are synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
4. negative control and positive control: with the negative contrast of deionized water, with the positive contrast of total RNA sample that contains m BCR fusion gene, adopt the test kit of the present invention providing in above-described embodiment 1 to form 1. RNA and extract reagent: Trizol reagent (Invitrogen company, product article No.: 15596-026/100ml), the ratio that adds 1ml Trizol reagent in every 1ml myeloid tissue, the acute lymphoblastic leukemia patient's of containing m BCR fusion gene that rapid extraction has been made a definite diagnosis myeloid tissue RNA, as positive control.
5. m BCR fusion gene fluorescence quantitative PCR reaction solution: formed by quantitative fluorescent PCR mixed solution and the primer, the probe that detect m BCR fusion gene: 1 * PCR premix (Qiagen company, 210212,2 * PCR Premix), the Mg of 2.5-4.0mM product article No.:
2+, the dNTPs of 0.2-0.4mM and the dUTP of 0.3-0.6mM, the UNG enzyme of the Taq enzyme of 0.2U/ μ L and 0.01-0.05U/ μ L, the m BCR fusion gene upstream primer (in sequence table, sequence 1) of 0.25pmol/ μ L, the m BCR fusion gene downstream primer (in sequence table, sequence 2) of 0.25pmol/ μ L, the m BCR fusion gene Taqman fluorescent probe (in sequence table, sequence 3) of 0.3pmol/ μ L, the positive control sequence upstream primer (in sequence table, sequence 8) in inside of 0.25pmol/ μ L, the positive control sequence downstream primer (in sequence table, sequence 9) in inside of 0.25pmol/ μ L, the positive control sequence Taqman fluorescent probe (in sequence table, sequence 10) (above all concentration all refers to the final concentration of PCR reaction system) in inside of 0.3pmol/ μ L.The template (comprising cDNA and the synthetic cDNA of inner positive control sequence RNA reverse transcription that sample RNA reverse transcription is synthetic) of conventionally getting 1-2 μ L, all the other are without RNase deionized water, reaction cumulative volume is generally 20 μ L.
6. ABL reference gene fluorescence quantitative PCR reaction solution: formed by quantitative fluorescent PCR mixed solution and the primer, the probe that detect ABL reference gene: 1 * PCR premix (Qiagen company, 210212,2 * PCR Premix), the Mg of 2.5-4.0mM product article No.:
2+, the dNTPs of 0.2-0.4mM, the ABL reference gene upstream primer (in sequence table, sequence 4) of the Taq enzyme of the dUTP of 0.3-0.6mM, 0.2U/ μ L and the UNG enzyme of 0.01-0.05U/ μ L, 0.25pmol/ μ L, the abl gene Taqman fluorescent probe (in sequence table, sequence 6) (above all concentration all refers to the final concentration of PCR reaction system) of the ABL reference gene downstream primer (in sequence table, sequence 5) of 0.25pmol/ μ L, 0.3pmol/ μ L.During detection, add abl gene standard substance template 2 μ L, all the other are without RNase deionized water, and reaction cumulative volume is generally 20 μ L.
7. inner positive control sequence fluorescence quantitative PCR reaction solution: formed by quantitative fluorescent PCR mixed solution and the primer, the probe that detect inner positive control sequence: 1 * PCR premix (Qiagen company, 210212,2 * PCR Premix), the Mg of 2.5-4.0mM product article No.:
2+, the dNTPs of 0.2-0.4mM and the Taq enzyme of the dUTP of 0.3-0.6mM, 0.2U/ μ L and the UNG enzyme of 0.01-0.05U/ μ L, the positive control sequence upstream primer (in sequence table, sequence 8) in inside of 0.25pmol/ μ L be, the positive control sequence Taqman fluorescent probe (in sequence table, sequence 10) (above all concentration all refers to the final concentration of PCR reaction system) in inside of the positive control sequence downstream primer (in sequence table, sequence 9) in the inside of 0.25pmol/ μ L, 0.3pmol/ μ L.During detection, conventionally get the template (the synthetic cDNA of inner positive control sequence RNA reverse transcription) of 1-2 μ L, all the other are without RNase deionized water, and reaction cumulative volume is generally 20 μ L.
3, the setting of pcr amplification program: on lightcycler instrument first through 50 ℃ of 10s, 95 ℃ of 10min, and then through 95 ℃ of 15s, 60 ℃ of 1min, totally 40 circulations.
The test kit of embodiment 2. use embodiment 1 preparations detects the expression amount of m BCR fusion gene mRNA
Take that to detect 30 routine acute lymphoblastic leukemia Bone Marrow of Patients tissue sample results be example.
The testing process that the test kit of the present invention providing with embodiment 1 detects m BCR fusion gene mRNA expression amount is: first according to gene order, design specific primer and fluorescent probe.Next obtains clinical leukaemic's myeloid tissue sample, and rapid extraction is organized RNA, carries out synthetic cDNA the first chain of reverse transcription PCR; First prepare the fluorescence quantitative PCR reaction solution of ABL reference gene and inner positive control sequence, it is 1.0x10 that the positive control sequence standard substance in inside and ABL standard substance are diluted to respectively to copy number/mL
3, 1.0x10
4, 1.0x10
5and 1.0x10
6make respectively inner positive control sequence standard substance typical curve (as shown in FIG. 1A and 1B) and abl gene standard substance typical curve (as shown in Figure 2 A and 2 B), and then preparation m BCR fusion gene fluorescence quantitative PCR reaction solution, carry out fluorescence quantitative PCR detection sample, in quantitative real time PCR Instrument data analysis system, read CT value result.After pcr amplification finishes, first analyze inner positive control sequence amplification, if its Ct value is less than 33, point out whole testing process effective, if its Ct value is greater than 35, prompting detects unsuccessfully, need to re-start detection, if its Ct value is between 33～35, need duplicate detection.When the positive control sequence Ct value in inside is less than 33, real-time fluorescence quantitative PCR the data obtained is calculated, calculate respectively the Ct value of m BCR fusion gene and abl gene, both differences are Δ Ct value.Finally, fluorescent quantitative PCR result adopts software analysis, and markization is calculated sampled data.
Concrete steps are as follows:
1. the extracting of the total RNA of acute lymphoblastic leukemia myeloid tissue: the total RNA that presses the method extracting acute lymphoblastic leukemia myeloid tissue sample of RNA extracting and purifying.The RNA extracting identifies its integrity through agarose gel electrophoresis, measures purity and the concentration of 260nm and 280nm optical density value calculating RNA by ultraviolet spectrophotometer, and the water of processing with 0.1%DEPC regulates each sample RNA of extracting to same concentrations.
2. cDNA is synthesized in reverse transcription: get the above-mentioned RNA extracting solution of 2 μ L, at 70 ℃ of insulation 10min, add subsequently the test kit of the present invention that embodiment 1 provides to form 2. cDNA the first chain synthetic agent box, that is: 25mmol/L MgC1
24 μ L, 10 * reversed transcriptive enzyme damping fluid, 2 μ L, 10mmol/L dNTPs2 μ L, RNA enzyme inhibitors 0.5 μ L, Oligo (dT)
150.5 μ g and AMV reversed transcriptive enzyme 1.5U, add without RNase deionized water to cumulative volume 20 μ L, in 42 ℃ of insulation 15min, carries out reverse transcription reaction, synthetic cDNA the first chain.After reaction finishes, be heated to 99 ℃, 5min is with deactivation reversed transcriptive enzyme, adds 20 μ L sterilized waters and mixes and put refrigerator-20 ℃ preservation.
Getting 1 μ L concentration is the inner positive control sequence RNA (in sequence table, sequence 7) of 2 μ g/ml, at 70 ℃ of insulation 10min, adds subsequently the test kit of the present invention that embodiment 1 provides to form 2. cDNA the first chain synthetic agent box, that is: 25mmol/L MgC1
24 μ L, 10 * reversed transcriptive enzyme damping fluid, 2 μ L, 10mmol/L dNTPs2 μ L, RNA enzyme inhibitors 0.5 μ L, Oligo (dT)
150.5 μ g and AMV reversed transcriptive enzyme 1.5U, add without RNase deionized water to cumulative volume 20 μ L, in 42 ℃ of insulation 15min, carries out reverse transcription reaction, synthetic cDNA.After reaction finishes, be heated to 99 ℃, 5min is with deactivation reversed transcriptive enzyme, adds 20 μ L sterilized waters and mixes and put refrigerator-20 ℃ preservation.
Getting 1 μ L concentration is the positive control RNA of 2 μ g/ml, at 70 ℃ of insulation 10min, adds subsequently the test kit of the present invention that embodiment 1 provides to form 2. cDNA the first chain synthetic agent box, that is: 25mmol/L MgC1
24 μ L, 10 * reversed transcriptive enzyme damping fluid, 2 μ L, 10mmol/L dNTPs2 μ L, RNA enzyme inhibitors 0.5 μ L, Oligo (dT)
150.5 μ g and AMV reversed transcriptive enzyme 1.5U, add without RNase deionized water to cumulative volume 20 μ L, in 42 ℃ of insulation 15min, carries out reverse transcription reaction, synthetic cDNA.After reaction finishes, be heated to 99 ℃, 5min is with deactivation reversed transcriptive enzyme, adds 20 μ L sterilized waters and mixes and put refrigerator-20 ℃ preservation.
3. inside positive controlling gene sequence standard substance and ABL standard substance being diluted to respectively to copy number/mL is 1.0x10
3, 1.0x10
4, 1.0x10
5and 1.0x10
6the positive control sequence in inside providing with embodiment 1 and the fluorescence quantitative PCR reaction solution of ABL reference gene, make respectively inner positive control sequence standard substance typical curve (as shown in FIG. 1A and 1B) and ABL standard substance typical curve (as shown in Figure 2 A and 2 B).
4. m BCR fusion gene fluorescent quantitative PCR: PCR reaction system is 20 μ L: containing 1 * PCR premix (Qiagen company, product article No.: 210212,2 * PCR Premix) 10.0 μ L, the Mg of 2.5-4.0mM
2+, the dNTPs of 0.2-0.4mM and the dUTP of 0.3-0.6mM, the UNG enzyme of the Taq enzyme of 0.2U/ μ L and 0.01-0.05U/ μ L, on m BCR fusion gene, downstream primer final concentration is 0.2 μ mol/L, m BCR fusion gene Taqman fluorescent probe final concentration 0.3 μ mol/L, the cDNA1.0 μ L that sample RNA reverse transcription is synthetic, the primer final concentration that simultaneously adds inner positive control sequence is 0.2 μ mol/L, the Taqman fluorescent probe final concentration 0.3 μ mol/L of inner positive control sequence, final concentration is the 2. middle synthetic cDNA of reverse transcription of the synthetic cDNA(of the inside positive control sequence RNA reverse transcription of 0.2 μ mol/L), add ultrapure water to cumulative volume 20 μ L.On lightcycler quantitative real time PCR Instrument, react: amplification condition: 95 ℃ of 10min denaturations, 95 ℃ of 15s then, 40 circulations of 60 ℃ of 1min amplifications, amplification procedure Instrumental is collected fluorescent signal automatically.
5. ABL reference gene fluorescent quantitative PCR: PCR reaction system is 20 μ L: containing 2 * PCR mixed solution (Qiagen company, product article No.: 210212,2 * PCR Premix) 10.0 μ L, the Mg of 2.5-4.0mM
2+, the dNTPs of 0.2-0.4mM and the Taq enzyme of the dUTP of 0.3-0.6mM, 0.2U/ μ L and the UNG enzyme of 0.01-0.05U/ μ L, abl gene upstream and downstream primer final concentration is 0.2 μ mol/L, the Taqman fluorescent probe final concentration 0.2 μ mol/L of abl gene, abl gene cDNA1.0 μ L, adds without RNase deionized water and mends to cumulative volume 20 μ L.On lightcycler quantitative real time PCR Instrument, react: amplification condition is 95 ℃ of 10min denaturations, 95 ℃ of 15s then, 40 circulations of 60 ℃ of 1min amplifications, amplification procedure Instrumental is collected fluorescent signal automatically.
6. positive control, negative control fluorescent quantitative PCR: PCR reaction system is 20 μ L: containing 2 * PCR mixed solution (Qiagen company, product article No.: 210212,2 * PCR Premix) 10.0 μ L, the Mg of 2.5-4.0mM
2+, the dNTPs of 0.2-0.4mM and the Taq enzyme of the dUTP of 0.3-0.6mM, 0.2U/ μ L and the UNG enzyme of 0.01-0.05U/ μ L, m BCR fusion gene upstream and downstream primer final concentration is 0.2 μ mol/L, m BCR fusion gene Taqman fluorescent probe final concentration 0.3 μ mol/L, the cDNA1.0 μ L that positive control RNA reverse transcription is synthetic or negative control deionized water 1.0 μ L, add without RNase deionized water and mend to cumulative volume 20 μ L.On lightcycler quantitative real time PCR Instrument, react: amplification condition is 95 ℃ of 10min denaturations, 95 ℃ of 15s then, 40 circulations of 60 ℃ of 1min amplifications, amplification procedure Instrumental is collected fluorescent signal automatically.
7. data collection process and analysis: after pcr amplification finishes, first analyze inner positive control sequence amplification, if its Ct value is less than 33, point out whole testing process effective, if its Ct value is greater than 35, need to re-start detection.When the positive control sequence Ct value in inside is less than 33, real-time fluorescence quantitative PCR the data obtained is calculated, draw after m BCR fusion gene is with respect to the relative expression quantity of ABL reference gene and carry out again statistical study, with ratio, be more than or equal to 0.0001 positive expression, be less than 0.0001 negative expression (specifically referring to table 1):
Table 1 is the expression of quantitative fluorescent PCR analysis m BCR fusion gene in acute lymphoblastic leukemia Bone Marrow of Patients
| Sample | M BCR template | ABL template | m?BCR/ABL |
| Acute lymphoblastic leukemia | 7758991 | 318823477 | 0.02434 |
| Acute lymphoblastic leukemia | 7718284 | 151500230 | 0.05095 |
| Acute lymphoblastic leukemia | 912788 | 278557499 | 0.00328 |
| Acute lymphoblastic leukemia | 28022 | 278124665 | 0.00010 |
| Acute lymphoblastic leukemia | 551243 | 216755774 | 0.00254 |
| Acute lymphoblastic leukemia | 27748 | 316657072 | 0.00009 |
| Acute lymphoblastic leukemia | 67013 | 312665502 | 0.00021 |
| Acute lymphoblastic leukemia | 14551 | 217254752 | 0.00007 |
| Acute lymphoblastic leukemia | 91724 | 331725548 | 0.00028 |
| Acute lymphoblastic leukemia | 612541 | 313254364 | 0.00196 |
| Acute lymphoblastic leukemia | 152514 | 244718275 | 0.00062 |
| Acute lymphoblastic leukemia | 7195462 | 301727641 | 0.02385 |
| Acute lymphoblastic leukemia | 6612745 | 212485613 | 0.03112 |
| Acute lymphoblastic leukemia | 12664861 | 301828591 | 0.04196 |
| Acute lymphoblastic leukemia | 264661 | 301857780 | 0.00088 |
| Acute lymphoblastic leukemia | 27712635 | 307764186 | 0.09005 |
| Acute lymphoblastic leukemia | 127356 | 208177583 | 0.00061 |
| Acute lymphoblastic leukemia | 41982465 | 271845588 | 0.15443 |
| Acute lymphoblastic leukemia | 26486 | 296162748 | 0.00009 |
| Acute lymphoblastic leukemia | 126658 | 261774550 | 0.00048 |
| Acute lymphoblastic leukemia | 7749 | 186566127 | 0.00004 |
| Acute lymphoblastic leukemia | 317486 | 261554585 | 0.00121 |
| Acute lymphoblastic leukemia | 754861 | 175542965 | 0.00430 |
| Acute lymphoblastic leukemia | 21274658 | 271655854 | 0.07831 |
| Acute lymphoblastic leukemia | 3758 | 218566596 | 0.00002 |
| Acute lymphoblastic leukemia | 86265911 | 310576652 | 0.27776 |
| Acute lymphoblastic leukemia | 91759560 | 319757691 | 0.28697 |
| Acute lymphoblastic leukemia | 47585 | 331768965 | 0.00014 |
| Acute lymphoblastic leukemia | 9275861 | 236518950 | 0.03922 |
| Acute lymphoblastic leukemia | 19055 | 201757785 | 0.00009 |
Numerical value in above-mentioned table in m BCR template and ABL template all represents fluorescence aggregate-value.
Test kit detectivity is evaluated:
With qualitative PCR method detection method as a comparison, above-mentioned 30 routine acute lymphoblastic leukemia myeloid tissue samples are detected simultaneously, comparative result shows, adopt this test kit of the present invention to detect its susceptibility, specificity and sensitivity more accurate compared with Immunohistochemical Method, meet current clinic diagnosis real requirement (specifically referring to table 2) completely:
Table 2 is that two kinds of different methods detect the comparison that in acute lymphoblastic leukemia, m BCR fusion gene is expressed
As seen from the above table, by qualitative PCR method check fluorescent quantitation, qualitative PCR method is as reference, it is positive that fluorescent quantitation and qualitative PCR method detect 22 examples simultaneously, and qualitative PCR method has detected 1 example feminine gender, draw thus, the positive predictive value of fluorescent quantitation is 95.7%; It is negative that fluorescent quantitation and qualitative PCR method detect 7 examples simultaneously, all do not detect the positive, draws thus, and the negative predictive value of fluorescent quantitation is 100%.
Wherein:
1. specificity: 87.5%;
2. sensitivity: 100%;
3. positive predictive value: positive predictive value reaches 95.7%;
4. negative predictive value: negative predictive value reaches 100%;
5. repeated: repeatedly to repeat experimental result consistent;
6. consuming time: be about 4h the detection time of a clinical samples, consuming time short, and Immunohistochemical Method about 72h consuming time.
Above-mentioned experiment can illustrate, adopt all higher fluorescence quantitative PCR detection m BCR fusion gene mRNA levels of susceptibility and specificity, specificity and the susceptibility of its detected result are significantly increased, adopt inner positive control sequence to monitor whole testing process, effectively guaranteed the quality of each detection.
The invention provides a kind of acute lymphoblastic leukemia m BCR fusion gene real-time fluorescence quantitative PCR detection kit that can monitor whole testing process, adopt artificial design and the positive control sequence in synthetic inside, the whole process that monitoring acute lymphoblastic leukemia m BCR fusion gene real-time fluorescence quantitative PCR detects, can effectively solve the false positive in current acute lymphoblastic leukemia m BCR fusion gene real-time fluorescence quantitative PCR testing process, Problem of False Negative, make detected result more reliable, the gene type that this test kit is acute lymphoblastic leukemia and chemotherapy and prognosis provide a kind of brand-new fast and convenient gene diagnosis technology.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (6)
1. a test kit that detects m BCR fusion gene mRNA expression amount, comprise and detecting with primer, fluorescent probe, cDNA the first chain synthetic agent, quantitative fluorescent PCR mixed solution, negative control and positive control, it is characterized in that, described for detection primer, fluorescent probe comprise m BCR fusion gene primer, reference gene ABL primer and Taqman fluorescent probe, described test kit also comprises the primer of inner positive control sequence, inner positive control sequence and the Taqman fluorescent probe of inner positive control sequence, wherein:
M BCR fusion gene upstream primer sequence is as shown in SEQ ID NO:1 in sequence table;
M BCR fusion gene downstream primer sequence is as shown in SEQ ID NO:2 in sequence table;
M BCR fusion gene Taqman fluorescent probe is as shown in SEQ ID NO:3 in sequence table;
Abl gene upstream primer sequence is as shown in SEQ ID NO:4 in sequence table;
Abl gene downstream primer sequence is as shown in SEQ ID NO:5 in sequence table;
Abl gene Taqman fluorescent probe is as shown in SEQ ID NO:6 in sequence table;
Inner positive control sequence is as shown in SEQ ID NO:7 in sequence table;
The upstream primer of inner positive control sequence is as shown in SEQ ID NO:8 in sequence table;
The downstream primer of inner positive control sequence is as shown in SEQ ID NO:9 in sequence table;
The Taqman fluorescent probe of inner positive control sequence is as shown in SEQ ID NO:10 in sequence table;
Described cDNA the first chain synthetic agent contains MgC1
2, reversed transcriptive enzyme damping fluid, dNTPs, RNA enzyme inhibitors, Oligo (dT)
15, AMV reversed transcriptive enzyme and without RNase deionized water; Described quantitative fluorescent PCR mixed solution contains PCR premix, Mg
2+, dNTPs, dUTP, Taq enzyme, UNG enzyme and without RNase deionized water.
2. test kit according to claim 1, it is characterized in that, 5 ' end of the Taqman fluorescent probe of described m BCR fusion gene and the Taqman fluorescent probe of abl gene is connected with fluorescence report group FAM, 3 ' end is all connected with fluorescent quenching group TAMRA, 5 ' end of the Taqman fluorescent probe of inner positive control sequence is connected with fluorescence report group TET, and 3 ' end is connected with fluorescent quenching group TAMRA.
3. test kit according to claim 1, is characterized in that, the nucleotide sequence of described reference gene ABL is as shown in SEQ ID NO:11 in sequence table.
4. test kit according to claim 1, is characterized in that, described negative control is deionized water; Described positive control is the total RNA sample that contains m BCR fusion gene.
5. test kit according to claim 1, is characterized in that, described cDNA the first chain synthetic agent is: 25mmol/L MgC1
24 μ L, 10 * reversed transcriptive enzyme damping fluid, 2 μ L, 10mmol/L dNTP2 μ L, RNA enzyme inhibitors 0.5 μ L, Oligo (dT)
150.5 μ g, AMV reversed transcriptive enzyme 1.5U and without RNase deionized water, cumulative volume 11 μ L.
6. test kit according to claim 1, is characterized in that, the mixed solution of described quantitative fluorescent PCR is: 1 * PCR premix, the Mg of 2.5-4.0mM
2+, the dNTPs of 0.2-0.4mM, the Taq enzyme of the dUTP of 0.3-0.6mM, 0.2U/ μ L, the UNG enzyme of 0.01-0.05U/ μ L and without RNase deionized water.
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