CN102925558A - Kit for detecting mRNA expression level of PML-RARa fusion gene - Google Patents
Kit for detecting mRNA expression level of PML-RARa fusion gene Download PDFInfo
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Abstract
The invention relates to a kit for the mRNA expression level of a PML-RARa fusion gene, and belongs to the field of biotechnology. The kit comprises a PML-RARa L fusion gene system, a PML-RARa V fusion gene system or a PML-RARa s fusion gene system and a reference gene system ABL, wherein each system comprises an upstream primer and a downstream primer and Taqman fluorescence probe. The PML-RARa fusion protein, as a variant retinoic acid receptor, has different DNA properties with a wild type RARa protein and is an intrinsic and effective repressor for retinoic acid (RA) signal which is the transcription factor of an intrinsic RARa target gene. Therefore, when the fluorescence quantitative polymerase chain reaction (PCR) method is used for detecting the mRNA expression level of the PML-RARa fusion gene, the detection result is more specific and sensible. The kit provides a novel quick and simple genetic diagnosis technology for predicting the prognosis of acute promyelocytic leukemia and determining chemotherapy regimens.
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 PML-RARa of detection fusion gene mRNA expression amount.
Background technology
Acute promyelocytic leukemia (Acute Promyelocytic Leukemia, APL) be acute myeloid leukaemia (Acute myeloid leukemia, AML) a kind of hypotype in, account for adult AML patient's 10% ~ 15%, patient is often young, age is many between 30 ~ 38 years old, and the person is rarer below 10 years old.According to statistics, it is Hesperian about 10% that domestic APL sickness rate is higher than, and accounts for 18.7% of AML, some areas such as the sickness rate in oil field, northeast then up to 20% ~ 30%.Therefore, there is the difference of age, race and region in APL.
In the recent decade because the application of all-trans-retinoic acid and white arsenic, adopt two inductive treatments can so that the initial stage inducer remission rate of APL reach more than 90%.After the drug-induced treatment, give SCC and in conjunction with keeping treatment with the medicine of vitamin A acid and white arsenic, can make patient finish treatment and clinical cure rate the time about 2 years up to 90%.
At present, the cause of disease of APL is still not clear, and still along with the fast development of the subjects such as molecular biology, cytogenetics, people have had more deep understanding for APL molecular biology pathogenesis.The key mechanism of the APL morbidity above 90% is t(15; 17) (q22; Q21), cause the RARa gene on PML gene on No. 15 karyomit(e) and No. 17 karyomit(e) to form the PML-RARa fusion gene.Transposition is reset PML gene and the RAR α gene generation interactivity on the 17q21 on the 15q22, forms PML-RARa fusion gene and RARa-PML fusion gene at No. 15 karyomit(e)s respectively.Studies show that patient's leukemia cell all has the expression of PML-RARa fusion gene, only have 70% ~ 80% patient to express simultaneously the PML-RARa fusion gene, illustrate that the PML-RARa fusion gene is the key of causing a disease.Position according to the PML breaking point is different, and the PML-RARa fusion gene has three kinds of hypotype bcr1, bcr2 and bcr3, is called elongated (L-type), anomaly (V-type) and short type (S type), and the probability of generation then is followed successively by 55%, 5% and 40%.The PML-RARa fusion rotein is as a kind of retinoid receptor of variation, has different DNA characteristics compared to wild-type RARa albumen, be the inhibition of vitamin A acid (retinoic acid, RA) signal, be called the inherence and effective RARa target gene transcription factor inhibition by different approach.
Therefore, when the patient detects the sudden change of PML-RARa fusion gene, can be used as the diagnosis basis of acute promyelocytic leukemia, also can be used as the molecular marker to all-trans-retinoic acid and arsenic agent treatment outcome prediction.
The method that can be used at present the PML-RARa fusion gene has probe hybridization method, gene chips, mass spectroscopy, gene sequencing method and immunofluorescence technique etc.Probe hybridization method false positive rate is higher; The gene chips cost is high, the preparation method is loaded down with trivial details; Mass spectroscopy is difficult to carry out in common medical institutions; Though gene sequencing method susceptibility and specificity are high, price comparison is expensive, and operation is more loaded down with trivial details, consuming time longer.But immuno-fluorescence assay PML-RARa fusion gene quantitative expression high-throughput is finished clinical sample and is detected, and specificity and susceptibility are all very high, and its susceptibility and specificity all are higher than the methods such as direct immunofluorescence detection.Real-time fluorescence quantitative PCR (Polymerase Chain Reaction, the polymerase chain reaction) compares with immunofluorescence technique and have that specificity strengthens, sensitivity improves and detect fast, reduced the characteristics such as pollutions, and real time fluorescence quantifying PCR method detection PML-RARa fusion gene is not yet arranged at present.
Summary of the invention
In order to solve the problem of prior art, the embodiment of the invention provides a kind of test kit that adopts fluorescent quantitative PCR technique to detect PML-RARa fusion gene mRNA expression amount.Described technical scheme is as follows:
A kind of test kit that detects PML-RARa 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 detection comprises at least a and reference gene ABL primer and Taqman fluorescent probe in PML-RARaL fusion gene primer, PML-RARaV fusion gene primer and the PML-RARa S fusion gene primer with primer, fluorescent probe, wherein:
PML-RARa L fusion gene upstream primer sequence is shown in SEQ ID NO:1 in the sequence table;
PML-RARa L fusion gene downstream primer sequence is shown in SEQ ID NO:2 in the sequence table;
PML-RARa L fusion gene Taqman fluorescent probe is shown in SEQ ID NO:3 in the sequence table;
PML-RARa V fusion gene upstream primer sequence is shown in SEQ ID NO:4 in the sequence table;
PML-RARa V fusion gene downstream primer sequence is shown in SEQ ID NO:5 in the sequence table;
PML-RARa V fusion gene Taqman fluorescent probe is shown in SEQ ID NO:6 in the sequence table;
PML-RARa S fusion gene upstream primer sequence is shown in SEQ ID NO:7 in the sequence table;
PML-RARa S fusion gene downstream primer sequence is shown in SEQ ID NO:8 in the sequence table;
PML-RARa S fusion gene Taqman fluorescent probe is shown in SEQ ID NO:9 in the sequence table;
Abl gene upstream primer sequence is shown in SEQ ID NO:10 in the sequence table;
Abl gene downstream primer sequence is shown in SEQ ID NO:11 in the sequence table;
Abl gene Taqman fluorescent probe is shown in SEQ ID NO:12 in the 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 the RNase deionized water; Described quantitative fluorescent PCR mixed solution contains PCR premix, Mg
2+, dNTPs, dUTP, Taq enzyme, UNG enzyme and without the RNase deionized water.
Further, described test kit also comprises primer and the Taqman fluorescent probe of inner positive control sequence, inner positive control sequence, and wherein: inner positive control sequence is shown in SEQ ID NO:13 in the sequence table; The upstream primer of inner positive control sequence is shown in SEQ ID NO:14 in the sequence table; The downstream primer of inner positive control sequence is shown in SEQ ID NO:15 in the sequence table; Inner positive control sequence Taqman fluorescent probe is shown in SEQ ID NO:16 in the sequence table.
Further, 5 ' end of the Taqman fluorescent probe of described PML-RARa L fusion gene Taqman fluorescent probe, PML-RARa V fusion gene Taqman fluorescent probe, PML-RARa S fusion gene Taqman fluorescent probe and abl gene is connected with fluorescence report group FAM, and 3 ' end all is connected with fluorescent quenching group TAMRA; 5 ' end of the Taqman fluorescent probe of the positive control sequence in described inside 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 shown in SEQ ID NO:17 in the sequence table.
Particularly, described negative control is deionized water; Described positive control is the total RNA sample that contains PML-RARa L fusion gene, PML-RARa V fusion gene and PML-RARa S fusion gene.
Particularly, described the first chain cDNA 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)
150.5ug, AMV reversed transcriptive enzyme 1.5U and without the RNase deionized water, cumulative volume 11 μ L.
Particularly, the mixed solution of described quantitative fluorescent PCR (representing take the reaction system final concentration) as: 1 * PCR premix (stoste is 2 * PCR Premix), the Mg of 2.5-4.0mM
2+, the Taq enzyme of dUTP, 0.2U/ μ L of dNTPs, 0.3-0.6mM of 0.2-0.4mM and 0.01-0.05U/ μ L the UNG enzyme and without the 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%, namely has one to contain the PML-RARa fusion gene and just can be detected in 10000 cells.
(2) high specificity: use specific probe that quantitative molecular is identified, accuracy is high.Simultaneously, target sequence is by primer and the dual control of probe, and specificity is good, false positive is low.
(3) handy and safe: simple to operate, safety, level of automation is high and prevent from polluting.Amplification and detection can detect in same pipe, do not need to uncap, and are difficult for contaminated; Increase simultaneously and detect one the step finish, do not need post-processed, need not worry radiocontamination.
(4) complete monitoring: the test kit that the embodiment of the invention provides has been introduced inner positive control quality control system, and testing process is carried out Complete Quality Supervision, effectively avoids false positive or false negative.
(5) quick: speed is fast, high-throughput, can finish at 3-4 hour.
Quick, accurate, the detection by quantitative PML-RARa fusion gene mRNA level of test kit energy of the present invention, false positive and false-negative generation have effectively been stopped, be used for the diagnosis of acute promyelocytic leukemia and the monitoring of therapeutic process minimal residual disease, for the diagnosis of acute promyelocytic leukemia, formulate treatment plan and therapeutic evaluation and prognosis important detection means is provided.
Description of drawings
In order to be illustrated more clearly in the technical scheme in the embodiment of the invention, the accompanying drawing of required use was done to introduce simply during the below will describe embodiment, apparently, accompanying drawing in the following describes only is some embodiments of the present invention, for those of ordinary skills, under the prerequisite of not paying 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 invention 2
6-1.0x10
3The fluorescence curve figure of the positive control sequence standard substance in the 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 invention 2
6-1.0x10
3The canonical plotting that the fluorescence curve figure of the positive control sequence standard substance in the 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 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 invention 2
6-1.0x10
3The canonical plotting that the ABL standard substance fluorescence curve figure of copy obtains.
Embodiment
For making the purpose, technical solutions and advantages of the present invention clearer, embodiment of the present invention is described further in detail below in conjunction with accompanying drawing.
The preparation of embodiment 1. test kits
1, the design of specific primer and fluorescent probe
(abl gene sequence, PML gene order and RARa 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 according to gene order; The PML gene I/D is respectively 5371, and reference sequences number is NG_029036.1; The RARa gene I/D is respectively 5914, and reference sequences number is NG_027701.1) 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: (Invitrogen company, the product article No.: 15596-026/100ml), every 1ml myeloid tissue adds 1mlTrizol rapid extraction acute promyelocytic leukemia Bone Marrow of Patients and organizes RNA Trizol reagent.
2. cDNA the first chain synthetic agent box (RTPCR) (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 the RNase deionized water.
3. primer, probe and standard substance: comprise PML-RARa fusion gene primer, inner positive control sequence, inner positive control sequence primer, reference gene ABL primer and the Taqman fluorescent probe corresponding with primer, specific as follows:
PML-RARa L fusion gene upstream primer sequence: 5 '-TCTTCCTGCCCAACAGCAA-3 ' (sequence 1 in the sequence table);
PML-RARa L fusion gene downstream primer sequence: 5 '-GCTGGGCACTATCTCTTCAGAAC-3 ' (sequence 2 in the sequence table);
PML-RARa L fusion gene Taqman fluorescent probe: sequence 3 in FAM5 '-CAGCCATGAGACCCA-3 ' TAMRA(sequence table);
PML-RARa V fusion gene upstream primer sequence: 5 '-GGACCTCAGCTCTTGCATCAC-3 ' (sequence 4 in the sequence table);
PML-RARaV fusion gene downstream primer sequence: 5 '-GCTGGGCACTATCTCTTCAGAAC-3 ' (sequence 5 in the sequence table);
PML-RARa V fusion gene Taqman fluorescent probe: sequence 6 in FAM5 '-AAGCCATTGAGACCCAG-3 ' TAMRA(sequence table);
PML-RARa S fusion gene upstream primer sequence: 5 '-GAAGGTCATCAAGATGGAGTCTGA-3 ' (sequence 7 in the sequence table);
PML-RARa S fusion gene downstream primer sequence: 5 '-TGCTGCTCTGGGTCTCAATG-3 ' (sequence 8 in the sequence table);
PML-RARa S fusion gene Taqman fluorescent probe: sequence 9 in FAM5 '-AAGGAGGCAAGGTTGG-3 ' TAMRA(sequence table);
Inner positive control sequence is: 5 '-UCUUCCUGCCGAACAGCUACCACGUGGCCAGUGGCGCCGGGGAGGCAGCGAUUCAG ACCCAGAGCAGCACUUGUGAAGAGAUAGUGCCCAGC-3 ' (sequence 13 in the sequence table);
Inner positive control sequence upstream primer sequence is: 5 '-TCTTCCTGCCGAACAGCTA-3 ' (sequence 14 in the sequence table);
Inner positive control sequence downstream primer sequence is: 5 '-GCTGGGCACTATCTCTTCACAAG-3 ' (sequence 15 in the sequence table);
Sequence 16 in inner positive control sequence Taqman fluorescent probe: TET5 '-CAGCGATTCAGACCCA-3 ' TAMRA(sequence table).
ABL gene sequence: 5'-CAGGGTCTGAGTGAAGCCGCTCGTTGGAACTCCAAGGAAAACCTTCTCGCTGGACCCAGTGAAAATGACCCCAACCTTTTCGTTGCACTGTATGATTTTGTGGCCAGTGGAGATAACACTCTAAGCATAACTAAAGGTGAAAAGCTCCGGGTCTTAGGCTATAATCACAATGGGGAATGGTGTGAAGCCCAAACCAAAAATGGCCAAGGCTGGGTCCCAAGCAACTACATCACGCCAGTCAACAGTCTGGAGAAACACTCCTGGTACCATGGGCCTGTGTCCCGCAATGCCGCTGAGTATCTGCTGAGCAGCGGGATCAATGGCAGCTTCTTGGTGCGTGAGAGTGAGAGCAGTCCTGGC-3’(the sequence 17 in the sequence table);
Abl gene upstream primer sequence is: 5 '-CCGGGTCTTAGGCTATAATCACA-3 ' (sequence 10 in the sequence table);
Abl gene downstream primer sequence is: 5 '-GCCTTGGCCATTTTTGGTT-3 ' (sequence 11 in the sequence table);
Sequence 12 in abl gene Taqman fluorescent probe: FAM5 '-TGGTGTGAAGCCC-3 ' TAMRA(sequence table);
Wherein, inner positive control sequence is artificial synthesized sequence, comprises the artificial composition sequence of the known PML-RARa 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 synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
4. negative control and positive control: with the negative contrast of deionized water, to contain the positive contrast of the total RNA sample of PML-RARa fusion gene, RNA extracts reagent to adopt the test kit of the present invention that provides in above-described embodiment 1 to form 1.: Trizol reagent (Invitrogen company, product article No.: 15596-026/100ml), the ratio that adds 1ml Trizol reagent in every 1ml myeloid tissue, the acute promyelocytic leukemia patient's of containing the PML-RARa fusion gene that rapid extraction has been made a definite diagnosis myeloid tissue RNA is as positive control.
5. PML-RARa L fusion gene fluorescence quantitative PCR reaction solution: by the quantitative fluorescent PCR mixed solution and detect the specific primer of MPL gene W515L site mutation, probe forms: 1 * PCR premix (Qiagen company, 210212,2 * PCR Premix), the Mg of 2.5-4.0mM product article No.:
2+0.2-0.4mM dNTPs and the dUTP of 0.3-0.6mM, 0.2U/ the UNG enzyme of the Taq enzyme of μ L and 0.01-0.05U/ μ L, 0.25pmol/ the PML-RARa L fusion gene upstream primer (sequence 1 in the sequence table) of μ L, 0.25pmol/ the PML-RARa L fusion gene downstream primer (sequence 2 in the sequence table) of μ L, 0.3pmol/ the PML-RARa L fusion gene Taqman fluorescent probe (sequence 3 in the sequence table) of μ L, 0.25pmol/ the positive control sequence upstream primer (sequence 14 in the sequence table) in the inside of μ L, 0.25pmol/ the positive control sequence downstream primer (sequence 15 in the sequence table) in the inside of μ L, 0.3pmol/ the positive control sequence Taqman fluorescent probe (sequence 16 in the sequence table) (above all concentration all refers to the final concentration of PCR reaction system) in the inside of μ L.Usually get the template (comprising cDNA and the synthetic cDNA of inner positive control sequence RNA reverse transcription that sample RNA reverse transcription is synthetic) of 1-2 μ L, all the other are without the RNase deionized water, and the reaction cumulative volume is generally 20 μ L.
6. PML-RARa V fusion gene fluorescence quantitative PCR reaction solution: by the quantitative fluorescent PCR mixed solution and detect the specific primer of MPL gene W515L site mutation, probe forms: 1 * PCR premix (Qiagen company, 210212,2 * PCR Premix), the Mg of 2.5-4.0mM product article No.:
2+0.2-0.4mM dNTPs and the dUTP of 0.3-0.6mM, 0.2U/ the UNG enzyme of the Taq enzyme of μ L and 0.01-0.05U/ μ L, 0.25pmol/ the PML-RARa V fusion gene upstream primer (sequence 4 in the sequence table) of μ L, 0.25pmol/ the PML-RARaV fusion gene downstream primer (sequence 5 in the sequence table) of μ L, 0.3pmol/ the PML-RARa V fusion gene Taqman fluorescent probe (sequence 6 in the sequence table) of μ L, 0.25pmol/ the positive control sequence upstream primer (sequence 14 in the sequence table) in the inside of μ L, 0.25pmol/ the positive control sequence downstream primer (sequence 15 in the sequence table) in the inside of μ L, 0.3pmol/ the positive control sequence Taqman fluorescent probe (sequence 16 in the sequence table) in the inside of μ L, 0.3pmol/ the positive control sequence Taqman fluorescent probe (sequence 13 in the sequence table) (above all concentration all refers to the final concentration of PCR reaction system) in the inside of μ L.Usually get the template (comprising cDNA and the synthetic cDNA of inner positive control sequence RNA reverse transcription that sample RNA reverse transcription is synthetic) of 1-2 μ L, all the other are without the RNase deionized water, and the reaction cumulative volume is generally 20 μ L.
7. PML-RARa S fusion gene fluorescence quantitative PCR reaction solution: by the quantitative fluorescent PCR mixed solution and detect the specific primer of MPL gene W515L site mutation, probe forms: 1 * PCR premix (Qiagen company, 210212,2 * PCR Premix), the Mg of 2.5-4.0mM product article No.:
2+0.2-0.4mM dNTPs and the dUTP of 0.3-0.6mM, 0.2U/ the UNG enzyme of the Taq enzyme of μ L and 0.01-0.05U/ μ L, 0.25pmol/ the PML-RARa S fusion gene upstream primer (sequence 7 in the sequence table) of μ L, 0.25pmol/ the PML-RARa S fusion gene downstream primer (sequence 8 in the sequence table) of μ L, 0.3pmol/ the PML-RARa S fusion gene Taqman fluorescent probe (sequence 9 in the sequence table) of μ L, 0.25pmol/ the positive control sequence upstream primer (sequence 14 in the sequence table) in the inside of μ L, 0.25pmol/ the positive control sequence downstream primer (sequence 15 in the sequence table) in the inside of μ L, 0.3pmol/ the positive control sequence Taqman fluorescent probe (sequence 16 in the sequence table) (above all concentration all refers to the final concentration of PCR reaction system) in the inside of μ L.Usually get the template (comprising cDNA and the synthetic cDNA of inner positive control sequence RNA reverse transcription that sample RNA reverse transcription is synthetic) of 1-2 μ L, all the other are without the RNase deionized water, and the reaction cumulative volume is generally 20 μ L.
8. ABL reference gene fluorescence quantitative PCR reaction solution: formed by quantitative fluorescent PCR mixed solution and the primer, the probe that detect the ABL reference gene: 1 * PCR premix (Qiagen company, 210212,2 * PCR Premix), the Mg of 2.5-4.0mM product article No.:
2+, the Taq enzyme of dUTP, 0.2U/ μ L of dNTPs, 0.3-0.6mM of 0.2-0.4mM and the UNG enzyme of 0.01-0.05U/ μ L, the ABL reference gene upstream primer (sequence 10 in the sequence table) of 0.25pmol/ μ L, the ABL reference gene downstream primer (sequence 11 in the sequence table) of 0.25pmol/ μ L, the abl gene Taqman fluorescent probe (sequence 12 in the sequence table) (above all concentration all refers to the final concentration of PCR reaction system) of 0.3pmol/ μ L.During detection, add abl gene standard substance template 2 μ L, the reaction cumulative volume is generally 20 μ L.
9. 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 Taq enzyme of dUTP, 0.2U/ μ L of the dNTPs of 0.2-0.4mM and 0.3-0.6mM and the UNG enzyme of 0.01-0.05U/ μ L, the positive control sequence upstream primer (sequence 11 in the sequence table) in inside of 0.25pmol/ μ L, the positive control sequence downstream primer (sequence 12 in the sequence table) in inside of 0.25pmol/ μ L, the positive control sequence Taqman fluorescent probe (sequence 13 in the sequence table) (above all concentration all refers to the final concentration of PCR reaction system) in inside of 0.3pmol/ μ L.During detection, usually get the template (cDNA that inner positive control sequence RNA reverse transcription is synthetic) of 1-2 μ L, all the other are without the RNase deionized water, and the reaction cumulative volume is generally 20 μ L.
3, the setting of pcr amplification program: on the 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. usefulness embodiment 1 preparation detects the expression amount of PML-RARa fusion gene mRNA
To detect 30 routine acute promyelocytic leukemia myeloid tissue sample results as example, wherein, the patient detects PML-RARa fusion gene form and comprises PML-RARa L fusion gene, PML-RARa V fusion gene and PML-RARa S fusion gene.The testing process that detects PML-RARa fusion gene mRNA expression amount with test kit of the present invention is: at first design specific primer and fluorescent probe according to gene order.Next obtains Clinical Acute promyelocytic leukemia Bone Marrow of Patients tissue samples, and rapid extraction is organized RNA, carries out synthetic cDNA the first chain of reverse transcription PCR; Prepare first 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 copy number/mL
3, 1.0x10
4, 1.0x10
5And 1.0x10
6Make respectively inner positive control sequence standard substance typical curve (shown in Figure 1A and Figure 1B) and ABL standard substance typical curve (shown in Fig. 2 A and Fig. 2 B), and then prepare PML-RARa fusion gene PCR reaction solution and carry out the fluorescence quantitative PCR detection sample, in the quantitative real time PCR Instrument data analysis system, read CT value result.Pcr amplification is at first analyzed inner positive control sequence amplification, if its Ct value is less than 33 after finishing, point out whole testing process effective, if its Ct value greater than 35, is pointed out and detected unsuccessfully, then need to re-start detection, if its Ct value is positioned between 33 ~ 35, need duplicate detection.When the positive control sequence Ct value in inside less than 33 the time, the real-time fluorescence quantitative PCR the data obtained is calculated, calculate respectively the Ct value of PML-RARa fusion gene and abl gene, both differences are Δ Ct value.At last, fluorescent quantitative PCR result adopts software analysis, and markization calculating sampled data.
Concrete steps are as follows:
1. the extracting of acute promyelocytic leukemia Bone Marrow of Patients total tissue RNA: the method extracting acute promyelocytic leukemia Bone Marrow of Patients total tissue RNA of pressing the RNA extracting and purifying.The RNA that extracts identifies its integrity through agarose gel electrophoresis, by purity and the concentration of ultraviolet spectrophotometer mensuration 260nm and 280nm optical density value calculating RNA, regulates the RNA of extracting to same concentrations with the water that 0.1%DEPC processes.
2. cDNA is synthesized in reverse transcription: get the above-mentioned RNA extracting solution of 2 μ L, at 70 ℃ of insulation 10min, the test kit of the present invention that adding subsequently embodiment 1 provides forms 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 the RNase deionized water to cumulative volume 20 μ L, in 42 ℃ of insulation 15min, carry out reverse transcription reaction, synthetic cDNA the first chain.Reaction is heated to 99 ℃ after finishing, and 5min adds 20 μ L sterilized water mixings and puts refrigerator-20 ℃ preservation with the deactivation reversed transcriptive enzyme.
Get 1 μ L concentration and be sequence 13 in the inner positive control sequence RNA(sequence table of 2 μ g/ml), the test kit of the present invention that adds subsequently embodiment 1 and provide at 70 ℃ of insulation 10min forms 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 the RNase deionized water to cumulative volume 20 μ L, in 42 ℃ of insulation 15min, carry out reverse transcription reaction, synthetic cDNA.Reaction is heated to 99 ℃ after finishing, and 5min adds 20 μ L sterilized water mixings and puts refrigerator-20 ℃ preservation with the deactivation reversed transcriptive enzyme.
Getting 1 μ L concentration is the positive control RNA of 2 μ g/ml, and at 70 ℃ of insulation 10min, the test kit of the present invention that adding subsequently embodiment 1 provides forms 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 the RNase deionized water to cumulative volume 20 μ L, in 42 ℃ of insulation 15min, carry out reverse transcription reaction, synthetic cDNA.Reaction is heated to 99 ℃ after finishing, and 5min adds 20 μ L sterilized water mixings and puts refrigerator-20 ℃ preservation with the deactivation reversed transcriptive enzyme.
3. inside positive controlling gene sequence standard substance and ABL standard substance being diluted to respectively copy number/mL is 1.0x10
3, 1.0x10
4, 1.0x10
5And 1.0x10
6The fluorescence quantitative PCR reaction solution of the positive control sequence in the inside that provides with embodiment 1 and ABL reference gene is made respectively inner positive control sequence standard substance typical curve (shown in Figure 1A and Figure 1B) and ABL standard substance typical curve (shown in Fig. 2 A and Fig. 2 B).
4. PML-RARa fusion gene fluorescent quantitative PCR: the PCR reaction system is 20 μ L:1 * PCR premix (Qiagen company, product article No.: 210212,2 * PCR Premix) 10.0 μ L, the Mg of 2.5-4.0mM
2+0.2-0.4mM dNTPs and the dUTP of 0.3-0.6mM, 0.2U/ the UNG enzyme of the Taq enzyme of μ L and 0.01-0.05U/ μ L, PML-RARa L fusion gene, on PML-RARa V fusion gene and the PML-RARa S fusion gene, each 0.2 μ mol/L of downstream primer final concentration, add corresponding PML-RARa L fusion gene, each Taqman fluorescent probe final concentration 0.3 μ mol/L of PML-RARa V fusion gene and PML-RARa S fusion gene, cDNA1.0 μ L, add simultaneously on the inner positive control sequence, downstream primer final concentration each 0.2 μ mol/L and inner positive control sequence Taqman fluorescent probe final concentration 0.3 μ mol/L, final concentration is the 2. synthetic cDNA of middle 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.React at the lightcycler quantitative real time PCR Instrument: amplification condition is 95 ℃ of 10min denaturations, 95 ℃ of 15s then, and 40 circulations of 60 ℃ of 1min amplifications, the amplification procedure Instrumental is collected fluorescent signal automatically.
5. ABL reference gene fluorescent quantitative PCR: the PCR reaction system is 20 μ L: contain 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 Taq enzyme of dUTP, 0.2U/ μ L of the dNTPs of 0.2-0.4mM and 0.3-0.6mM and the UNG enzyme of 0.01-0.05U/ μ L, abl gene primer final concentration 0.2 μ mol/L, probe final concentration 0.2 μ mol/L, abl gene cDNA1.0 μ L adds and mends to cumulative volume 20 μ L without the RNase deionized water.React at the lightcycler quantitative real time PCR Instrument: amplification condition is 95 ℃ of 10min denaturations, 95 ℃ of 15s then, and 40 circulations of 60 ℃ of 1min amplifications, the amplification procedure Instrumental is collected fluorescent signal automatically.
6. positive control, negative control fluorescent quantitative PCR: the PCR reaction system is 20 μ L: contain 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 Taq enzyme of dUTP, 0.2U/ μ L of the dNTPs of 0.2-0.4mM and 0.3-0.6mM and the UNG enzyme of 0.01-0.05U/ μ L, PML-RARa fusion gene primer final concentration 0.2 μ mol/L, probe final concentration 0.3 μ mol/L, cDNA1.0 μ L or negative control deionized water 1.0 μ L that the positive control RNA reverse transcription is synthetic add and mend to cumulative volume 20 μ L without the RNase deionized water.React at the lightcycler quantitative real time PCR Instrument: amplification condition is 95 ℃ of 10min denaturations, 95 ℃ of 15s then, and 40 circulations of 60 ℃ of 1min amplifications, the amplification procedure Instrumental is collected fluorescent signal automatically.
7. data collection process and analysis: pcr amplification is at first analyzed inner positive control sequence amplification, if its Ct value less than 33, points out whole testing process effective, if its Ct value then needs to re-start detection greater than 35 after finishing.When the positive control sequence Ct value in inside less than 33 the time, the real-time fluorescence quantitative PCR the data obtained is calculated, draw PML-RARa fusion gene (PML-RARa L fusion gene, PML-RARa V fusion gene or PML-RARa S fusion gene) with respect to carrying out again statistical study behind the relative expression quantity of ABL reference gene, with ratio more than or equal to 0.0001 positive expression, less than 0.0001 negative expression (specifically referring to table 1):
Table 1 is analyzed the expression of PML-RARa fusion gene in acute promyelocytic leukemia for quantitative fluorescent PCR
| Sample | The PML-RARa template | The ABL template | PML-RARa/ABL |
| Acute promyelocytic leukemia | 19967554 | 200865446 | 0.09941 |
| Acute promyelocytic leukemia | 44865765 | 330864244 | 0.13560 |
| Acute promyelocytic leukemia | 28423 | 309977622 | 0.00009 |
| Acute promyelocytic leukemia | 4144333 | 318654644 | 0.01301 |
| Acute promyelocytic leukemia | 80434 | 308754678 | 0.00026 |
| Acute promyelocytic leukemia | 478534 | 209976585 | 0.00228 |
| Acute promyelocytic leukemia | 39861 | 339075566 | 0.00012 |
| Acute promyelocytic leukemia | 24412 | 339976577 | 0.00007 |
| Acute promyelocytic leukemia | 3097642 | 293332112 | 0.01056 |
| Acute promyelocytic leukemia | 4505433 | 243222123 | 0.01852 |
| Acute promyelocytic leukemia | 48134543 | 307643888 | 0.15646 |
| Acute promyelocytic leukemia | 3075443 | 349976543 | 0.00879 |
| Acute promyelocytic leukemia | 44975331 | 198002344 | 0.22715 |
| Acute promyelocytic leukemia | 4974481 | 176643332 | 0.02816 |
| Acute promyelocytic leukemia | 3765432 | 347775442 | 0.01083 |
| Acute promyelocytic leukemia | 13223976 | 310089922 | 0.04265 |
| Acute promyelocytic leukemia | 29865 | 320075566 | 0.00009 |
| Acute promyelocytic leukemia | 488009 | 276654444 | 0.00176 |
| Acute promyelocytic leukemia | 2330018 | 309987766 | 0.00752 |
| Acute promyelocytic leukemia | 48646441 | 209333273 | 0.23239 |
| Acute promyelocytic leukemia | 9754452 | 309765468 | 0.03149 |
| Acute promyelocytic leukemia | 4097669 | 308766221 | 0.01327 |
| Acute promyelocytic leukemia | 34354 | 396000331 | 0.00009 |
| Acute promyelocytic leukemia | 4674564 | 197433455 | 0.02368 |
| Acute promyelocytic leukemia | 754434 | 287765567 | 0.00262 |
| Acute promyelocytic leukemia | 474367 | 167754322 | 0.00283 |
| Acute promyelocytic leukemia | 1339802 | 303655433 | 0.00441 |
| Acute promyelocytic leukemia | 30764 | 397558543 | 0.00008 |
| Acute promyelocytic leukemia | 88975446 | 318896650 | 0.27901 |
| Acute promyelocytic leukemia | 47188660 | 177700123 | 0.26555 |
Numerical value in the above-mentioned table in PML-RARa template and the ABL template all represents the fluorescence aggregate-value.
The test kit detectivity is estimated:
With immunofluorescence technique detection method as a comparison, simultaneously above-mentioned 30 routine acute promyelocytic leukemia Bone Marrow of Patients tissue samples are detected, comparative result shows, it is more accurate than Immunohistochemical Method to adopt this test kit of the present invention to detect its susceptibility, specificity and sensitivity, meets present clinic diagnosis real requirement (specifically referring to table 2) fully:
Table 2 is that two kinds of different methods detect the comparison that the PML-RARa fusion gene is expressed in the acute promyelocytic leukemia
As seen from the above table, by immunofluorescence technique check fluorescent quantitation, immunofluorescence technique is as reference, fluorescent quantitation and immunofluorescence technique detect 17 routine high expression levels simultaneously, and immuno-fluorescence assay draws thus to the low expression of 2 examples, and the positive predictive value of fluorescent quantitation is 89.5%; Fluorescent quantitation and immunofluorescence technique detect the low expression of 11 examples simultaneously, all do not detect low the expression, draw thus, and the negative predictive value of fluorescent quantitation is 100%.
Wherein:
1. specificity: 84.6%;
2. sensitivity: 100%;
3. positive predictive value: positive predictive value reaches 89.5%;
4. negative predictive value: negative predictive value reaches 100%;
5. repeated: repeatedly the repeated experiments result is consistent;
6. consuming time: as to be about 4h the detection time of a clinical samples, weak point consuming time, and Immunohistochemical Method about 72h consuming time.
Above-mentioned experiment can illustrate, adopt all higher fluorescence quantitative PCR detection PML-RARa 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 promyelocytic leukemia fusion gene PML-RARa real-time fluorescence quantitative PCR detection kit that can monitor whole testing process, adopt artificial design and the positive control sequence in synthetic inside, monitor the whole process that white acute promyelocytic leukemia fusion gene PML-RARa real-time fluorescence quantitative PCR detects, can effectively solve the false positive in the present acute promyelocytic leukemia fusion gene PML-RARa real-time fluorescence quantitative PCR testing process, Problem of False Negative, so that detected result is more reliable, the test kit that the embodiment of the invention provides provides a kind of brand-new fast and convenient gene diagnosis technology for the gene type of acute promyelocytic leukemia and chemotherapy and prognosis.
The above only is preferred embodiment of the present invention, and is in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Sequence table
Claims (7)
1. test kit that detects PML-RARa 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 detection comprises at least a and reference gene ABL primer and Taqman fluorescent probe in PML-RARaL fusion gene primer, PML-RARa V fusion gene primer and the PML-RARa S fusion gene primer with primer, fluorescent probe, wherein:
PML-RARa L fusion gene upstream primer sequence is shown in SEQ ID NO:1 in the sequence table;
PML-RARa L fusion gene downstream primer sequence is shown in SEQ ID NO:2 in the sequence table;
PML-RARa L fusion gene Taqman fluorescent probe is shown in SEQ ID NO:3 in the sequence table;
PML-RARa V fusion gene upstream primer sequence is shown in SEQ ID NO:4 in the sequence table;
PML-RARa V fusion gene downstream primer sequence is shown in SEQ ID NO:5 in the sequence table;
PML-RARa V fusion gene Taqman fluorescent probe is shown in SEQ ID NO:6 in the sequence table;
PML-RARa S fusion gene upstream primer sequence is shown in SEQ ID NO:7 in the sequence table;
PML-RARa S fusion gene downstream primer sequence is shown in SEQ ID NO:8 in the sequence table;
PML-RARa S fusion gene Taqman fluorescent probe is shown in SEQ ID NO:9 in the sequence table;
Abl gene upstream primer sequence is shown in SEQ ID NO:10 in the sequence table;
Abl gene downstream primer sequence is shown in SEQ ID NO:11 in the sequence table;
Abl gene Taqman fluorescent probe is shown in SEQ ID NO:12 in the 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 the RNase deionized water; Described quantitative fluorescent PCR mixed solution contains PCR premix, Mg
2+, dNTPs, dUTP, Taq enzyme, UNG enzyme and without the RNase deionized water.
2. test kit according to claim 1, it is characterized in that, described test kit also comprises primer and the Taqman fluorescent probe of inner positive control sequence, inner positive control sequence, and wherein: inner positive control sequence is shown in SEQ ID NO:13 in the sequence table; The upstream primer of inner positive control sequence is shown in SEQ ID NO:14 in the sequence table; The downstream primer of inner positive control sequence is shown in SEQ ID NO:15 in the sequence table; Inner positive control sequence Taqman fluorescent probe is shown in SEQ ID NO:16 in the sequence table.
3. test kit according to claim 2, it is characterized in that, 5 ' end of the Taqman fluorescent probe of described PML-RARa L fusion gene Taqman fluorescent probe, PML-RARa V fusion gene Taqman fluorescent probe, PML-RARa S fusion gene Taqman fluorescent probe and abl gene is connected with fluorescence report group FAM, and 3 ' end all is connected with fluorescent quenching group TAMRA; 5 ' end of the Taqman fluorescent probe of the positive control sequence in described inside is connected with fluorescence report group TET, and 3 ' end is connected with fluorescent quenching group TAMRA.
4. test kit according to claim 1 is characterized in that, the nucleotide sequence of described reference gene ABL is shown in SEQ ID NO:17 in the sequence table.
5. 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 PML-RARa L fusion gene, PML-RARa V fusion gene and PML-RARa S fusion gene.
6. 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 the RNase deionized water, cumulative volume 11 μ L.
7. 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+, 0.2-0.4mM dNTPs, 0.3-0.6mM dUTP, 0.2U/ μ L Taq enzyme, 0.01-0.05U/ μ L the UNG enzyme and without the RNase deionized water.
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| CN113652481A (en) * | 2021-07-30 | 2021-11-16 | 广州达安基因股份有限公司 | Primer, probe and kit for quantitatively detecting PML-RARA fusion gene |
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