CN105950775B - A kind of kit detecting NPM1 gene mutation typing - Google Patents

A kind of kit detecting NPM1 gene mutation typing Download PDF

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CN105950775B
CN105950775B CN201610549977.8A CN201610549977A CN105950775B CN 105950775 B CN105950775 B CN 105950775B CN 201610549977 A CN201610549977 A CN 201610549977A CN 105950775 B CN105950775 B CN 105950775B
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CN105950775A (en
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李明
何皖平
刘悦
蒋析文
高秀洁
温楚茵
张文苑
邱秋淳
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Guangzhou Da'an Gene Co ltd
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Daan Gene Co Ltd Zhongshan University
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Abstract

The present invention relates to a kind of kits for detecting NPM1 gene mutation typing, including RT-PCR reaction solution, primed probe mixed liquor, RT-PCR to react enzyme system, DEPC H2O and separation simultaneously concentrate the packing box for packing these reagent bottles or pipe.Since this kit applies one-step method real-time fluorescent PCR reaction pattern, and LNA modification is carried out to specific probe, 12 exons mutation of NPM1 gene in the super quick quick detection patients with acute myeloid leukemia marrow of energy and peripheral blood sample, limit of identification is 0.2ng, simultaneously parting can be carried out to A type, Type B, the mutation of D type according to different channel signal values, internal standard control system is added simultaneously can detect sample extraction effect, can be widely applied to estimating for acute myelogenous leukemia after chemotherapy effect.

Description

A kind of kit detecting NPM1 gene mutation typing
Technical field
The present invention relates to a kind of super quick parting kits for detecting NPM1 gene mutation nucleic acid, more particularly to a kind of utilization It is prominent that multiple real time fluorescence polymerase chain reaction technology and lock nucleic acid modification one tube reaction of probe can detect NPM1 gene simultaneously Become the kit of A type, Type B, D type.
Background technique
Acute myeloid leukemia (Acute myelogenous leukemia, AML) is initiated by hematopoietic stem/progenitor Malignant clone hematologic disease, it is clinical using original and inmature marrow cell paraplasm in marrow and peripheral blood as main feature Using the leukemiacell infiltration of low-heat, metabolic disorder anaemia, bleeding, infection and internal organs as main feature.Epidemiological survey is aobvious Show, AML is the most common adult acute leukemia, and Annual occurence rate is 3/100000 or so.Nucleophosmin (nucleophosmin, NPM1) is one of the major protein molecule positioned at pars granulosa, is positioned at No. 5 chromosome long arm Upper (5q35), is widely expressed in various types of cells.NPM1 high be expressed in be proliferated active cell include tumour cell and Stem cell plays a significant role in neoplastic process.NPM1 mutation, has now been found that about more than 50 variants, most common Type be 12 exons occur four bases repetition cause PROTEIN C end structure change, it is most frequent for be mutated A, account for 77%-80% case is inserted into tetra- nucleotide of TCTG on 956~959 positions of wild type NPM1 nucleotide sequence and repeats structure At.In addition, mutation B, D account for 10%, 5% respectively, it is to be inserted into tetra- nucleotide of CATG, CCTG in identical gene loci respectively.
According to US National comprehensive cancer network (National Comprehensive Cancer Network) 2016 Send out the NCCN clinical practice guideline mended: it is prognosis bona that NPM1 mutation is explicitly shown in acute myeloid leukaemia (2016.V1) Index can not benefit from Allogeneic Hematopoietic Stem Cell Transplantation.Therefore to NPM1 mutation detection be clear AML sufferer whether into The important determinant of row bone-marrow transplantation.
The method of detection NPM1 mutation includes: Sanger PCR sequencing PCR, CAPs method, RT-qPCR etc. at present.Sanger PCR sequencing PCR Also known as generation PCR sequencing PCR, using dideoxy nucleotide, as chain termination reagent, (dideoxy nucleotide is on deoxyribose without poly- 3-OH group required for synthase extended chain, so being used as chain termination reagent) pass through the primer extend generation one of polymerase The method separated again after series molecule of different sizes, is the goldstandard for detection of nucleic acids, but its disadvantage is also more clear: Low, cumbersome, the easy pollution of sensitivity.CAPs is also known as restriction fragment length polymorphism polymerase chain reaction, basic principle It is to use PCR amplification target DNA, amplified production is cut into different size segment with specificity endonuclease digestion again, directly in gel It is differentiated on electrophoresis.The restriction enzyme site of iso-allele is not distributed different, generates the DNA fragmentation band of different length, this Method does not need that cost is relatively low and interpretation is simple, but cumbersome and sensitivity is low.RT-qPCR is also known as one-step method reverse transcription fluorescence Reverse transcription and quantitative fluorescent PCR are reached the technology of step completion, operating process stopped pipe by quantitative PCR method by the adjustment of temperature It carries out, not will cause sample contamination, while detection sensitivity is high, has a wide range of application, but detection site needs clear mutation type.
Summary of the invention
The purpose of the present invention is to provide a kind of using multiple real time fluorescence polymerase chain reaction technology to NPM1 gene Mutation carries out the highly sensitive kit of parting, can accurately detect NPM1 Gene A type, Type B, D type using this kit and be mutated.
According to NPM1 Gene A type, Type B, the D type mutant gene sequence in GenBank, using Primer Express3.0 The primer and probe of software gene mutation area design specificity, and it is preliminary to its specificity progress by the BLAST function of NCBI Identification.Three Genotyping probes with FAM, TEXAS RED and CY5 and joined lock nucleic acid (Locked nucleic respectively Acid, LNA) modification, specificity and sensitivity are increased, different channel fluorescence labels can divide the gene mutation detected Type, internal standard probe are marked with HEX, can be monitored to sample quality and extraction effect.These primed probes are containing heat-resistant dna Polymerase, reverse transcriptase, high quality deoxyribonucleoside triphosphate (dNTPs) RT-PCR reaction enzyme system and contain Mg2+Deng In the RT-PCR reaction solution of composition, the cyclic amplification of beyond body nucleic acid is realized by fluorescent PCR instrument.
Kit according to the present invention specifically includes that 1) RT-PCR reaction solution, primed probe mixed liquor, RT-PCR reaction Enzyme system, DEPC H2O, negative quality-control product, positive quality control product and the packing box for 2) separating and concentrating these reagent bottles of packaging or pipe.
A preferred embodiment of the invention is forward and reverse primer of the primed probe mixed liquor by NPM1 gene specific And the mutation typing probes composition of specificity, typing probes have carried out LNA modification simultaneously and have improved sensitivity and specificity, NPM1 The sequence of the forward and reverse primer of gene specific is 5 '-GGTGGTTCTCTTCCCAAAG-3 ' respectively, 5 '- CTGTTACAGAAATGAAATAAGACG-3'；The sequence for being mutated A type specificity lock nucleic acid probe is 5 '-CAAGATCTCTGT+CT + GGCAGTGG-3 ', the 5 ' of probe and 3 ' end respectively in connection with have fluorescence occur group FAM and fluorescent quenching group BHQ1；It is mutated B The sequence of type specificity lock nucleic acid probe is that the end 5 '-CAAGATCTCTGC+AT+GGCAGTGG-3 ', the 5 ' of probe and 3 ' is tied respectively Conjunction has fluorescence that group TEXASRED and fluorescent quenching group BHQ2 occurs；The sequence for being mutated D type specificity lock nucleic acid probe is 5 '- Group CY5 and fluorescent quenching occur respectively in connection with there is fluorescence for the end CAAGATCTCTGC+CT+GGCAGTGG-3 ', the 5 ' of probe and 3 ' Group BHQ2；The sequence of the forward and reverse primer of internal standard TBP gene specific is 5 '-respectively CTAAAGACCATTGCACTTCGT-3 ', 5 '-ATCAGTGCCGTGGTTCGTG-3 '；The probe sequence of internal standard TBP gene specific Column be 5 '-AATCCCAAGCGGTTTGCTGCGGTAA-3 ', the 5 ' of probe and 3 ' end respectively in connection with have fluorescence occur group HEX and Fluorescent quenching group BHQ1.(wherein "+" indicates to carry out LNA modification to right side base)
Another preferred embodiment of the invention is that primer concentration is 0.1~0.5 μm of ol/L in primed probe mixed liquor, Concentration and probe concentration is 0.1~0.25 μm of ol/L.
Another preferred embodiment of the invention be RT-PCR reaction solution by Tris-HCl (50mmol/L, pH8.0~ 8.8)、MgCl2(3~8mmol/L), KCl (150~350mmol/L) composition.
Another preferred embodiment of the invention be RT-PCR reaction enzyme system by hot start Taq polymerase, reverse transcriptase, DNTPs composition.Commercial product can be used in hot start Taq polymerase, reverse transcriptase, dNTPs, such as the product of Sheng Gong biotech firm, In in every person-portion RT-PCR reaction enzyme system the dosage of hot start Taq polymerase be 5~10U, reverse transcriptase dosage is 1.5~5U, dNTPs Dosage is 6~12mmol.
A preferred embodiment of the invention is that kit provides quality-control product, respectively negative quality-control product and positive matter Control product.Negative quality-control product is sterilizing pure water, and positive quality control product is artificial synthesized containing NPM1 mutation A type, Type B, D type gene RNA is transcribed in vitro.Samples detection to be checked is carried out in kit can carry out the detection of two special quality control product simultaneously, only work as positive quality control product FAM, TEXAS RED, CY5, HEX channel fluorescence signal are positive when detection, and negative quality-control product detects 4 channel fluorescence signals When being negative, the testing result of sample to be checked is just effective.
A preferred embodiment of the invention be operation whole flow process be using nucleic acid extraction kit extract whole blood or RNA in sample of bone marrow；The condition of kit PCR amplification are as follows: 50 DEG C 15 minutes, 94 DEG C 2 minutes；94 DEG C 15 seconds, 58 DEG C 35 seconds, 40 circulations (fluorescence signal is collected when annealing)；The judgement of PCR result is determining by each sense channel Ct value, internal standard channel The testing result of sample of the Ct value less than 35 be considered as effectively, other sense channels Ct value is considered as the Air conduct measurement gene less than 37 Saltant type is positive.
Kit of the invention expands sample to be checked and is automatically performed by commercially available fluorescence quantitative PCR instrument, easy to operate, time-consuming It is few, and reduce the generation of pollution to the maximum extent.In accurate detection patients with acute myeloid leukemia marrow and peripheral blood sample 12 exons mutation of NPM1 gene, while according to different channel signal values can to A type, Type B, D type mutation carry out parting, simultaneously Internal standard control system, which is added, can detect sample extraction effect, can be widely applied to estimating for acute myelogenous leukemia after chemotherapy effect.
Present invention advantage compared with prior art are as follows: 1. NPM1 is mutated and carries out parting detection, can be reflected in sample The specific type of the mutation of NPM1 gene, while detection sensitivity greatly promotes, and can be used for acute myeloid leukemia chemotherapy effect It estimates and minimal residual tracks；2. respectively for NPM1 Gene A type, Type B, D type mutation specific sequence design primer special, spy Needle guarantees the specificity and accuracy of detection, it may have higher flux, the advantages of easy to operate, relative reduction cost；3. a Sample carries out specific detections to three kinds of types of NPM1 gene mutation simultaneously, and detection process only needs 1.5h, compared to one sample into Row 3 times detections, greatly reduce workload, improve detection efficiency；Relative to existing nucleic acid Sanger PCR sequencing PCR, institute is detected It takes time and greatly reduces；4. reagent using yin and yang attribute quality-control product as Quality Control, can the quality to reagent carry out stringent control； 5. reagent is suitble to a variety of fluorescence using the big packaging setting of RT-PCR reaction solution, primed probe mixed liquor, RT-PCR reaction enzyme system Detection device has the characteristics that applicability is more extensive.
Detailed description of the invention
Fig. 1 shows that the amplification curve of RNA is transcribed in vitro in the mutation of NPM1 Gene A type.The amplification curve in the channel FAM is S in figure Type illustrates that kit has good linear dependence, and can detect the concentration samples of 0.2ng.
Fig. 2 shows that the amplification curve of RNA is transcribed in vitro in the mutation of NPM1 gene Type B.The amplification in the channel TEXAS RED is bent in figure Line is S type, illustrates that kit has good linear dependence, and can detect the concentration samples of 0.2ng.
Fig. 3 shows that the amplification curve of RNA is transcribed in vitro in the mutation of NPM1 gene D type.The amplification curve in the channel CY5 is S in figure Type illustrates that kit has good linear dependence, and can detect the concentration samples of 0.2ng.
Fig. 4 shows the amplification curve of NPM1 Gene A type sudden change sample nucleic acid.The amplification curve in the channel FAM and HEX is in figure S type, TEXAS RED and CY5 channel fluorescence signal are negative.
Fig. 5 shows the amplification curve of NPM1 gene Type B sudden change sample nucleic acid.The amplification in the channel TEXAS RED and HEX in figure Curve is S type, FAM and CY5 channel fluorescence signal is negative.
Fig. 6 shows the amplification curve of NPM1 gene D type sudden change sample nucleic acid.The amplification curve in the channel CY5 and HEX is in figure S type, FAM and TEXAS RED channel fluorescence signal are negative.
The amplification curve of Fig. 7 visualizingre agent box feminine gender quality-control product.There is no S type amplification curve in figure, illustrates FAM, TEXAS RED, CY5 and HEX channel fluorescence signal are negative.
The amplification curve of Fig. 8 visualizingre agent box positive quality control product.The expansion of FAM, TEXAS RED, the channel CY5 and HEX in figure Increasing curve is S type, illustrates that the fluorescence signal in the channel is positive.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.
The preparation and its use of the kit of 1 NPM1 gene mutation typing of embodiment
1, preparation includes the kit of following constituent
Primed probe mixed liquor (25 μ l/ pipe) 1 is managed, RT-PCR reaction solution (125 μ l/ pipe), and RT-PCR reacts enzyme system (75 μ L/ pipe) 1 pipe, the pipe of positive quality control product (200 μ l/ pipe) 1, the negative pipe of quality-control product (200 μ l/ pipe) 1, DEPC H2O (2000 μ l/ pipe) 1 Pipe.
2, sample extraction
Extract the in-vitro transcription RNA of NPM1 Gene A type mutation, number 1 (0.2ng), No. 2 (2ng), No. 3 (20ng), 4 Number (200ng), the in-vitro transcription RNA of NPM1 gene Type B mutation, number 5 (0.2ng), No. 6 (2ng), No. 7 (20ng), No. 8 (200ng), the in-vitro transcription RNA of NPM1 gene D type mutation, number 9 (0.2ng), No. 10 (2ng), No. 11 (20ng), No. 12 (200ng), while extracting the clinical sample of 3 Sanger PCR sequencing PCR confirmations, number 13 (mutation of A type), No. 14 (Type Bs Mutation), No. 15 (mutation of D type), by above 15 samples using Da'an Gene Company, Zhongshan University production nucleic acid Extracts kit carries out nucleic acid extraction.
3, real-time fluorescence quantitative PCR amplification and detection
3.1 reagents prepare:
Take in proportion corresponding amount 1 μ l of primed probe mixed liquor, 5 μ l of RT-PCR reaction solution, RT-PCR react 3 μ l of enzyme system, DEPC H211 μ l of O, mixes well rear spare.
3.2 sample-addings:
Into PCR reaction tube, it is separately added into 5 μ l of nucleic acid solution after positive and negative quality-control product, sample extraction, pipe lid is covered tightly, puts Enter instrument sample slot.
3.3 editors' (7500 fluorescence quantitative PCR instrument of ABI Prism)
Setup window is opened, by corresponding sequence setting positive and negative quality-control product and sample to be measured, and is arranged in the column Name Sample ID.All setting sample wells are chosen, are double-clicked, Add Detector is selected, selects Reporter for FAM and Quencher For none, reselection Reporter be HEX and Quencher is none；Reselection Reporter be Texas Red and Quencher is none；Then select Reporter for Cy5 and Quencher be none after close window.In Passive (none) is selected in Reference.Open instrument window be arranged cycling condition: 50 DEG C 15 minutes, 94 DEG C 2 minutes； 94 DEG C 15 seconds, 58 DEG C 35 seconds, 40 circulation.It is all be provided with after save file, run.
3.4 interpretations of result:
Detection data file is saved after reaction.Ampplot window is opened at Results.The purpose of selection analysis Sample position.Baseline numerical value is changed to start:3, stop:10, and opens manual setting Threshold:1.5 ±100000.It double-clicks numerical value on Rn coordinate and opens Graph settings window, Log in Post Run Settings is changed to Analysis preferences window is opened after Linear, OK, selects Analyze to automatically analyze knot under Analysis menu Fruit.The judgement of PCR result determines that the testing result of the sample of the Ct value less than 35 in internal standard channel is considered as by each sense channel Ct value Effectively, it is positive to be considered as the Air conduct measurement genic mutation type less than 37 for other sense channels Ct value.
4, testing result
To 1,2, No. 3 sample of number, there is S type curve in the channel FAM, other channels do not have curve.To 4,5, No. 6 samples of number This, there is S type curve in the channel TEXAS RED, other channels do not have curve.To 7,8, No. 9 samples of number, the channel CY5 has S type bent Line, other channels do not have curve.To No. 10 samples of number, there is S type curve in the channel FAM, HEX, other channels do not have curve.To volume Number No. 11 samples, TEXAS RED, the channel HEX have S type curve, other channels do not have curve.To No. 12 samples of number, CY5, HEX There is S type curve in channel, other channels do not have curve.The present invention can carry out parting to NPM1 gene mutation, have good special Property and higher sensitivity, can detecte the sample down to 0.2ng, it was demonstrated that method of the invention is feasible, accurate, reliable.

Claims (6)

1. a kind of kit for detecting NPM1 gene mutation typing, comprising: 1) RT-PCR reaction solution, primed probe mixed liquor, RT- PCR reacts enzyme system, DEPC H2O, negative quality-control product and positive quality control product and 2) separate and concentrate pack these reagent bottles or The packing box of pipe；Wherein primed probe mixed liquor is mutated A type, primer, probe and the internal standard of Type B, D type gene specific by NPM1 Composition, as follows respectively:
1) sequence of the forward and reverse primer of NPM1 gene specific is respectively: 5 '-GGTGGTTCTCTTCCCAAAG-3 ' and 5'-CTGTTACAGAAATGAAATAAGACG-3'；
2) being mutated the sequence of A type specificity lock nucleic acid probe is 5 '-CAAGATCTCTGT+CT+GGCAGTGG-3 ', the 5 ' of probe With 3 ' end respectively in connection with have fluorescence occur group FAM and fluorescent quenching group BHQ1；
3) being mutated the sequence of Type B specificity lock nucleic acid probe is 5 '-CAAGATCTCTGC+AT+GGCAGTGG-3 ', the 5 ' of probe With 3 ' end respectively in connection with have fluorescence occur group TEXASRED and fluorescent quenching group BHQ2；
4) being mutated the sequence of D type specificity lock nucleic acid probe is 5 '-CAAGATCTCTGC+CT+GGCAGTGG-3 ', the 5 ' of probe With 3 ' end respectively in connection with have fluorescence occur group CY5 and fluorescent quenching group BHQ2；
5) sequence of the forward and reverse primer of internal standard TBP gene specific is 5 '-CTAAAGACCATTGCACTTCGT- respectively 3 ', 5 '-ATCAGTGCCGTGGTTCGTG-3 '；
6) probe sequence of internal standard TBP gene specific is 5 '-AATCCCAAGCGGTTTGCTGCGGTAA-3 ', probe 5 ' and 3 ' end respectively in connection with have fluorescence occur group HEX and fluorescent quenching group BHQ1；
Wherein each sequence kind "+" indicates to carry out LNA modification to right side base.
2. kit according to claim 1, it is further characterized in that in primed probe mixed liquor primer concentration be 0.1~ 0.5 μm of ol/L, concentration and probe concentration are 0.1~0.25 μm of ol/L.
3. kit according to claim 1, it is further characterized in that RT-PCR reaction solution is 8.0~8.8 by pH value 50mmol/L Tris-HCl, 3~8mmol/L MgCl2, 150~350mmol/L KCl composition.
4. kit according to claim 1, wherein RT-PCR react enzyme system by hot start Taq polymerase, reverse transcriptase, DNTPs composition, it is characterised in that the dosage of hot start Taq polymerase is 5~10U in every person-portion RT-PCR reaction enzyme system；Reverse transcriptase Dosage is 1.5~5U；DNTPs is 6~12mmol.
5. kit according to claim 1, it is characterised in that negative quality-control product is sterilizing pure water, and positive quality control product is artificial closes At containing NPM1 mutation A type, Type B, D type gene in-vitro transcription RNA.
6. kit according to claim 1, it is further characterized in that the use of the kit the following steps are included:
1) sample extraction: the RNA in whole blood or sample of bone marrow is extracted using nucleic acid extraction kit；
2) PCR amplification: condition be 50 DEG C 15 minutes, 94 DEG C 2 minutes；94 DEG C 15 seconds, 58 DEG C 35 seconds, 40 circulation；
3) testing result determines: the judgement of PCR result is determining by each sense channel Ct value, the sample of the Ct value less than 35 in internal standard channel This testing result is considered as effectively, and it is positive that other sense channels Ct value is considered as the Air conduct measurement genic mutation type less than 37.
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CN110669840B (en) * 2019-10-30 2021-03-19 宁波胤瑞生物医学仪器有限责任公司 NPM1 mutation detection kit
CN111394516A (en) * 2020-03-19 2020-07-10 申联生物医药（上海）股份有限公司 Internal reference gene for respiratory tract RNA virus PCR detection and detection product thereof
CN113249475B (en) * 2021-04-30 2022-02-11 镇江市第一人民医院 Drop-off ddPCR method and kit for quantitatively detecting NPM1 gene mutation
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