CN112011621B - Primer combination and method for screening high-risk subtype of acute lymphocytic leukemia - Google Patents
Primer combination and method for screening high-risk subtype of acute lymphocytic leukemia Download PDFInfo
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Abstract
The invention discloses a primer combination and a method for screening high-risk subtypes of acute lymphoblastic leukemia, wherein the primer combination comprises 55 pairs of Taqman probes and primers for detecting fusion genes and 125 pairs of primers for detecting 206 mutation sites; the method is used for screening the high-risk subtype of the acute lymphocytic leukemia by adopting a multiplex PCR technology and a high-throughput sequencing technology. The invention aims at common and latest molecular abnormalities of high-risk subtypes of acute lymphocytic leukemia, simultaneously performs fusion gene and gene mutation detection, has the advantages of comprehensive range, high accuracy and strong specificity, requires low amount of RNA and DNA for detection, can meet the requirement by one-time blood collection, has strong clinical operability, can quickly and sensitively detect the relevant molecular abnormalities of the high-risk subtypes of the acute lymphocytic leukemia, and guides clinical screening.
Description
Technical Field
The invention relates to the field of biological pharmacy, in particular to a primer combination and a method for screening high-risk subtypes of acute lymphocytic leukemia.
Background
Acute Lymphoblastic Leukemia (ALL) is a type of hematological malignancy that is highly dangerous to human health, and can be further classified into B-cell Acute Lymphoblastic Leukemia (B-ALL) and T-cell Acute Lymphoblastic Leukemia (T-ALL) according to their specific cell origins, with B-ALL and T-ALL accounting for about 80% and 20% of ALL ALLs. Traditional morphology in combination with flow immunotyping has failed to meet the demand for accurate medical age diagnosis of ALL patients. With the development of high-throughput sequencing technologies, more and more molecular abnormalities associated with ALL patient disease are gradually discovered. Patients with these molecular abnormalities often have difficulty in clinical treatment, easy recurrence and poor prognosis, and belong to the high-risk subtype of acute lymphocytic leukemia. Therefore, the primer combination and the method for screening the high-risk subtype of the acute lymphocytic leukemia have great clinical value and application prospect.
At present, the detection of fusion genes in clinic mainly depends on a real-time fluorescent quantitative PCR technology. Whether the corresponding fusion gene exists in a patient is judged by designing specific primers at two ends of the fusion site for PCR amplification and collecting a fluorescent signal, and the detection result is sensitive and the specificity is high. However, because the high-risk subtypes of acute lymphoblastic leukemia involve many fusion genes, designing primers for all fusion sites respectively to perform single detection would not only greatly increase the workload of the detector and the consumption of detection reagents, but also increase the amount of specimens required for detection, increase the detection period, and fail to meet the requirements of screening the high-risk subtypes in early clinical stage, rapidly and accurately obtaining detection results.
On the other hand, the detection of gene mutation in clinic at present mainly depends on PCR amplification technology combined with sequencing interpretation, the number of mutation sites related to acute lymphoblastic leukemia is larger than that of fusion genes, the detection of each site alone is not in accordance with clinical practice, clinicians often select the mutation site most related to diseases according to experience in work for detection, but the probability of false negative occurrence, particularly the missed diagnosis of the newly discovered mutation site related to diseases is greatly increased. The whole genome sequencing or whole exon sequencing based on the high-throughput sequencing technology can well overcome the occurrence of false negative, however, the characteristics of high cost, high difficulty and long period of the whole genome sequencing and the whole exon sequencing cause that the whole genome sequencing and the whole exon sequencing are not suitable for the early screening of high-risk subtypes of acute lymphocytic leukemia and have limited clinical application.
The multiplex PCR technology can add a plurality of pairs of primers into a PCR reaction system for amplification, and can be used for detecting fusion genes and sequencing after amplification of mutation sites. Theoretically, the addition of the primers for the multiple sites to be detected in a PCR reaction system can greatly reduce the workload and the reagent cost, and simultaneously can meet the requirements of clinical hope of comprehensiveness, accuracy and rapid detection result acquisition, thereby really realizing the accurate and timely screening of high-risk subtypes of each acute lymphocytic leukemia patient. However, adding multiple pairs of primers to a PCR reaction system tends to increase the probability of primer dimer formation, resulting in a decrease in detection efficiency. Therefore, the design and optimization of the multiplex PCR primers are the key and difficult points of the technology, and the more the primers are, the greater the design difficulty is. In view of the above difficulties, the multiplex PCR technology has not been widely applied to screening of high-risk subtypes of acute lymphocytic leukemia.
Disclosure of Invention
The invention aims to provide a primer combination and a method for screening high-risk subtypes of acute lymphocytic leukemia, and aims to solve the problems of difficulty in screening high-risk subtypes of acute lymphocytic leukemia, time consumption, high omission factor and the like.
The purpose of the invention can be realized by the following technical scheme:
a primer combination for screening high-risk subtypes of acute lymphocytic leukemia comprises a primer combination of related fusion genes and a primer combination of related gene mutation, wherein the primer combination of related fusion genes comprises 66 pairs of primers for detecting 55 fusion genes, and totally 12 primer pools, the sequences of the primers are shown as R1-R12, the primer combination of related gene mutation comprises 125 pairs of primers for 206 detection sites, and totally 2 primer pools, namely a first primer pool M1 and a second primer pool M2, and the information of 55 fusion genes is as follows:
| NUP214-ABL1 | ETV6-ABL1 | SNX2-ABL1 | ZMIZ1-ABL1 |
| RCSD1-ABL1 | RANBP2-ABL1 | ETV6-JAK2 | TERF2-JAK2 |
| TPR-JAK2 | PAX5-JAK2 | ATF7IP-JAK2 | BCR-JAK2 |
| SIL-TAL1 | RCSD1-ABL2 | PAG1-ABL2 | ZC3HAV1-ABL2 |
| BCR-ABL1 | AML1-EVI1 | AML1-CLCA2 | AML1-MTG16 |
| TEL-AML1 | MLL-AF4 | PML-RaRa | P2RY8-CRLF2 |
| CBFB-MYH11 | EBF1-PDGFRB | TNIP1-PDGFRB | SSBP2-PDGFRB |
| ZEB2-PDGFRB | E2A-PBX1 | MLL-AF9 | ETV6-NTRK3 |
| NPM1-RaRa | STA5B-RaRa | BCOR-RaRa | PLZF-RaRa |
| AML1-ETO | MYB-TYK | MLL-AF6 | MLL-AF17 |
| MLL-ELL | MLL-ENL | MYH9-ILZRB | NUP98-HOXA9 |
| NUP98-HOXA11 | NUP98-HOXA13 | NUP98-HOXC11 | NUP98-HOXD13 |
| NUP98-OMX1 | NUP98-top1 | SSBP2-CSF1R | MLL-AFX |
| MLL-AF1q | MLL-AF1p | MLL-AF10 |
the sequence information of the 66 pairs of primers is shown in the following table:
the information of 206 mutation sites is shown in the following table:
further, in the 125 pairs of primers in the primer combination of related gene mutation, 65 pairs of primers form a first primer pool M1, the sequence of the first primer pool M1 comprises M1-1 f-M1-65 f and M1-1 r-M1-65r, 60 pairs of primers form a second primer pool M2, the sequence of the second primer pool M2 comprises M2-1 f-M2-60 f and M2-1 r-M2-60r, and the information of the sequence of 125 pairs of primers is as follows:
a test method for screening primer combination of high risk subtype of acute lymphoblastic leukemia, a detection method for 66 pairs of primers in the related fusion gene primer combination and 125 pairs of primers in the related gene mutation primer combination:
s1, separating leukemia cells of acute lymphocytic leukemia patients;
s2, extracting total RNA of the cells and preparing cDNA;
s3, extracting cell genome DNA;
s4, preparing fusion gene detection primers and probes;
and S5, constructing a DNA library and sequencing.
Further, the separation method in step S1:
1.1, placing the blood collection tube in a low-speed centrifuge after balancing, centrifuging at 2500rpm multiplied by 5min, slowly absorbing and removing upper plasma, diluting the remaining blood cells to 5mL by using sterile normal saline, fully blowing, beating and uniformly mixing;
1.2, taking a 15mL centrifuge tube, marking the name of a patient, adding 5mL human lymphocyte separation liquid, and slowly dripping the diluted marrow liquid on the lymphocyte separation liquid along the tube wall to form layering so as to avoid mixing the two;
1.3, adopting a density gradient centrifugation method, placing a centrifuge tube in a low-speed centrifuge at 2000rpm for 20min, after centrifugation, dividing liquid in the tube into three layers, wherein an upper clear layer is diluted plasma and platelets, a middle white membrane layer is mononuclear cells, and a lower red layer is granulocytes and erythrocytes;
1.4, slowly sucking the middle mononuclear cell layer, placing the middle mononuclear cell layer in another clean centrifuge tube for marking the name of a patient, adding 10mL of sterile physiological saline, blowing, uniformly mixing, placing the mixture in a low-speed centrifuge for centrifugation at 2000rpm multiplied by 5min, and discarding the supernatant;
1.5, resuspending the washed cell mass with 1mL of sterile physiological saline, counting the cell mass by using a cell counting plate under a high power microscope, calculating the total cell amount, wherein the cell counting solution is 1% of glacial acetic acid, 20 mu L of cell suspension is added into 380 mu L of 1% glacial acetic acid, uniformly blowing and stirring, sucking 20 mu L of cell suspension and adding the cell suspension into the cell counting plate, counting the number n of cells in 16 lattices under a microscope, and then the total cell amount is n multiplied by 10000 multiplied by 20;
1.6 dilution of the cell suspension to 1X 10 7 1.5mL of EP tubes are filled with cell suspension, each tube is 1mL, after balancing, the mixture is subjected to high-speed instantaneous centrifugation at 8000rpm, supernatant is carefully sucked and discarded without touching cell sediment at the bottom of the tube, one tube is marked with a serial number and a patient name on the tube wall, and the other tube is added with 1mL of Trizol after the serial number and the patient name are marked on the tube wall for uniformly mixing;
further, the method for extracting total RNA of the cells and preparing cDNA in the step S2 comprises the following steps:
2.1, putting 500 mu L Trizol mixed solution into another 1.5mL EP tube without RNase, adding 200 mu L chloroform, violently shaking to mix uniformly, standing at room temperature for 10min, centrifuging at 4 ℃, multiplying by 15min at 12000rpm, dividing the solution after centrifugation into three layers, wherein the upper layer is a clear transparent colorless aqueous phase, the middle white membrane layer is mainly protein and DNA, and the lower layer is an organic phase;
2.2, slowly absorbing 200 mu L of the upper layer solution into a new 0.6ml of EP tube without RNase, slowly moving a suction head downwards along with the liquid level to avoid sucking the intermediate protein and DNA layer, adding equal volume of isopropanol precooled at-20 ℃, covering a tube cover, then gently turning upside down and uniformly mixing, standing at-20 ℃ for 10min to fully precipitate RNA, centrifuging at 4 ℃, and multiplying by 12000rpm for 10min, and discarding the supernatant to obtain a precipitate;
2.3, adding 500 mu L of 75% ethanol precooled at the temperature of 20 ℃, mixing the absolute ethanol with DEPC water according to the ratio of 3, centrifuging at the temperature of 1,4 ℃, slowly discarding supernatant, and carefully absorbing residual ethanol by using a pipette;
2.4, placing on a super clean bench, standing at room temperature until ethanol which is not completely absorbed volatilizes and the white RNA precipitate is semitransparent;
2.5, adding 20 mu L DEPC water to the bottom of the tube, gently blowing and beating, fully dissolving the precipitate, carrying out low-speed instantaneous separation, detecting the RNA concentration and purity by using an ultraviolet spectrophotometer, wherein the RNA quality with the A260/280 ratio of 1.8-2.0 meets the requirement, and then carrying out reverse transcription on the RNA to form cDNA;
2.6, carrying out reverse transcription reaction by adopting a Japanese TaKaRa reverse transcription kit (RR 036A) to prepare a 20 mu L reverse transcription system: 5 × PrimeScript RT Master Mix 4 μ L, total RNA (x) μ L (total amount of RNA 1 μ g, x =1000/RNA concentration), DEPC water make-up to 20 μ L, reverse transcription reaction conditions: 37 ℃ of: 15min, inactivating reaction conditions of reverse transcriptase: 85 ℃:5s, and storing at 4 ℃, and performing reverse transcription on the obtained cDNA to detect the multiple PCR fusion gene.
Further, the extraction and purification of the DNA in the step S3 is performed by using a reagent purchased from Qiagen corporation of QIAamp DNA Blood Mini Kit (QIAamp DNA Blood Mini Kit) of Germany, and the method is a silicon membrane adsorption method, and comprises the following steps:
3.1 separating the resulting 1X 10 7 Dissolving leukemia cells of an acute lymphoblastic leukemia patient in 200 mu L of PBS, and adding 20 mu L of proteinase K into the bottom of the tube;
3.2, slowly adding 200 mu L of oily lysate AL, shaking, uniformly mixing, and then quickly separating at a low speed;
3.3, carrying out water bath at 56 ℃ for 20min, after the oily mixed liquid becomes clear, wiping off the liquid on the outer wall, centrifuging the liquid adhered to the inner wall and the tube cover to the bottom of the tube at a low speed;
3.4, adding 200 mu L of absolute ethyl alcohol, reversing, uniformly mixing, and instantly separating at a low speed;
3.5, carefully transferring the obtained mixed solution to a QIAamp Mini adsorption column, placing the adsorption column on a 2mL collection tube provided by the kit, covering a tube cover, marking, centrifuging at high speed of 12000rpm for 3min, and discarding the collection tube and the filtrate;
3.6, putting the adsorption column into a new collection tube, adding 500 mu L of buffer AW1 into the adsorption column, covering a tube cover, centrifuging at 12000rpm multiplied by 3min, and discarding the collection tube and filtrate;
3.7, putting the adsorption column into a new collecting pipe, adding 500 mu L of buffer AW2, covering a pipe cover, centrifuging at 12000rpm multiplied by 3min, and discarding the collecting pipe and filtrate;
3.8, placing the adsorption column in a self-prepared clean sterile 1.5mL EP tube, opening the tube cover of the adsorption column, centrifuging at 12000rpm for 3min to fully volatilize the ethanol, and discarding the EP tube and the filtrate;
3.9, placing the adsorption column in a self-prepared clean sterile 1.5mL EP tube, suspending and dropwise adding 100 mu L of an elution buffer solution AE, carefully dropwise adding the AE in the middle of an adsorption film of the adsorption column, incubating at room temperature for 3min to fully elute DNA in the adsorption film, and centrifuging at 12000rpm for 3min, wherein the filtrate in the EP tube is a genome DNA solution;
3.10, in order to improve the collection efficiency of the DNA, repeating the step 3.9, adding the DNA solution collected in the EP tube into the middle part of the adsorption membrane of the adsorption column again, incubating at room temperature for 3min to fully dissolve the DNA in the adsorption membrane, centrifuging at 12000rpm multiplied by 3min, and collecting the DNA solution again;
3.11, detecting the concentration and the purity of the genome DNA by using an ultraviolet spectrophotometer, wherein the DNA quality with the A260/280 ratio of 1.8-2.0 meets the requirement, and the DNA library is used for constructing a DNA library and carrying out high-throughput sequencing.
Further, in the step S5, the DNA library is constructed and sequenced, and the steps are as follows:
p1, PCR amplification of a detection site; the reagent is Multiplex PCR Assay Kit of TaKaRa company, and the PCR amplification reaction system is as follows:
| reagent | Volume of |
| 2×Multiplexbuffer | 10μL |
| Taq | 0.1μL |
| DNA template | 1μL |
| Primer(M1，M2) | 0.5μL |
| Deionized water | 8.5μL |
| TotalVolume | 20μL |
Mixing, placing on a PCR instrument, and operating:
p2, ultrasonically breaking DNA; the Covaris S220 instrument is selected for ultrasonic DNA fragmentation, the water bath temperature is set to be 4 ℃, and the SonoLab is started TM Software and setting a bubble removing program in water bath for 30min, diluting a PCR product obtained by amplification to 50 mu L by using enzyme-free deionized water and meeting the requirement that the final concentration is 30 ng/mu L, and breaking a system by 50 mu LTransferring the mixture into a special reaction tube of Covaris S220, setting interruption parameter Intensity as 175, duty cycle as 10%, cycles per Burst as 200 and time as 430S;
p3, DNA end repair;
p4, adding dA tail;
p5, connecting a joint;
p6, PCR amplification;
and P7, quantifying the amount of the Qubit and sequencing.
Further, the operation method of DNA end repair in step P3 is as follows:
4.1, taking 9.5 mu L of the solution after DNA ultrasonic disruption, adding a reagent:
| reagent | Volume of |
| 10×EndRepairbuffer | 1.25μL |
| dNTPmix(1mM) | 1.25μL |
| BluntEnzyme | 0.5μL |
| TotalVolume | 12.5μL |
4.2, mixing uniformly, placing on a PCR instrument, and operating: 30min at 25 ℃;
4.3, purifying the reaction solution with the repaired DNA tail end to 20.5 mu L by adopting 1.8X Agencour AMPure XP Beads, fully and uniformly mixing magnetic Beads, sucking 22.5 mu L of magnetic Beads into the reaction solution, gently blowing and sucking for 10 times, uniformly mixing, and standing for 5min at room temperature;
4.4, placing the EP tube on a magnetic frame for 5min until the liquid is clear, carefully absorbing the supernatant, and avoiding magnetic beads absorbed on the tube wall;
4.5, keeping the EP tube on a magnetic frame, adding 200 mu L of 80% ethanol along the tube wall, standing at room temperature for 30 seconds, discarding the supernatant, and cutting the magnetic beads not to be absorbed on the tube wall;
4.6, repeating the 80% ethanol washing once;
4.7, keeping the EP tube on a magnetic rack for drying for 2-5min, observing that the surface of the magnetic bead does not reflect light any more, and avoiding excessive drying;
4.8, taking the EP tube off the magnetic frame, adding 22 mu L DEPC water, gently blowing and beating the mixture evenly, standing the mixture at room temperature for 2min, and then placing the EP tube on the magnetic frame until the liquid is clear;
4.9 carefully pipette 20.5. Mu.L of supernatant into a new PCR tube, and do not pipette down the beads on the tube wall.
The operation method for adding dA tail in the step P4 comprises the following steps:
5.1, adding a reagent into 20.5 mu L of reaction liquid obtained after DNA end repair purification in the step P3:
| reagent | Volume of |
| NEbuffer2 | 2.5μL |
| dATP(10mM) | 0.5μL |
| KlenowExo- | 1.5μL |
| TotalVolume | 25μL |
5.2, uniformly mixing, placing on a PCR instrument, and operating: 30min at 37 ℃;
5.3, purifying the reaction solution added with the dA tail to 10 mu L by adopting 1.8 XAgencour AMPure XP Beads, fully and uniformly mixing magnetic Beads, sucking 45 mu L (1.8X) of magnetic Beads into the reaction solution, gently blowing and sucking for 10 times, uniformly mixing, and standing for 5min at room temperature;
5.4, placing the EP tube on a magnetic frame for 5min until the liquid is clear, carefully absorbing the supernatant, and avoiding magnetic beads absorbed on the tube wall;
5.5, keeping the EP tube on a magnetic frame, adding 200 mu L of 80% ethanol along the tube wall, standing at room temperature for 30 seconds, removing supernatant, and cutting the magnetic beads which are not absorbed on the tube wall;
5.6, repeating the washing once by 80 percent ethanol;
5.7, keeping the EP tube on a magnetic rack for drying for 2-5min, observing that the surface of the magnetic bead does not reflect light any more, and avoiding excessive drying;
5.8, taking the EP tube off the magnetic frame, adding 11 mu L DEPC water, gently blowing and beating the mixture evenly, standing the mixture at room temperature for 2min, and then placing the EP tube on the magnetic frame until the liquid is clear;
5.9 carefully pipette 10. Mu.L of the supernatant into a new PCR tube, and do not pipette down the beads on the tube wall.
Further, the operation method of the joint connection in the step P5 is as follows:
6.1, taking 10 mu L of reaction liquid obtained after dA tail addition purification in the step P4, adding reagents:
| reagent | Volume of |
| 2×QuickLigationbuffer | 10μL |
| Adaptor(10μM) | 0.2μL |
| QuickLigation | 1μL |
| TotalVolume | 21.2μL |
The joint was synthesized by Shanghai Producer, inc., with the specific sequence:
6.2, mixing uniformly, placing on a PCR instrument, and operating: 15min at 25 ℃;
6.3, purifying the reaction liquid after the joint connection to 23 mu L by adopting Agencour AMPure XP Beads, adding 28.8 mu L of non-enzyme water to the reaction liquid after the joint connection to ensure that the total volume is 50 mu L, fully and uniformly mixing magnetic Beads, sucking 30 mu L (0.6 x) of magnetic Beads into the reaction liquid, gently blowing and sucking for 10 times, uniformly mixing, and standing at room temperature for 5min;
6.4, the EP tube is placed on a magnetic frame for 5min until the liquid is clear, the supernatant is carefully sucked to a new tube, and the magnetic beads on the tube wall are not sucked;
6.5, adding 7.5 mu L (0.15 x) of magnetic beads into the reaction solution, gently blowing, stirring and mixing uniformly, and standing at room temperature for 5min;
6.6, placing the EP tube on a magnetic frame for 5min until the liquid is clear, carefully absorbing the supernatant, and avoiding absorbing the magnetic beads on the tube wall;
6.7, keeping the EP tube on a magnetic frame, adding 200 mu L of 80% ethanol along the tube wall, standing at room temperature for 30 seconds, removing supernatant, and cutting magnetic beads which are not absorbed on the tube wall;
6.8, repeating the 80% ethanol washing once;
6.9, keeping the EP tube on a magnetic rack for drying for 2-5min, observing that the surface of the magnetic bead does not reflect light any more, and avoiding excessive drying;
6.10, taking the EP tube off the magnetic frame, adding 24 mu L DEPC water, gently blowing and beating the mixture evenly, standing the mixture at room temperature for 2min, and then placing the EP tube on the magnetic frame until the liquid is clear;
6.11 carefully pipette 23. Mu.L of supernatant into a new PCR tube, and do not pipette into the magnetic beads on the tube wall;
further, the operation method of PCR amplification in step P6:
7.1, taking 23 mu L of the reaction liquid obtained after the joint connection purification in the step P5, adding a reagent:
| reagent | Volume of |
| 2×PCRbuffer | 25μL |
| LP1(10μM) | 1μL |
| LP2(10μM) | 1μL |
| TotalVolume | 50μL |
7.2, mixing uniformly, and placing on a PCR instrument for operation;
7.3, purifying the reaction solution after PCR amplification to 20 mu L by adopting 0.9 XAgencourt AMPure XP Beads, fully and uniformly mixing magnetic Beads, sucking 45 mu L (0.9X) of magnetic Beads into the reaction solution, gently and uniformly sucking for 10 times, and standing for 5min at room temperature;
7.4, placing the EP tube on a magnetic frame for 5min until the liquid is clear, carefully absorbing the supernatant, and avoiding magnetic beads absorbed on the tube wall;
7.5, keeping the EP tube on a magnetic frame, adding 200 mu L of 80% ethanol along the tube wall, standing at room temperature for 30 seconds, removing supernatant, and cutting the magnetic beads which are not absorbed on the tube wall;
7.6, repeating the 80% ethanol washing once;
7.7, keeping the EP tube on a magnetic frame for drying for 2-5min, observing that the surface of the magnetic bead does not reflect light any more, and keeping the EP tube from being dried excessively;
7.8, taking the EP tube off the magnetic frame, adding 21 mu L DEPC water, gently blowing and beating the mixture evenly, standing the mixture at room temperature for 2min, and then placing the EP tube on the magnetic frame until the liquid is clear;
7.9 carefully pipette 20. Mu.L of the supernatant into a new PCR tube, and do not pipette down the beads on the tube wall.
The invention has the beneficial effects that:
1. the primer combination is comprehensive and accurate, the fusion gene and the gene mutation existing in the acute lymphocytic leukemia patient can be rapidly detected, the problems of complexity, time consumption, non-standardization and the like of the traditional high-risk subtype screening method for the acute lymphocytic leukemia are effectively solved, and the reagent cost and the time cost are reduced;
2. the detection method of the primer combination can complete the detection of the fusion gene and gene mutation related to the high-risk subtype of the acute lymphoblastic leukemia through clinical blood collection for one time, quickly and accurately obtain molecular detection information required by screening of the high-risk subtype of the clinical acute lymphoblastic leukemia, has great significance for guiding the screening of the high-risk subtype of the acute lymphoblastic leukemia and guiding clinical treatment, and has high clinical practical value.
Drawings
The invention will be further described with reference to the accompanying drawings.
FIG. 1 is a schematic diagram showing the results of multiplex PCR amplification according to the present invention;
FIG. 2 is a schematic representation of the results of electrophoresis according to the present invention;
FIG. 3 is a schematic representation of the results of electrophoresis according to the present invention;
FIG. 4 is a schematic diagram showing the results of multiplex PCR amplification according to the present invention;
FIG. 5 is a schematic diagram showing the results of multiplex PCR amplification according to the present invention;
FIG. 6 is a schematic diagram showing the result of electrophoresis in accordance with the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A primer combination and a method for screening high-risk subtypes of acute lymphoblastic leukemia verify the specificity and sensitivity of the primer combination, the patient is a relapse-refractory Ph chromosome negative B-ALL patient, the MLL-AF4 rearrangement of the patient is found through FISH detection, and the CRLF2_ p.A11A mutation of the patient is found through first-generation sequencing detection.
The detection method comprises the following steps:
1. separating by density gradient centrifugation to obtain patient leukemia cells, and separating human peripheral blood lymphocyte separation solution from Tianjin sea company;
2. extracting nucleic acid: the method comprises the following steps of (1) extracting RNA by a chloroform extraction method, carrying out reverse transcription on the RNA with qualified concentration and purity to obtain cDNA, selecting a PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) Kit from TaKaRa company, and selecting a QIAamp DNA Blood Mini Kit from Qiagen company for extracting the DNA;
3. detection of fusion gene: as shown in FIG. 1, the internal reference GAPDH peaks at 17 cycles, which indicates that the extraction and reverse transcription of the RNA of the sample are successful, the sample is not degraded, and the MLL-AF4 peaks at 26 cycles, which indicates that the MLL-AF4 fusion gene exists in the sample, and is consistent with the FISH detection result of the patient at the early stage.
4. Detection of gene mutation: wherein, after PCR amplification is carried out by using two primer pools of M1 and M2, 2 percent agarose gel electrophoresis is carried out, a DNA indicator is selected as DL1000 of TaKaRa company, the electrophoresis result is shown in figure 2, and the 2 nd Lane and the 3 rd Lane are observed to find that products amplified by the primer pools of M1 and M2 are mainly concentrated between 300 and 400bp and are consistent with the theoretical amplification length of the primer design;
performing 2% agarose gel electrophoresis for one time after the PCR amplification of the added joint, wherein the electrophoresis result is shown in figure 3, comparing the lane 2 (before the amplification without the added joint) with the lane 3 (after the amplification with the added joint) to find that the amplification product after the added joint is about 100bp longer than the amplification product before the amplification without the added joint, which indicates that the joint connection is successful, sending the amplification product to a sequencing company for high-throughput sequencing after the library is built, and reading sequencing data to find that the patient has NF1_ p.R1241 mutation besides the CRLF2_ p.A11A mutation found by the first-generation sequencing;
5. the primer combination and the detection method successfully detect MLL-AF4 rearrangement and CRLF2_ p.A11A mutation which are proved to exist in a patient by a clinical classical detection method (FISH and first-generation sequencing), and discover that the patient has unknown NF1_ p.R1241 mutation on the basis, so that the operation feasibility is high, and the result reliability is high.
Example 2
A primer combination and a method for screening high-risk subtypes of acute lymphocytic leukemia verify the specificity and sensitivity of the primer combination, the patient is a T-ALL patient with relapse difficulty, SIL-TAL1 fusion genes exist in the patient through q-PCR detection, and abnormality does not exist in the patient through first-generation sequencing detection.
The specific detection method comprises the following steps:
1. separating by density gradient centrifugation to obtain patient leukemia cells, and separating human peripheral blood lymphocyte separation solution from Tianjin sea company;
2. extracting nucleic acid: the extraction of RNA adopts a chloroform extraction method, the concentration and the purity of the obtained RNA are qualified, then reverse transcription can be carried out to obtain cDNA, the PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) Kit of TaKaRa company is suggested to be used, and the QIAamp DNA Blood Mini Kit of Qiagen company is suggested to be used for extracting DNA;
3. detection of fusion gene: the result of multiplex PCR amplification, as shown in FIG. 4, the internal reference GAPDH starts a peak at 16 cycles, which indicates that the RNA extraction and reverse transcription of the sample are successful, the sample is not degraded, and SIL-TAL1 starts a peak at 26 cycles, which indicates that SIL-TAL1 fusion gene exists in the sample, and is consistent with the early q-PCR detection result of the patient;
4. detection of gene mutation: performing PCR amplification by using two primer pools of M1 and M2, performing 2% agarose gel electrophoresis, selecting DL1000 of TaKaRa company as a DNA indicator, and observing an electrophoresis result shown in figure 2 of the specification, wherein a 4 th lane and a 5 th lane show that products amplified by the primer pools of M1 and M2 are mainly concentrated between 300 and 400bp and are consistent with a theoretical amplification length designed by the primers, performing 2% agarose gel electrophoresis after joint PCR amplification, and an electrophoresis result shown in figure 3 of the specification, comparing the amplification product after joint addition (before no joint amplification) with the amplification product after the joint addition (after joint amplification) of the 4 th lane and the 5 th lane (after joint amplification) to show that the amplification product after joint addition is about 100bp longer than that before joint amplification, prompting that the joint connection is successful, sending the obtained result to a sequencing company for high-throughput sequencing after the steps are completed, and sequencing data show that the patient has LEF1_ p.106PL and CH1_ p.RnT1598P mutations;
5. the primer combination and the detection method successfully detect the SIL-TAL1 fusion gene which is proved to exist in a patient by a clinical classical method (q-PCR), and discover that the patient has the unknown LEF1_ p.P106L and NOTCH1_ p.R1598P mutation, the two mutation sites are both related to poor disease prognosis of the T-ALL patient, and the clinical treatment of the patient returns and verifies the detection result.
Example 3
A primer combination and a method for screening high-risk subtypes of acute lymphocytic leukemia are provided, and the effect of the primer combination in the screening of the high-risk subtypes of the acute lymphocytic leukemia is detected.
The patient was a relapsed refractory Ph chromosome negative B-ALL patient who relapsed only 4 months after induction of remission and died 1 month after relapse, and for economic reasons the patient was not tested molecularly during clinical treatment.
The specific detection method comprises the following steps:
1. separating leukemia cells of patient by density gradient centrifugation, and recommending to use separation solution of peripheral blood lymphocytes from Tianjin junction company;
2. extracting nucleic acid, wherein the extraction of RNA adopts a chloroform extraction method, the concentration and purity of the obtained RNA can be subjected to reverse transcription to obtain cDNA, the PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) Kit of TaKaRa company is recommended to be used, and the QIAamp DNA Blood Mini Kit of Qiagen company is recommended to be used for extracting DNA;
3. detection of fusion gene: the result of the multiplex PCR amplification is shown in FIG. 5, the internal reference GAPDH starts to peak at 18 cycles, which indicates that the RNA extraction and reverse transcription of the sample are successful, the sample is not degraded, and the E2A-PBX1 starts to peak at 22 cycles, which indicates that the E2A-PBX1 fusion gene exists in the sample;
4. detection of gene mutation: performing 2% agarose gel electrophoresis after the joint PCR amplification, wherein the electrophoresis result is shown in the figure 6 of the specification, comparing the lane 2 (before the joint amplification) with the lane 3 (after the joint amplification), and finding that the amplification product after the joint is added is about 100bp longer than the amplification product before the joint amplification, so as to prompt the successful connection of the joint, sending the joint to a sequencing company for high-throughput sequencing after the steps are completed, and reading sequencing data to find that the CRLF2_ p.R186S mutation exists in the patient;
5. the primer combination and the method successfully detect that the E2A-PBX1 fusion gene and the CRLF2_ p.R186S mutation exist in the patient, and previous researches show that the prognosis of the patient with the E2A-PBX1 fusion gene or the CRLF2_ p.R186S mutation is poor, and the clinical treatment of the patient returns to verify the detection result.
The results of the embodiment 1, the embodiment 2 and the embodiment 3 show that the primer combination and the detection method provided by the invention are comprehensive, accurate and rapid in detecting whether the acute lymphoblastic leukemia patient has the fusion gene and gene mutation, so that the reagent cost and the time cost are reduced, and the primer combination and the detection method have great significance for clinically screening the high-risk subtype of acute lymphoblastic leukemia and guiding clinical treatment and high clinical practical value.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed.
Sequence listing
<110> university of southeast
<120> primer combination and method for screening high-risk subtype of acute lymphocytic leukemia
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aaagttgaag aaacccccta caga 24
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cttggttccc gatatggatg a 21
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cccttcagcg gccagtagca tctga 25
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gcttcactct gaccatcact gtct 24
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tagctttcca tgtggcaggt att 23
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aggctgcaca tctggttctg t 21
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cccagggcac ctatcctcat a 21
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ctctgtctcc ccgcctgaa 19
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cgcctcagcc acctactaca g 21
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cgccaagaaa agaagttccc aaaaccact 29
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aggagaatgc aggcactttg a 21
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catcctcagc actctctcca atggcaata 29
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gcggccatga atgggtc 17
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tggcgatctg gttctctttc a 21
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ggtgccttcc caggtgatg 19
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ccagcctcat gcacaacca 19
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ccctccctga cctgtctcgg cc 22
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tcacgatctg ctgcagaatg t 21
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agccctgagt acaagctgag caagctcc 28
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cccactcctc cacctttgac 20
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actgctccac ctctgattcc a 21
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ggaatacttt gagaggttgg agaa 24
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cacccaaccc agtggttaaa g 21
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agacccagag cagcagttct gaagaga 27
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cgcctcagcc acctactaca g 21
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<400> 89
cgccaagaaa agaagttccc aaaaccact 29
<210> 90
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<400> 90
aggagaatgc aggcactttg a 21
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<400> 91
catcctcagc actctctcca atggcaata 29
<210> 92
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tcatcactcc atggaactcc aa 22
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gaaatcggat agatggcctc agt 23
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ggagttggac gggcttgac 19
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gtagcaaggt tccacagaaa aaagt 25
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atactgagca agtctcttcc aaagg 25
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agatgagtcc attcttgcac accgagaa 28
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cctgggactc ttggaactgg 20
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gcccctggat ttaatactac gacagcca 28
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ccttcgctgg gttgtttttc 20
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tgaagaagaa ctcccgttcc a 21
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ataaggcacg cgcttctttc 20
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gcagaggaaa cctcggattc t 21
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ctggaaactc tatacacaca cagccggagg 30
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<212> DNA
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cgcctcagcc acctactaca g 21
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<400> 113
cgccaagaaa agaagttccc aaaaccact 29
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aggagaatgc aggcactttg a 21
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catcctcagc actctctcca atggcaata 29
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cccttccagt attgcctgta tca 23
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cttgtaggtg gctgtatctg acaga 25
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aactgctgtt gcctggttga t 21
<210> 120
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 120
ttccactaga ggtgtgtgca gag 23
<210> 121
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 121
ggcaaactga gcgcatgtta c 21
<210> 122
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 122
aaaatggatc cagacaactg ttcaaac 27
<210> 123
<211> 29
<212> DNA
<213> (Artificial sequence)
<400> 123
ctactttagc ttttaatcct ccacatgga 29
<210> 124
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 124
caccatcaga agagtgggca tt 22
<210> 125
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 125
gtgacaaggc tatgggacac t 21
<210> 126
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 126
<210> 127
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 127
ccatcaggcc tctgtgagtc ta 22
<210> 128
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 128
ctctgaatgc ctttccttct ggt 23
<210> 129
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 129
ggttgggttt gggttcctga tc 22
<210> 130
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 130
tctctttgca gcagaagacc c 21
<210> 131
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 131
cccattccac agctcagtaa cg 22
<210> 132
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 132
ggatatgcat ctctacttac tggcatag 28
<210> 133
<211> 26
<212> DNA
<213> (Artificial sequence)
<400> 133
cctttctctc tttctttctg cctcat 26
<210> 134
<211> 29
<212> DNA
<213> (Artificial sequence)
<400> 134
caattttaag acaaaacgct atggctttc 29
<210> 135
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 135
cccaaagtgt caggttgcaa aa 22
<210> 136
<211> 25
<212> DNA
<213> (Artificial sequence)
<400> 136
gagtgctcag tgtctaattc cactt 25
<210> 137
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 137
cattcaagcg agcctggttt aa 22
<210> 138
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 138
cgaaatgatg aagtcccacg tgat 24
<210> 139
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 139
gccagtaatg ttaaagtaga gactcagagt 30
<210> 140
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 140
gctcttcctg gatcacgtca tg 22
<210> 141
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 141
ctttctcgta gcatcgtcct cat 23
<210> 142
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 142
aggtatatgc atcccagcag agaa 24
<210> 143
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 143
ctgcctacag ggtctctcaa aa 22
<210> 144
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 144
tccctccata aagctgtcaa atatgtc 27
<210> 145
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 145
cgtggaggta aagtgcctga at 22
<210> 146
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 146
cccgtgaaga gattcaaacc ca 22
<210> 147
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 147
tccaaatgtt ctaaaccttc tctaaccttg 30
<210> 148
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 148
gctcagtccc tcaatctcct caa 23
<210> 149
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 149
aattgtttag actcctactc ttgctgt 27
<210> 150
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 150
tttttctcaa tgcatgcctc caa 23
<210> 151
<211> 29
<212> DNA
<213> (Artificial sequence)
<400> 151
ttttctcatc agtttatttt ggtttgcct 29
<210> 152
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 152
aggcctgatt attcaaatga tttgaacttt 30
<210> 153
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 153
gctgtcatat agcggctcag aa 22
<210> 154
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 154
ccttttcttc cctaacccac ctt 23
<210> 155
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 155
gcaatgccct ctcaagagac aa 22
<210> 156
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 156
catgtactgg tccctcattg ca 22
<210> 157
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 157
ttgcacttct gaacataatt tgcaaca 27
<210> 158
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 158
aaatttttgg tgcatgttgc caaattac 28
<210> 159
<211> 29
<212> DNA
<213> (Artificial sequence)
<400> 159
cagtcatcat ttgccttaat ttagcaagt 29
<210> 160
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 160
tgaagcaagg agcattaata caatgtatct 30
<210> 161
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 161
gcttgaagac cacgttggtg t 21
<210> 162
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 162
<210> 163
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 163
gtccacaggc gaggagtagc t 21
<210> 164
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 164
<210> 165
<211> 26
<212> DNA
<213> (Artificial sequence)
<400> 165
agctctatct tccctagtgt ggtaac 26
<210> 166
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 166
tccatcttga ggacagctct ga 22
<210> 167
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 167
<210> 168
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 168
cccaggcctc ataaccttgt taa 23
<210> 169
<211> 26
<212> DNA
<213> (Artificial sequence)
<400> 169
tgaaacaaaa tgccaccatg attctc 26
<210> 170
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 170
gggcttatca aagtatggtt taagttgc 28
<210> 171
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 171
atttatctga aacattgggt ggctttattg 30
<210> 172
<211> 29
<212> DNA
<213> (Artificial sequence)
<400> 172
aattaacatt gtcgcccttc ttattctct 29
<210> 173
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 173
aaagctcaaa tacgagagaa accttca 27
<210> 174
<211> 29
<212> DNA
<213> (Artificial sequence)
<400> 174
ttgaaatttt cattgcttaa agcaggcta 29
<210> 175
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 175
tttcagttga tttgcttgag atcaagattg 30
<210> 176
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 176
tcgttccttg ggtttctttc aaca 24
<210> 177
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 177
ttcaacactg tagccattgc aac 23
<210> 178
<211> 29
<212> DNA
<213> (Artificial sequence)
<400> 178
atggaagtgt ttccacattt ttatgaaca 29
<210> 179
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 179
cagttccagg aggtctacct ga 22
<210> 180
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 180
ttctctggtg gcagtagtat gact 24
<210> 181
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 181
<210> 182
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 182
gcagcacaga ttcccttaac ca 22
<210> 183
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 183
ccaccagctt ctctaggaag ga 22
<210> 184
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 184
catgcttctt gggatctcag tgt 23
<210> 185
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 185
caactttggg acaggagtca ga 22
<210> 186
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 186
gtcaaggatg aacacagaaa ccca 24
<210> 187
<211> 26
<212> DNA
<213> (Artificial sequence)
<400> 187
tcacctcatc ctaacacatt tcaagc 26
<210> 188
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 188
tgtaagagtc ttcccgtgag gtt 23
<210> 189
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 189
aaatgaggga gactggttag ggat 24
<210> 190
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 190
tacatcacta atcattgtgt gtgtgtgt 28
<210> 191
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 191
cttgggctgt gtcctgtttc 20
<210> 192
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 192
tccttgtacc aggacagtga ct 22
<210> 193
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 193
gtgacttgga accaaggatg ct 22
<210> 194
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 194
taacatggcc tgaaaatgac ctgt 24
<210> 195
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 195
attcaggatg gtgtcaaggg ttc 23
<210> 196
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 196
cctcatggtt cgtgttgatg tagg 24
<210> 197
<211> 26
<212> DNA
<213> (Artificial sequence)
<400> 197
ttcccatttt aatcacagag ctagca 26
<210> 198
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 198
tcaaactggc tcatgctagc at 22
<210> 199
<211> 29
<212> DNA
<213> (Artificial sequence)
<400> 199
atcatcttaa gtgtttttcc agtgtctga 29
<210> 200
<211> 29
<212> DNA
<213> (Artificial sequence)
<400> 200
ggagcatatg attttatggt aaaggtgtg 29
<210> 201
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 201
cagattgact ctgagctgag aaaaatttta 30
<210> 202
<211> 29
<212> DNA
<213> (Artificial sequence)
<400> 202
actctgtttt attaaaacct agggcaact 29
<210> 203
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 203
gtggttggtc aggtagatga gac 23
<210> 204
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 204
ctaccagtca attagaaaca tgctcaga 28
<210> 205
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 205
gccctgcctc taaaaataaa aatcttttca 30
<210> 206
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 206
ttagccagca aggacacaat cc 22
<210> 207
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 207
ccagttgagg gaacacaatg ga 22
<210> 208
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 208
gtgggatctc atattctgga tcctatg 27
<210> 209
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 209
tgggcactaa attcgtgaaa tgc 23
<210> 210
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 210
cactgcagga aagtataaat ttcaagcttt 30
<210> 211
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 211
agtgacagta attgaatccc taggagat 28
<210> 212
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 212
tacaatctaa agtgacttta gcaatgtgct 30
<210> 213
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 213
aaggccagag gattgatgtt cag 23
<210> 214
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 214
aggttgtact acttactaga aaatgcatgg 30
<210> 215
<211> 29
<212> DNA
<213> (Artificial sequence)
<400> 215
aattcaagga aaattaacaa catgccctt 29
<210> 216
<211> 29
<212> DNA
<213> (Artificial sequence)
<400> 216
atgtgtactg cagaagtttt atgtgatct 29
<210> 217
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 217
gccacaccaa ccttcttttt aaaattagat 30
<210> 218
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 218
gtagtgctgt gtgcactaat gg 22
<210> 219
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 219
gggccactct tctttgcaga a 21
<210> 220
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 220
gtggacaggt tttgaaagat atttgtgtta 30
<210> 221
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 221
caggaacaat gtcttttcaa gtcctttg 28
<210> 222
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 222
tggtttgctg ctaagctatt taagagaata 30
<210> 223
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 223
tcaattcctg ttaagtcaac tgggaaa 27
<210> 224
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 224
cacacacata cacacaaaat gaatgataca 30
<210> 225
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 225
atctccaaca aagcttctgt gact 24
<210> 226
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 226
ccagtactgc aaggaccact t 21
<210> 227
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 227
tatctgggat gagccgtgat ca 22
<210> 228
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 228
ttacagatgc agcagcagaa cct 23
<210> 229
<211> 18
<212> DNA
<213> (Artificial sequence)
<400> 229
<210> 230
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 230
gttagatgct tatttaacct tggcaatagc 30
<210> 231
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 231
tctggatgct tgtctatttc taaaagcc 28
<210> 232
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 232
taagcgaaag cccttcctgt c 21
<210> 233
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 233
gcacaggcat ggactaactc ag 22
<210> 234
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 234
cggtgcttct cctatgtgac tg 22
<210> 235
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 235
ctaaacacca tttaagaaca gagactgagt 30
<210> 236
<211> 26
<212> DNA
<213> (Artificial sequence)
<400> 236
catggtgcaa caaaagtaag aatcca 26
<210> 237
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 237
tggcatagca ttagtgataa accatttctt 30
<210> 238
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 238
gctaaatgat gtaccttcgg agttatgt 28
<210> 239
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 239
cctcgtttaa tctcctgaag tacagtttta 30
<210> 240
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 240
ttctcccaat gaaagtaaag tacaaacctt 30
<210> 241
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 241
atttctgaca ctcagggcac aa 22
<210> 242
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 242
cctgctcaaa aggagagcgt at 22
<210> 243
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 243
cacaggcagc agggatatag tatc 24
<210> 244
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 244
cttctaggca gcaagaagag ct 22
<210> 245
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 245
aatagcagac ttgttggaat ctcttcaa 28
<210> 246
<211> 19
<212> DNA
<213> (Artificial sequence)
<400> 246
cgggcagggc ttacttacc 19
<210> 247
<211> 26
<212> DNA
<213> (Artificial sequence)
<400> 247
gaaagttgaa agtgagcacg tattcc 26
<210> 248
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 248
cagcaacact atgagaaaac aagatgag 28
<210> 249
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 249
ggttgggtct gctgtatgtg tatg 24
<210> 250
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 250
gtccagatga agctcccaga atg 23
<210> 251
<211> 26
<212> DNA
<213> (Artificial sequence)
<400> 251
cagatggtca tggtcaaata cctagc 26
<210> 252
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 252
tgagaccttc aatgactttc tagtaactca 30
<210> 253
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 253
gttctagcaa tgctggatac ttacatca 28
<210> 254
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 254
aaaagataac ctcacaccag aaattcca 28
<210> 255
<211> 26
<212> DNA
<213> (Artificial sequence)
<400> 255
aaagaagaaa gaaacctcca tgctca 26
<210> 256
<211> 19
<212> DNA
<213> (Artificial sequence)
<400> 256
ggcagtggct atgcctact 19
<210> 257
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 257
cgccctgtga gagatgtttt tc 22
<210> 258
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 258
caacaacatc agcctgatga aagg 24
<210> 259
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 259
ccagcctcat ctgtttgtct gta 23
<210> 260
<211> 29
<212> DNA
<213> (Artificial sequence)
<400> 260
gacattttgc tgctgaaaaa tggtaaaag 29
<210> 261
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 261
cgtgggccag gctttattct c 21
<210> 262
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 262
cccagattct tctgctgatc ga 22
<210> 263
<211> 25
<212> DNA
<213> (Artificial sequence)
<400> 263
gggagaaagg tcctttacac atacc 25
<210> 264
<211> 25
<212> DNA
<213> (Artificial sequence)
<400> 264
ggcccaaatt caccaataat agagg 25
<210> 265
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 265
caacgtgtgt agacaggttt cag 23
<210> 266
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 266
gcagaaaaac cttttaagca taagtaagca 30
<210> 267
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 267
tgcgttcatc acttttccaa aagc 24
<210> 268
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 268
gtcagtatgg tgcaggtgtg at 22
<210> 269
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 269
cctatttgat tgtctttttg ctgctgt 27
<210> 270
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 270
caaatgccac aacacgcaaa att 23
<210> 271
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 271
<210> 272
<211> 27
<212> DNA
<213> (Artificial sequence)
<400> 272
ccaagattct gtaaacgttg actcctt 27
<210> 273
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 273
ccagagaaag ccagtctctt gac 23
<210> 274
<211> 25
<212> DNA
<213> (Artificial sequence)
<400> 274
gtccctaatt ttgctgttga ctcct 25
<210> 275
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 275
ggtcaactgg cctgacctaa ac 22
<210> 276
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 276
ggtccacttc agttgctggt tt 22
<210> 277
<211> 25
<212> DNA
<213> (Artificial sequence)
<400> 277
actaaacaga actcatgtga gcaca 25
<210> 278
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 278
gtgcgtttaa ctctaatagg aagaaaacac 30
<210> 279
<211> 25
<212> DNA
<213> (Artificial sequence)
<400> 279
aaaagacagt ctgctaattc cagct 25
<210> 280
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 280
atggaaactt gaagttgcta aacagttg 28
<210> 281
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 281
tggaagttta cgagactatc ttcaaaaaca 30
<210> 282
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 282
atcccagccc aagcgagaca ta 22
<210> 283
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 283
gcccagctgt gagatgtact tg 22
<210> 284
<211> 26
<212> DNA
<213> (Artificial sequence)
<400> 284
aacttgaaac ccaaggtaca tttcag 26
<210> 285
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 285
ggagtcaaac aggcctaggt ttc 23
<210> 286
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 286
ggtaagccat aagtttcctg cgtt 24
<210> 287
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 287
gcctgattct aggtaatagt ctttaccttt 30
<210> 288
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 288
agagttttta tgcaaagttt gacctttgaa 30
<210> 289
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 289
<210> 290
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 290
gtttacttga aggcctccgg aat 23
<210> 291
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 291
gcggagtgcc attcagaaaa tt 22
<210> 292
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 292
ctttgctgct gctggatgtt t 21
<210> 293
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 293
aagcttattg cataactgaa tgtataccca 30
<210> 294
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 294
<210> 295
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 295
ggcctgtttg agtgacagtc aa 22
<210> 296
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 296
gagaatgaca aagaaggcgc att 23
<210> 297
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 297
tggaacagat gaaatggaaa gtagttcc 28
<210> 298
<211> 29
<212> DNA
<213> (Artificial sequence)
<400> 298
ttgttttcct tgacagaaat tgatatgcc 29
<210> 299
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 299
tttttatagg gtctattgcc gaaaacac 28
<210> 300
<211> 26
<212> DNA
<213> (Artificial sequence)
<400> 300
caacaacatc aagagcagaa tttgga 26
<210> 301
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 301
agaaaatagc tgctgttttc ttgaaatacg 30
<210> 302
<211> 29
<212> DNA
<213> (Artificial sequence)
<400> 302
atcgtttttg acagtttgac agttaaagg 29
<210> 303
<211> 29
<212> DNA
<213> (Artificial sequence)
<400> 303
ttgcttacct gggctttaat ttttatgtg 29
<210> 304
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 304
gcacgaaata gacctaaaat caaagttgaa 30
<210> 305
<211> 26
<212> DNA
<213> (Artificial sequence)
<400> 305
ttgcttgtag tcatccatag gtagga 26
<210> 306
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 306
gcttgagatg cctgacaacc tt 22
<210> 307
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 307
cacccaaggt aagtaagccc t 21
<210> 308
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 308
ccagaagacg gaccatttcc tg 22
<210> 309
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 309
cctaagctcc agctccaggt a 21
<210> 310
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 310
cctgggtctt cagtgaacca tt 22
<210> 311
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 311
cccaaaatca tacacatgct ggaa 24
<210> 312
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 312
gctacagtga aatctcgatg gagt 24
<210> 313
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 313
gcagctttgg cagtattgga tttttaaatt 30
<210> 314
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 314
ccatcatgtc tttttgtttg aagaactagt 30
<210> 315
<211> 26
<212> DNA
<213> (Artificial sequence)
<400> 315
cctgcctgta atcatttccc tagttt 26
<210> 316
<211> 25
<212> DNA
<213> (Artificial sequence)
<400> 316
gtgtgtgtat gtttctccag tccaa 25
<210> 317
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 317
tctgccgagg agtataggta gg 22
<210> 318
<211> 26
<212> DNA
<213> (Artificial sequence)
<400> 318
cgagttacag tcctaatggt gactca 26
<210> 319
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 319
gtcgatgagg gtcaagttca aca 23
<210> 320
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 320
ccacaagttc aggcctagag gta 23
<210> 321
<211> 19
<212> DNA
<213> (Artificial sequence)
<400> 321
ggcctagtcg agcagggat 19
<210> 322
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 322
cctctcagag aatcccaact cagt 24
<210> 323
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 323
gtaggactcc tggtggttgt tc 22
<210> 324
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 324
acggatgtaa tattttctga agagccaa 28
<210> 325
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 325
cttttgaaaa tggttgttgc tgtgtaaaaa 30
<210> 326
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 326
cggtacctcc tactgaagtt gag 23
<210> 327
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 327
tttgtcacaa gtacaaaaag gtaaaagcaa 30
<210> 328
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 328
aggtagttga tggcgttgtt ga 22
<210> 329
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 329
ggaaaaactg agccaggcct ta 22
<210> 330
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 330
ggagagctgg tgagaactaa actc 24
<210> 331
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 331
gcacttgtac accttcatct gct 23
<210> 332
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 332
agtgggtcca tgaatccctt ct 22
<210> 333
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 333
ggtcttagga gggaccctag tt 22
<210> 334
<211> 25
<212> DNA
<213> (Artificial sequence)
<400> 334
gagggaacaa aaactctacc accat 25
<210> 335
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 335
cacttaagct gatagagaca tgatgtaacc 30
<210> 336
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 336
ggctgagaag tttgtaggtg gt 22
<210> 337
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 337
ccaggttcca gacatggcta tt 22
<210> 338
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 338
ctcacagtcc atggttatat gcttataaga 30
<210> 339
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 339
ggatgccaaa cacatacctt gaatttaaaa 30
<210> 340
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 340
cagactattt tacatgaatt ggcatccaat 30
<210> 341
<211> 28
<212> DNA
<213> (Artificial sequence)
<400> 341
ggttgcttca tctacagcaa ataatcag 28
<210> 342
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 342
tagagggtac ctcaaactaa ggca 24
<210> 343
<211> 26
<212> DNA
<213> (Artificial sequence)
<400> 343
ccataatgca cagagagggt caatat 26
<210> 344
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 344
aaaggtgagt ttgtattaaa aggtactggt 30
<210> 345
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 345
gatattctcg acacagcagg tcaa 24
<210> 346
<211> 29
<212> DNA
<213> (Artificial sequence)
<400> 346
aacactagcg tatgtctctc agagtataa 29
<210> 347
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 347
tggcaccaga taaatatgtg caca 24
<210> 348
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 348
gcacataact gaaaaccata gggtatttca 30
<210> 349
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 349
<210> 350
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 350
<210> 351
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 351
cgtctacctg gagattgaca acc 23
<210> 352
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 352
gccgaaccaa tacaaccctc t 21
<210> 353
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 353
caaatggaag gtcacactag ggtt 24
<210> 354
<211> 18
<212> DNA
<213> (Artificial sequence)
<400> 354
<210> 355
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 355
gctcaccctg tgatttgttg ct 22
<210> 356
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 356
cctgattcca gcctgggttt at 22
<210> 357
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 357
aaaaacaaaa ttgggcttaa aagaaccatg 30
<210> 358
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 358
atcaaaaaca ccctttatga tgatgatgaa 30
<210> 359
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 359
gaagttcaga tctttttcac tatgcacatt 30
<210> 360
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 360
agaaataagt ttcagtaaca tcagcatcct 30
<210> 361
<211> 29
<212> DNA
<213> (Artificial sequence)
<400> 361
tagtcagagg aaaatgccaa ttgtagtac 29
<210> 362
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 362
caaaacacct gcagatctaa tagaaaacaa 30
<210> 363
<211> 24
<212> DNA
<213> (Artificial sequence)
<400> 363
gcacttggtt tcaaattcag gcta 24
<210> 364
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 364
aaagcaaatc aatcaaatat accatgtgca 30
<210> 365
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 365
ccaaaagaat ctaatgagat ttggcacata 30
<210> 366
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 366
ggctctaggg ctgagggaat at 22
<210> 367
<211> 21
<212> DNA
<213> (Artificial sequence)
<400> 367
gttccctctg gacactctgt g 21
<210> 368
<211> 22
<212> DNA
<213> (Artificial sequence)
<400> 368
atgtccacga ccgagggaaa gt 22
<210> 369
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 369
<210> 370
<211> 25
<212> DNA
<213> (Artificial sequence)
<400> 370
ccctctgagt caggaaacat tttca 25
<210> 371
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 371
gtgaacaagg aggaggtgag caa 23
<210> 372
<211> 58
<212> DNA
<213> (Artificial sequence)
<400> 372
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatct 58
<210> 373
<211> 65
<212> DNA
<213> (Artificial sequence)
<400> 373
gatcggaaga gcacacgtct gaactccagt cacattactc gatctcgtat gccgtcttct 60
gcttg 65
<210> 374
<211> 65
<212> DNA
<213> (Artificial sequence)
<400> 374
gatcggaaga gcacacgtct gaactccagt cactccggag aatctcgtat gccgtcttct 60
gcttg 65
<210> 375
<211> 65
<212> DNA
<213> (Artificial sequence)
<400> 375
gatcggaaga gcacacgtct gaactccagt caccgctcat tatctcgtat gccgtcttct 60
gcttg 65
<210> 376
<211> 65
<212> DNA
<213> (Artificial sequence)
<400> 376
gatcggaaga gcacacgtct gaactccagt cacgagattc catctcgtat gccgtcttct 60
gcttg 65
<210> 377
<211> 65
<212> DNA
<213> (Artificial sequence)
<400> 377
gatcggaaga gcacacgtct gaactccagt cacattcaga aatctcgtat gccgtcttct 60
gcttg 65
<210> 378
<211> 65
<212> DNA
<213> (Artificial sequence)
<400> 378
gatcggaaga gcacacgtct gaactccagt cacgaattcg tatctcgtat gccgtcttct 60
gcttg 65
<210> 379
<211> 65
<212> DNA
<213> (Artificial sequence)
<400> 379
gatcggaaga gcacacgtct gaactccagt cacctgaagc tatctcgtat gccgtcttct 60
gcttg 65
<210> 380
<211> 65
<212> DNA
<213> (Artificial sequence)
<400> 380
gatcggaaga gcacacgtct gaactccagt cactaatgcg catctcgtat gccgtcttct 60
gcttg 65
<210> 381
<211> 65
<212> DNA
<213> (Artificial sequence)
<400> 381
gatcggaaga gcacacgtct gaactccagt caccggctat gatctcgtat gccgtcttct 60
gcttg 65
<210> 382
<211> 65
<212> DNA
<213> (Artificial sequence)
<400> 382
gatcggaaga gcacacgtct gaactccagt cactccgcga aatctcgtat gccgtcttct 60
gcttg 65
<210> 383
<211> 65
<212> DNA
<213> (Artificial sequence)
<400> 383
gatcggaaga gcacacgtct gaactccagt cactctcgcg catctcgtat gccgtcttct 60
gcttg 65
<210> 384
<211> 65
<212> DNA
<213> (Artificial sequence)
<400> 384
gatcggaaga gcacacgtct gaactccagt cacagcgata gatctcgtat gccgtcttct 60
gcttg 65
Claims (9)
1. A primer combination for screening high-risk subtypes of acute lymphocytic leukemia comprises a primer combination of related fusion genes and a primer combination of related gene mutation, and is characterized in that the primer combination of related fusion genes comprises 66 pairs of primers for detecting 55 fusion genes, and 12 primer pools in total, wherein the sequences are shown as R1-R12, the primer combination of related gene mutation comprises 125 pairs of primers for 206 detection sites, and 2 primer pools in total are respectively a first primer pool M1 and a second primer pool M2;
in the 125 pairs of primers in the related gene mutation primer combination, 65 pairs of primers form a first primer pool M1, the sequence of the first primer pool M1 comprises M1-1 f-M1-65 f and M1-1R-M1-65R, 60 pairs of primers form a second primer pool M2, and the sequence of the second primer pool M2 comprises M2-1 f-M2-60 f and M2-1R-M2-60R.
2. The method for detecting the primer combination for screening the high-risk subtype of acute lymphoblastic leukemia according to claim 1, wherein the method is a method for non-diagnosis, and the method for detecting the 66 pairs of primers in the primer combination of related fusion genes and the 125 pairs of primers in the primer combination of related gene mutation comprises the following steps:
s1, separating leukemia cells of acute lymphocytic leukemia patients;
s2, extracting total RNA of the cells and preparing cDNA;
s3, extracting cell genome DNA;
s4, preparing fusion gene detection primers and probes;
and S5, constructing a DNA library and sequencing.
3. The test method for screening the primer combination of the high-risk subtype of acute lymphoblastic leukemia according to claim 2, wherein the separation method in the step S1 comprises the following steps:
1.1, placing the blood collection tube in a low-speed centrifuge after balancing, centrifuging at 2500rpm multiplied by 5min, slowly absorbing and removing upper plasma, diluting the remaining blood cells to 5mL by using sterile normal saline, fully blowing, beating and uniformly mixing;
1.2, taking a 15mL centrifuge tube, marking the name of a patient, adding 5mL human lymphocyte separation liquid, and slowly dripping the diluted marrow liquid on the lymphocyte separation liquid along the tube wall to form layering so as to avoid mixing the two;
1.3, adopting a density gradient centrifugation method, placing a centrifuge tube in a low-speed centrifuge at 2000rpm for 20min, after centrifugation, dividing liquid in the tube into three layers, wherein an upper clear layer is diluted plasma and platelets, a middle white membrane layer is mononuclear cells, and a lower red layer is granulocytes and erythrocytes;
1.4, slowly sucking the middle mononuclear cell layer, placing the middle mononuclear cell layer into another clean centrifuge tube for marking the name of a patient, adding 10mL of sterile physiological saline, blowing, beating and uniformly mixing, placing the mixture into a low-speed centrifuge for centrifugation at 2000rpm for 5min, and discarding the supernatant;
1.5, resuspending the washed cell mass with 1mL of sterile physiological saline, counting the cell mass by using a cell counting plate under a high power microscope, calculating the total cell amount, wherein the cell counting solution is 1% of glacial acetic acid, 20 mu L of cell suspension is added into 380 mu L of 1% glacial acetic acid, uniformly blowing and stirring, sucking 20 mu L of cell suspension and adding the cell suspension into the cell counting plate, counting the number n of cells in 16 lattices under a microscope, and then the total cell amount is n multiplied by 10000 multiplied by 20;
1.6 dilution of the cell suspension to 1X 10 7 Single monocyte/mL, 1.5mL EP tube in cell suspension, each tube of 1mL, after trimming, 8000rpm high speed instantaneous centrifugation, carefully suction to discard the supernatant, do not touch the tube bottom cell precipitation, a tube in the tubeThe number and the patient name are marked on the wall, and 1mL Trizol is added into the other tube after the number and the patient name are marked on the tube wall for mixing.
4. The method for testing the primer combination for screening the high-risk subtype of acute lymphoblastic leukemia according to claim 2, wherein the preparation method in step S2 comprises:
2.1, putting 500 mu L Trizol mixed solution into another 1.5mL EP tube without RNase, adding 200 mu L chloroform, violently shaking to mix uniformly, standing at room temperature for 10min, centrifuging at 4 ℃, multiplying by 15min at 12000rpm, dividing the solution after centrifugation into three layers, wherein the upper layer is a clear transparent colorless aqueous phase, the middle white membrane layer is mainly protein and DNA, and the lower layer is an organic phase;
2.2, slowly absorbing 200 mu L of the upper layer solution into a new 0.6ml of EP tube without RNase, slowly moving a suction head downwards along with the liquid level to avoid sucking the intermediate protein and DNA layer, adding equal volume of isopropanol precooled at-20 ℃, covering a tube cover, then gently turning upside down and uniformly mixing, standing at-20 ℃ for 10min to fully precipitate RNA, centrifuging at 4 ℃, and multiplying by 12000rpm for 10min, and discarding the supernatant to obtain a precipitate;
2.3, adding 500 mu L of 75% ethanol precooled at the temperature of 20 ℃, mixing the absolute ethanol with DEPC water according to the ratio of 3, centrifuging at the temperature of 1,4 ℃, slowly discarding supernatant, and carefully absorbing residual ethanol by using a pipette;
2.4, placing the mixture on a super clean bench, standing the mixture at room temperature until ethanol which is not completely absorbed volatilizes and the white RNA precipitate is semitransparent;
2.5, adding 20 mu L DEPC water to the bottom of the tube, gently blowing and beating, fully dissolving the precipitate, carrying out low-speed instantaneous separation, detecting the RNA concentration and purity by using an ultraviolet spectrophotometer, wherein the RNA quality with the A260/280 ratio of 1.8-2.0 meets the requirement, and then carrying out reverse transcription on the RNA to form cDNA;
2.6, carrying out reverse transcription reaction by using a reverse transcription kit, and preparing a 20 mu L reverse transcription system: DEPC water make up to 20 μ L, reverse transcription reaction conditions: 37 ℃ of: 15min, inactivating reaction conditions of reverse transcriptase: 85℃:5s,4 ℃, and the cDNA obtained by reverse transcription is used for detecting the multiple PCR fusion gene.
5. The method for detecting the primer combination for screening the high-risk subtype of acute lymphoblastic leukemia according to claim 3, wherein the method for extracting and purifying the DNA in the step S3 is a silicon membrane adsorption method, and comprises the following steps:
3.1 separating the resulting 1X 10 7 Dissolving leukemia cells of acute lymphoblastic leukemia patients in 200 μ L PBS, and adding 20 μ L proteinase K to the bottom of the tube;
3.2, slowly adding 200 mu L of oily lysate AL, shaking, uniformly mixing, and then quickly separating at a low speed;
3.3, carrying out water bath at 56 ℃ for 20min, after the oily mixed liquid becomes clear, wiping off the liquid on the outer wall, centrifuging the liquid adhered to the inner wall and the tube cover to the bottom of the tube at a low speed;
3.4, adding 200 mu L of absolute ethyl alcohol, reversing, uniformly mixing, and instantly separating at a low speed;
3.5, transferring the mixed solution into an adsorption column, placing the adsorption column on a 2mL collection tube provided by the kit, covering a tube cover, marking, centrifuging at high speed of 12000rpm for 3min, and discarding the collection tube and filtrate;
3.6, putting the adsorption column into a new collection tube, adding 500 mu L of buffer AW1 into the adsorption column, covering a tube cover, centrifuging at 12000rpm multiplied by 3min, and discarding the collection tube and filtrate;
3.7, putting the adsorption column into a new collecting pipe, adding 500 mu L of buffer AW2, covering a pipe cover, centrifuging at 12000rpm multiplied by 3min, and discarding the collecting pipe and filtrate;
3.8, placing the adsorption column in a self-prepared clean sterile 1.5mL EP tube, opening the tube cover of the adsorption column, centrifuging at 12000rpm for 3min to fully volatilize the ethanol, and discarding the EP tube and the filtrate;
3.9, placing the adsorption column in a self-prepared clean and sterile 1.5mL EP tube, suspending and dropwise adding 100 mu L of elution buffer AE, carefully dropwise adding the AE in the middle of an adsorption membrane of the adsorption column, incubating at room temperature for 3min to sufficiently elute DNA in the adsorption membrane, and centrifuging at 12000rpm multiplied by 3min, wherein the filtrate in the EP tube is a genome DNA solution;
3.10, in order to improve the collection efficiency of the DNA, repeating the step 3.9, adding the DNA solution collected in the EP tube into the middle part of the adsorption membrane of the adsorption column again, incubating at room temperature for 3min to fully dissolve the DNA in the adsorption membrane, centrifuging at 12000rpm multiplied by 3min, and collecting the DNA solution again;
3.11, detecting the concentration and the purity of the genome DNA by using an ultraviolet spectrophotometer, wherein the DNA quality with the A260/280 ratio of 1.8-2.0 meets the requirement, and the DNA library is used for constructing a DNA library and carrying out high-throughput sequencing.
6. The method for detecting the primer combination for screening the high-risk subtype of acute lymphoblastic leukemia according to claim 5, wherein the DNA library is constructed and sequenced in the step S5, and the steps are as follows:
p1, PCR amplification of a detection site;
p2, carrying out ultrasonic DNA crushing;
p3, DNA end repair;
p4, adding dA tail;
p5, connecting a joint;
p6, PCR amplification;
and P7, quantifying the amount of the Qubit and sequencing.
7. The method for detecting the primer combination for screening the high-risk subtype of acute lymphoblastic leukemia according to claim 6, wherein the operation method of DNA end repair in step P3 is as follows:
4.1, taking 9.5 mu L of the solution after the DNA is ultrasonically crushed, and adding a reagent;
4.2, mixing uniformly, placing on a PCR instrument, and operating: 30min at 25 ℃;
4.3, purifying the reaction solution after the DNA tail end is repaired to 20.5 mu L, fully and uniformly mixing magnetic beads, sucking 22.5 mu L of magnetic beads into the reaction solution, gently blowing and sucking for 10 times, uniformly mixing, and standing at room temperature for 5min;
4.4, placing the EP tube on a magnetic frame for 5min until the liquid is clear, carefully absorbing the supernatant, and avoiding absorbing the magnetic beads on the tube wall;
4.5, keeping the EP tube on a magnetic frame, adding 200 mu L of 80% ethanol along the tube wall, standing at room temperature for 30 seconds, removing supernatant, and cutting the magnetic beads which are not absorbed on the tube wall;
4.6, repeating the 80% ethanol washing once;
4.7, keeping the EP tube on a magnetic rack for drying for 2-5min, observing that the surface of the magnetic bead does not reflect light any more, and avoiding excessive drying;
4.8, taking the EP tube off the magnetic frame, adding 22 mu L DEPC water, gently blowing and beating the mixture evenly, standing the mixture at room temperature for 2min, and then placing the EP tube on the magnetic frame until the liquid is clear;
4.9 carefully pipette 20.5. Mu.L of supernatant into a new PCR tube, and do not pipette the beads onto the tube wall;
the operation method for adding dA tail in the step P4 comprises the following steps:
5.1, adding a reagent into 20.5 mu L of reaction liquid obtained after DNA end repair purification in the step P3;
5.2, uniformly mixing, placing on a PCR instrument, and operating: 30min at 37 ℃;
5.3, purifying the reaction solution added with the dA tail to 10 mu L, fully and uniformly mixing magnetic beads, sucking 45 mu L of magnetic beads into the reaction solution, softly sucking for 10 times, uniformly mixing, and standing at room temperature for 5min;
5.4, placing the EP tube on a magnetic frame for 5min until the liquid is clear, carefully absorbing the supernatant, and avoiding absorbing the magnetic beads on the tube wall;
5.5, keeping the EP tube on a magnetic frame, adding 200 mu L of 80% ethanol along the tube wall, standing at room temperature for 30 seconds, removing supernatant, and cutting the magnetic beads which are not absorbed on the tube wall;
5.6, repeating the washing once by 80 percent ethanol;
5.7, keeping the EP tube on a magnetic rack for drying for 2-5min, observing that the surface of the magnetic bead does not reflect light any more, and avoiding excessive drying;
5.8, taking the EP tube off the magnetic frame, adding 11 mu L DEPC water, gently blowing and beating the mixture evenly, standing the mixture at room temperature for 2min, and then placing the EP tube on the magnetic frame until the liquid is clear;
5.9 carefully pipette 10. Mu.L of the supernatant into a new PCR tube, and do not pipette down the beads on the tube wall.
8. The method for testing the primer combination for screening the high-risk subtype of acute lymphoblastic leukemia according to claim 7, wherein the operation method of the linker connection in the step P5 is as follows:
6.1, taking 10 mu L of reaction liquid obtained by adding dA tail in the step P4 and adding a reagent;
6.2, mixing uniformly, placing on a PCR instrument, and operating: 15min at 25 ℃;
6.3, purifying the reaction liquid after the joint connection to 23 mu L, adding 28.8 mu L of non-enzyme water to the reaction liquid after the joint connection to ensure that the total volume is 50 mu L, fully and uniformly mixing the magnetic beads, sucking 30 mu L (0.6 x) of the magnetic beads into the reaction liquid, softly blowing and sucking for 10 times for uniformly mixing, and standing at room temperature for 5min;
6.4, the EP tube is placed on a magnetic frame for 5min until the liquid is clear, the supernatant is carefully sucked to a new tube, and the magnetic beads on the tube wall are not sucked;
6.5, adding 7.5 mu L (0.15 x) of magnetic beads into the reaction solution, gently blowing, stirring and mixing uniformly, and standing at room temperature for 5min;
6.6, placing the EP tube on a magnetic frame for 5min until the liquid is clear, carefully absorbing the supernatant, and avoiding magnetic beads absorbed on the tube wall;
6.7, keeping the EP tube on a magnetic frame, adding 200 mu L of 80% ethanol along the tube wall, standing at room temperature for 30 seconds, removing supernatant, and cutting magnetic beads which are not absorbed on the tube wall;
6.8, repeating the 80% ethanol washing once;
6.9, keeping the EP tube on a magnetic frame for drying for 2-5min, observing that the surface of the magnetic bead does not reflect light any more, and keeping the EP tube from being dried excessively;
6.10, taking the EP tube off the magnetic frame, adding 24 mu L DEPC water, gently blowing and beating the mixture evenly, standing the mixture at room temperature for 2min, and then placing the EP tube on the magnetic frame until the liquid is clear;
6.11 carefully pipette 23. Mu.L of the supernatant into a new PCR tube, and do not pipette down the beads on the tube wall.
9. The method for testing the primer combination for screening the high-risk subtype of acute lymphoblastic leukemia according to claim 8, wherein the PCR amplification operation method in the step P6 is as follows:
7.1, taking 23 mu L of the reaction liquid obtained by connecting and purifying the joint in the step P5, and adding a reagent;
7.2, mixing uniformly, and placing on a PCR instrument for operation;
7.3, purifying the reaction solution after PCR amplification to 20 μ L, fully mixing the magnetic beads, sucking 45 μ L (0.9X) of the magnetic beads into the reaction solution, gently blowing and sucking for 10 times, mixing the mixture uniformly, and standing the mixture at room temperature for 5min;
7.4, placing the EP tube on a magnetic frame for 5min until the liquid is clear, carefully absorbing the supernatant, and avoiding absorbing the magnetic beads on the tube wall;
7.5, keeping the EP tube on a magnetic frame, adding 200 mu L of 80% ethanol along the tube wall, standing at room temperature for 30 seconds, discarding the supernatant, and cutting the magnetic beads not to be absorbed on the tube wall;
7.6, repeating the washing with 80% ethanol once;
7.7, keeping the EP tube on a magnetic rack for drying for 2-5min, observing that the surface of the magnetic bead does not reflect light any more, and keeping the EP tube from being excessively dried;
7.8, taking the EP tube off the magnetic frame, adding 21 mu L DEPC water, gently blowing and beating the mixture evenly, standing the mixture at room temperature for 2min, and then placing the EP tube on the magnetic frame until the liquid is clear;
7.9 carefully pipette 20. Mu.L of the supernatant into a new PCR tube, and do not pipette down the beads on the tube wall.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040203011A1 (en) * | 2001-04-27 | 2004-10-14 | Morris Stephen W. | Fusion genes associated with acute megakaryoblastoc leukemias |
| CN105838792A (en) * | 2016-04-22 | 2016-08-10 | 上海荻硕贝肯生物科技有限公司 | Primer, probe, kit and method for qualitatively detecting fusion genes of leukemia |
| CN111575373A (en) * | 2020-04-28 | 2020-08-25 | 南京实践医学检验有限公司 | Kit and method for detecting acute lymphocytic leukemia gene mutation by targeting high-throughput sequencing based on multiple PCR |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040203011A1 (en) * | 2001-04-27 | 2004-10-14 | Morris Stephen W. | Fusion genes associated with acute megakaryoblastoc leukemias |
| CN105838792A (en) * | 2016-04-22 | 2016-08-10 | 上海荻硕贝肯生物科技有限公司 | Primer, probe, kit and method for qualitatively detecting fusion genes of leukemia |
| CN111575373A (en) * | 2020-04-28 | 2020-08-25 | 南京实践医学检验有限公司 | Kit and method for detecting acute lymphocytic leukemia gene mutation by targeting high-throughput sequencing based on multiple PCR |
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