CN110656177A - Primer, probe, method and kit for detecting AF1Q gene relative expression level - Google Patents
Primer, probe, method and kit for detecting AF1Q gene relative expression level Download PDFInfo
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Abstract
The invention discloses a primer and a probe for detecting the relative expression quantity of an AF1Q gene, a method and a kit, which comprise an upstream primer and a downstream probe aiming at AF1Q, and an upstream primer and a downstream probe aiming at an ABL reference gene. The invention can quickly detect whether the AF1Q gene exists in the sample and the expression quantity of the AF1Q gene. The detection result completed by the invention is accurate and sensitive, and can be used for leukemia auxiliary diagnosis and MRD monitoring.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a kit for detecting the expression level of AF1Q gene.
Background
AML (acute myeloid leukemia) is a clinical molecular heterogeneous disease. Among the mortality rates of malignant tumors in China, leukemia highly ranks first. Based on this situation, a timely and accurate diagnosis is very critical as a precondition for targeted therapy.
The AF1q gene is a major member of a fusion gene with the MLL gene in acute leukemia. Plays an important regulatory role in the human hematopoietic system. It was originally found in MLL-AF1Q carried by a child patient with acute leukemia, and is one of more than 50 fusion genes related to MLL, and the expression of the fusion genes has important significance for the development of leukemia, treatment and prognosis. In recent years, studies have also shown that AF1Q itself, as an independent influencing factor, has a significant influence on leukemia.
The AF1Q gene is highly expressed in hematopoietic stem progenitor cells of CD34+, the expression level is reduced along with the maturation of the cells, and the expression level is low or no in peripheral blood cells. It is shown that AF1q gene is highly expressed in NB4 cell, and its expression level is reduced to different extent after ATRA induced differentiation.
The expression level of the AF1Q gene is closely related to acute leukemia and MDS prognosis. The patients with acute leukemia with high AF1Q gene expression have high recurrence rate and death rate, poor treatment effect, poor prognosis and higher proportion of AML-M1 subtype according to the FAB diagnosis standard, which indicates that the patients possibly represent more original genetic phenotype or directly cause the leukemia gene to appear. The AF1Q gene is highly expressed in high-risk MDS. And the higher the expression level of AF1Q gene in a patient who has received hematopoietic stem cell transplantation, the higher the risk of relapse. In addition, chromosome aberration caused by AF1Q gene can also cause NHL (non Hodgkin lymphoma) and is closely related to the histological classification and tumor progression of lymphoma.
In conclusion, the AF1Q gene can be a new index for judging prognosis and treatment effect of leukemia, can be used as a very valuable molecular biological tool for diagnosis, risk classification and monitoring of minimal residual disease of leukemia, and detection of the AF1Q gene can provide a better basis for targeted treatment of malignant hematological diseases.
Disclosure of Invention
The invention designs the primer and probe sequence of AF1Q gene and ABL gene, and uses the fluorescent quantitative PCR technology to detect the AF1Q gene expression level. A PCR reaction system and reaction conditions are optimized by adjusting the concentrations and the proportions of two gene primers and probes, and an AF1Q gene expression level detection method is developed, and is used for leukemia auxiliary diagnosis and MRD monitoring by taking the AF1Q expression level of a healthy donor as a reference.
The primer and the probe for detecting the relative expression quantity of the AF1Q gene are characterized by comprising the following components in parts by weight:
(1) an upstream primer AF1Q-F, AF1Q-R and a Probe AF1Q-Probe for detecting the AF1Q gene,
AF1Q-F：CGAGGCACTCCCTCCATCTT
AF1Q-R：CCTTGTAGGTGGCTGTATCTGAC
AF1Q-Probe：FAM-ACGCCAGTAATTGATTGATAACAGGAAGC–BHQ；
(2) an upstream primer ABL-F, a downstream primer ABL-R and a Probe ABL-Probe for detecting the internal reference gene ABL,
ABL-F：GTGAGTGACATAGCCTCATGTTC
ABL-R：GCAGGCGTGCTCTGTGAAAT
ABL–Probe：FAM-TCAGGGAGGGTTAGGAAAACCAC-BHQ。
the invention also provides a method for detecting the relative expression quantity of the AF1Q gene in a sample, which is characterized by comprising the following steps:
(1) extracting total RNA in a bone marrow specimen;
(2) reverse transcribing the total RNA into cDNA;
(3) respectively amplifying the cDNA in the step (2) by using the upstream and downstream primers and probes of the AF1Q gene and the ABL gene; respectively detecting fluorescent signals;
AF1Q-F：CGAGGCACTCCCTCCATCTT
AF1Q-R：CCTTGTAGGTGGCTGTATCTGAC
AF1Q-Probe：FAM-ACGCCAGTAATTGATTGATAACAGGAAGC–BHQ；
ABL-F：GTGAGTGACATAGCCTCATGTTC
ABL-R：GCAGGCGTGCTCTGTGAAAT
ABL–Probe：FAM-TCAGGGAGGGTTAGGAAAACCAC-BHQ；
(4) and determining the relative expression amount of the AF1Q gene in the sample according to the fluorescence signal of the AF1Q gene and the fluorescence signal of the ABL reference gene.
The invention finally provides a kit for detecting the relative expression quantity of the AF1Q gene, which comprises a detection system PCR reaction solution, and is characterized in that the detection system PCR reaction solution comprises:
(1) an upstream primer AF1Q-F, AF1Q-R and a Probe AF1Q-Probe for detecting the AF1Q gene,
AF1Q-F：CGAGGCACTCCCTCCATCTT
AF1Q-R：CCTTGTAGGTGGCTGTATCTGAC
AF1Q-Probe：FAM-ACGCCAGTAATTGATTGATAACAGGAAGC–BHQ；
(2) an upstream primer ABL-F, a downstream primer ABL-R and a Probe ABL-Probe for detecting the internal reference gene ABL,
ABL-F：GTGAGTGACATAGCCTCATGTTC
ABL-R：GCAGGCGTGCTCTGTGAAAT
ABL–Probe：FAM-TCAGGGAGGGTTAGGAAAACCAC-BHQ。
(3) a positive control, a negative control and a blank control.
Furthermore, the kit also comprises reagents such as erythrocyte lysate, Trizol, chloroform, isopropanol, absolute ethyl alcohol, DEPC water and the like, and a TOYOBO reverse transcription reagent.
In the technical scheme of the invention, the tumor is preferably cervical cancer, pancreatic cancer, papillary thyroid cancer, breast cancer, intestinal cancer, head and neck squamous cell carcinoma, oral squamous cell carcinoma and malignant hematological tumor.
The invention has the beneficial effects that: the quantitative standard curves of the target gene AF1Q and the reference gene ABL are respectively constructed by combining the qRT-PCR technology with a Taqman probe and utilizing a double-standard curve method, and the expression level of the marrow AF1Q of a detected person relative to the reference gene ABL is detected. Compared with the delta CT method which is popular at present, the kit and the detection method have the advantages of high result accuracy and the like. In addition, the kit reasonably optimizes the concentration and proportion of primers and probes required by a reaction system, and promotes the experimental conditions to be optimal, thereby saving a great deal of time for groping the conditions and improving the experimental efficiency. The detection kit is convenient to use and simple to operate.
Drawings
FIG. 1 is a standard curve diagram of AF1Q gene;
FIG. 2 is a standard graph of the ABL gene;
FIG. 3 is a representation of the results of a test on a healthy human bone marrow specimen;
FIG. 4 is a representation of the results of a test on a bone marrow specimen from a leukemia patient.
Detailed Description
The invention will be further elucidated with reference to the specific embodiments and the accompanying drawings.
EXAMPLE 1 preparation of the kit
The kit mainly comprises the following reagents: erythrocyte lysate, Trizol; chloroform; isopropyl alcohol; absolute ethyl alcohol; detection system PCR reaction solution: TOYOBO qPCR MIX, detecting an upstream primer AF1Q-F, AF1Q-R and a Probe AF1Q-Probe of a target gene AF1Q, detecting a primer ABL-F, ABL-R and a Probe ABL-Probe for reference gene ABL, and detecting a positive control and a negative control. Wherein, the primers and the probes for detecting the AF1Q gene are respectively as follows:
AF1Q-F：CGAGGCACTCCCTCCATCTT
AF1Q-R：CCTTGTAGGTGGCTGTATCTGAC
AF1Q-Probe：FAM-ACGCCAGTAATTGATTGATAACAGGAAGC-BHQ
the primers and the probes for detecting the ABL reference gene respectively comprise:
ABL-F：GTGAGTGACATAGCCTCATGTTC
ABL-R：GCAGGCGTGCTCTGTGAAAT
ABL–Probe：FAM-TCAGGGAGGGTTAGGAAAACCAC-BHQ
positive control: a solution containing the AF1Q genome;
negative control: solutions without AF1Q genome.
Example 2 operational procedure for the Process of the invention
(1) Extracting total RNA in bone marrow: adding 8ml of erythrocyte lysate into a clean 15ml centrifuge tube, taking 2ml of anticoagulated bone marrow sample, adding the anticoagulated bone marrow sample and mixing the anticoagulated bone marrow sample and the erythrocyte lysate with the anticoagulated bone marrow sample. Standing at 4 ℃ for 10min, centrifuging at 1500rpm for 5min, discarding the supernatant, adding erythrocyte lysate into the cell precipitate again to lyse erythrocytes, and repeating the operation until the erythrocytes are completely lysed; to the resulting cells, 1ml of Trizol was added and the pipetting was repeated until the pellet was completely dissolved, and the resulting liquid was transferred to a clean 1.5ml EP tube. Standing at room temperature for 10min, adding 0.2ml chloroform, shaking thoroughly, mixing, standing at 4 deg.C for 10min, and centrifuging at 12000rpm for 15 min; gently taking the supernatant to another new EP tube, adding isopropanol with the same volume, fully and uniformly mixing, standing at 4 ℃ for 10min, and centrifuging at 12000rpm for 10 min; discarding the supernatant, adding 1ml of 75% ethanol, washing the precipitate, centrifuging at 7500rpm for 10min, and discarding the supernatant; drying at room temperature for 5-10min, adding 20 μ l DEPC water to dissolve precipitate completely.
(2) RNA was reverse transcribed into cDNA with reference to TOYOBO reverse transcription kit instructions.
(3) Reagent preparation: preparing X mul of PCR reaction liquid of a detection system according to the parts of detected people, and subpackaging 18 mul of each part: x ═ 18 μ l reaction solution X (4 parts of target gene (standard curve) +4 parts of internal reference (standard curve) + n parts of specimen +1 part of positive control +1 part of negative control);
(4) sample adding: adding 2 mul cDNA into PCR reaction solution; directly adding 2 mul of positive control substance and negative control substance into the positive control substance and the negative control substance; blank control with 2. mu.l DEPC water;
(5) and (3) detection: the detection is carried out by using a real-time fluorescent quantitative PCR instrument, and available instruments comprise ABI7500, ABI7300 and the like. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 30 s; the fluorescence signal was collected at 95 ℃ for 5s, 55 ℃ for 30s, and 72 ℃ for 30s for 40 cycles, and at 72 ℃ for 30 s.
And (5) judging a result: the threshold line is adjusted to be above the background signal and the negative amplification line, and the system automatically calculates the copy number according to the standard curve and the CT value. When the internal reference is positive, the detection result is valid. The result is credible when the internal reference CT < 36; CT is more than or equal to 36 and less than or equal to 38, and needs to be detected again; CT >38 indicates that the detection result is wrong and is not credible.
EXAMPLE 3 establishment of AF1Q Gene Standard Curve
With the concentration of AF1Q plasmid being 107、106、105、104、103、102Experiments were carried out with 10 copies/. mu.l as template, and 3 replicates per concentration, and a standard curve for AF1Q gene detection was established, with the results shown in FIG. 1.
Example 4 establishment of ABL Gene Standard Curve
With ABL plasmid concentration of 107、106、105、104、103、10210 copies/. mu.l were used as a template for the experiment, and each concentration was repeated 3 times to establish a standard curve for ABL gene detection, and the results are shown in FIG. 2.
Example 5 detection of samples of healthy people Using the detection method of the present invention
Mu.l of each sample is added into the PCR reaction solution of the detection system, and positive and negative controls are simultaneously carried out. Each sample was repeated 3 times, one positive control and one negative control. The results are shown in Table 1. The results of the test are demonstrated in FIG. 3.
TABLE 110 healthy specimens AF1Q Gene expression levels
Example 6 detection of clinical specimens of leukemia Using the detection method of the present invention
10 clinical samples to be detected are taken, total RNA is extracted, and reagents are prepared and detected according to the method described in the embodiment 2. Mu.l of each sample is added into the PCR reaction solution of the detection system, and positive and negative controls are simultaneously carried out. Each sample was repeated 3 times, one positive control and one negative control. The results of the experiment are shown in table 2 below. The results of the test are demonstrated in FIG. 4.
TABLE 210 clinical specimens AF1Q Gene expression levels
Paired t-test of copy number ratios in tables 1 and 2 revealed that AF1Q expression was significantly higher in the blood of patients with white blood than in the normal population, P < 0.004. Thus, the method can simply and rapidly detect and judge the increase and decrease of the expression of AF 1Q.
Sequence listing
<110> Yidekang laboratory Co., Ltd for medical examination of Hefei Aidikang
<120> primers, probes, method and kit for detecting relative expression of AF1Q gene
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cgaggcactc cctccatctt 20
<210> 2
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ccttgtaggt ggctgtatct gac 23
<210> 3
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
acgccagtaa ttgattgata acaggaagc 29
<210> 4
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
<210> 6
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tcagggaggg ttaggaaaac cac 23
Claims (4)
1. The primer and the probe for detecting the relative expression quantity of the AF1Q gene are characterized by comprising the following components in parts by weight:
(1) an upstream primer AF1Q-F, AF1Q-R and a Probe AF1Q-Probe for detecting the AF1Q gene,
AF1Q-F：CGAGGCACTCCCTCCATCTT
AF1Q-R：CCTTGTAGGTGGCTGTATCTGAC
AF1Q-Probe：FAM-ACGCCAGTAATTGATTGATAACAGGAAGC–BHQ；
(2) an upstream primer ABL-F, a downstream primer ABL-R and a Probe ABL-Probe for detecting the internal reference gene ABL,
ABL-F：GTGAGTGACATAGCCTCATGTTC
ABL-R：GCAGGCGTGCTCTGTGAAAT
ABL–Probe：FAM-TCAGGGAGGGTTAGGAAAACCAC-BHQ。
2. a method for detecting the relative expression level of an AF1Q gene in a sample, which is characterized by comprising the following steps:
(1) extracting total RNA in a bone marrow specimen;
(2) reverse transcribing the total RNA into cDNA;
(3) respectively amplifying the cDNA in the step (2) by using the upstream and downstream primers and probes of the AF1Q gene and the ABL gene; respectively detecting fluorescent signals;
AF1Q-F：CGAGGCACTCCCTCCATCTT
AF1Q-R：CCTTGTAGGTGGCTGTATCTGAC
AF1Q-Probe：FAM-ACGCCAGTAATTGATTGATAACAGGAAGC–BHQ；
ABL-F：GTGAGTGACATAGCCTCATGTTC
ABL-R：GCAGGCGTGCTCTGTGAAAT
ABL–Probe：FAM-TCAGGGAGGGTTAGGAAAACCAC-BHQ；
(4) and determining the relative expression amount of the AF1Q gene in the sample according to the fluorescence signal of the AF1Q gene and the fluorescence signal of the ABL reference gene.
3. The kit for detecting the relative expression quantity of the AF1Q gene comprises a detection system PCR reaction solution, and is characterized in that the detection system PCR reaction solution comprises:
(1) an upstream primer AF1Q-F, AF1Q-R and a Probe AF1Q-Probe for detecting the AF1Q gene,
AF1Q-F：CGAGGCACTCCCTCCATCTT
AF1Q-R：CCTTGTAGGTGGCTGTATCTGAC
AF1Q-Probe：FAM-ACGCCAGTAATTGATTGATAACAGGAAGC–BHQ；
(2) an upstream primer ABL-F, a downstream primer ABL-R and a Probe ABL-Probe for detecting the internal reference gene ABL,
ABL-F：GTGAGTGACATAGCCTCATGTTC
ABL-R：GCAGGCGTGCTCTGTGAAAT
ABL–Probe：FAM-TCAGGGAGGGTTAGGAAAACCAC-BHQ。
(3) a positive control, a negative control and a blank control.
4. The kit of claim 3, further comprising reagents for red blood cell lysis, Trizol, chloroform, isopropanol, absolute ethanol, DEPC water, TOYOBO reverse transcription reagent.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2167858A1 (en) * | 1995-01-23 | 1996-07-24 | Amos Cohen | Sequence of af1q cdna |
| WO2008024642A2 (en) * | 2006-08-11 | 2008-02-28 | Baylor Research Institute | Gene expression signatures in blood leukocytes permit differential diagnosis of acute infections |
| 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 |
| CN105969866A (en) * | 2016-05-20 | 2016-09-28 | 武汉艾迪康医学检验所有限公司 | Primer, probe, composition and method for screening and identifying MLL rearrangement correlated fusion genes by utilizing multi-fluorescent polymerase chain reaction (PCR) technology |
-
2019
- 2019-10-15 CN CN201910978176.7A patent/CN110656177A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2167858A1 (en) * | 1995-01-23 | 1996-07-24 | Amos Cohen | Sequence of af1q cdna |
| WO2008024642A2 (en) * | 2006-08-11 | 2008-02-28 | Baylor Research Institute | Gene expression signatures in blood leukocytes permit differential diagnosis of acute infections |
| 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 |
| CN105969866A (en) * | 2016-05-20 | 2016-09-28 | 武汉艾迪康医学检验所有限公司 | Primer, probe, composition and method for screening and identifying MLL rearrangement correlated fusion genes by utilizing multi-fluorescent polymerase chain reaction (PCR) technology |
Non-Patent Citations (2)
| Title |
|---|
| WEI LI ET AL.: ""Novel AF1q/MLLT11 favorably affects imatinib resistance and cell survival in chronic myeloid leukemia"", 《CELL DEATH AND DISEASE》 * |
| WILLIAM TSE ET AL.: ""Elevated expression of the AF1q gene, an MLL fusion partner, is an independent adverse prognostic factor in pediatric acute myeloid leukemia"", 《BLOOD》 * |
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