CN111850131B - LAMP primer group, kit and detection method for detecting PML-RARA fusion gene - Google Patents
LAMP primer group, kit and detection method for detecting PML-RARA fusion gene Download PDFInfo
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Abstract
The invention relates to an LAMP primer group, a kit and a detection method for detecting a PML-RARA fusion gene, wherein the primer group comprises an LAMP primer group for detecting a bcr1 gene, an LAMP primer group for detecting a bcr2 gene and an LAMP primer group for detecting a bcr3 gene, and the sequences of the LAMP primer groups are shown as SEQ ID NO. 1-17. The detection method comprises the following steps: and (3) taking the DNA of the sample to be detected as a template, carrying out loop-mediated isothermal amplification by adopting the LAMP primer group for detecting the bcr1 gene, the LAMP primer group for detecting the bcr2 gene and the LAMP primer group for detecting the bcr3 gene, detecting an amplified product, and judging according to a detection result. The detection method can rapidly, sensitively and specifically detect three types of PML-RARA fusion genes, is simple to operate, does not need complex instruments, and has low detection cost.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an LAMP primer group, a kit and a detection method for detecting a PML-RARA fusion gene.
Background
Acute myeloid leukemia (Acute myeloid leukemia, AML) is a class of malignant tumors caused by a blocked differentiation of hematopoietic stem cells, which is a leading group of both domestic and global morbidity and mortality. Acute promyelocytic leukemia (Acute promyelocytic leukemia, APL) is a subtype of AML, accounting for about 10% -15% of total AML patients. In recent years, after the diagnosis of APL patients is confirmed, the initial induction remission rate of more than 90% can be achieved by taking all-trans retinoic acid and arsenic trioxide medicaments in time, and more than 70% of patients can survive for a long time. It was found that about 95% of APL patients had t (15; 17) chromosomal translocation, resulting in fusion of the PML gene with the RARA gene to form a PML-RARA fusion gene. Therefore, the PML-RARA fusion gene is an ideal molecular marker for diagnosing APL diseases, evaluating curative effects and detecting the tiny residual state of leukemia.
Depending on the location of the PML gene disruption, the PML-RARA fusion gene has mainly three subtypes in APL patients, namely bcr1, bcr2 and bcr3, which are respectively called long form (L-form), variant form (V-form) and short form (S-form), and the occurrence probability in APL patients is 55%,5% and 40%, respectively.
The current method for detecting the PML-RARA fusion gene mainly comprises a fluorescence in-situ hybridization method, a fluorescence quantitative PCR method, a gene chip method, a PCR sequencing method and the like. However, the methods have the defects of equipment dependence, high cost, complicated experimental method, long time and the like.
After the Loop-mediated isothermal amplification (Loop-mediated isothermal amplification, LAMP) method has been developed in recent years, the Loop-mediated isothermal amplification (Loop-mediated isothermal amplification, LAMP) method has been widely used for identifying virus, bacteria and other species in recent years due to the characteristics of rapidness (15-60 minutes), simplicity (no special equipment), sensitivity, specificity and the like, and has made a great contribution to large-scale rapid detection of the species.
Disclosure of Invention
The invention aims to solve the defects of a method for detecting a PML-RARA fusion gene in the prior art and provides an LAMP primer group, a kit and a detection method for detecting the PML-RARA fusion gene.
Technical proposal
A LAMP primer group for detecting PML-RARA fusion gene comprises a LAMP primer group for detecting bcr1 gene, a LAMP primer group for detecting bcr2 gene and a LAMP primer group for detecting bcr3 gene,
the LAMP primer group for detecting the bcr1 gene comprises a bcr1-F3 primer, a bcr1-B3 primer, a bcr1-FIP primer, a bcr1-BIP primer, a bcr1-LF primer and a bcr1-LB primer, wherein the nucleotide sequence of the bcr1-F3 primer is shown as SEQ ID NO. 1, the nucleotide sequence of the bcr1-B3 primer is shown as SEQ ID NO. 2, the nucleotide sequence of the bcr1-FIP primer is shown as SEQ ID NO. 3, the nucleotide sequence of the bcr1-BIP primer is shown as SEQ ID NO. 4, the nucleotide sequence of the bcr1-LF primer is shown as SEQ ID NO. 5, and the nucleotide sequence of the bcr1-LB primer is shown as SEQ ID NO. 6;
the LAMP primer group for detecting the bcr2 gene comprises a bcr2-F3 primer, a bcr2-B3 primer, a bcr2-FIP primer, a bcr2-BIP primer and a bcr2-LF primer, wherein the nucleotide sequence of the bcr2-F3 primer is shown as SEQ ID NO. 7, the nucleotide sequence of the bcr2-B3 primer is shown as SEQ ID NO. 8, the nucleotide sequence of the bcr2-FIP primer is shown as SEQ ID NO. 9, the nucleotide sequence of the bcr2-BIP primer is shown as SEQ ID NO. 10, and the nucleotide sequence of the bcr2-LF primer is shown as SEQ ID NO. 11;
the LAMP primer group for detecting the bcr3 gene comprises a bcr3-F3 primer, a bcr3-B3 primer, a bcr3-FIP primer, a bcr3-BIP primer, a bcr3-LF primer and a bcr3-LB primer, wherein the nucleotide sequence of the bcr3-F3 primer is shown as SEQ ID NO. 12, the nucleotide sequence of the bcr3-B3 primer is shown as SEQ ID NO. 13, the nucleotide sequence of the bcr3-FIP primer is shown as SEQ ID NO. 14, the nucleotide sequence of the bcr3-BIP primer is shown as SEQ ID NO. 165, the nucleotide sequence of the bcr3-LF primer is shown as SEQ ID NO. 16, and the nucleotide sequence of the bcr3-LB primer is shown as SEQ ID NO. 17.
A kit for detecting a PML-RARA fusion gene comprises the LAMP primer group for detecting the bcr1 gene, the LAMP primer group for detecting the bcr2 gene and the LAMP primer group for detecting the bcr3 gene.
Further, the kit also comprises a 2x reaction buffer, a fluorescent visual detection solution and Bst DNA polymerase.
A method for detecting a PML-RARA fusion gene: using the sample DNA to be detected as a template, carrying out loop-mediated isothermal amplification by using the LAMP primer group for detecting bcr1 genes, the LAMP primer group for detecting bcr2 genes and the LAMP primer group for detecting bcr3 genes according to claim 1 to obtain amplification products, detecting the amplification products, and judging according to the detection results.
Further, the reaction system for detecting bcr1 gene is:
the reaction system for detecting bcr2 gene is:
the reaction system for detecting bcr3 gene is:
further, the reaction conditions of the loop-mediated isothermal amplification are as follows: the reaction was carried out at a constant temperature of 63℃for 30min.
The invention has the beneficial effects that: the invention provides an LAMP primer group and a kit for detecting PML-RARA fusion genes, which can rapidly (15-60 min), sensitively and specifically detect three types of PML-RARA fusion genes, and have the advantages of simple operation, no need of complex instruments and low detection cost.
Drawings
FIG. 1 shows the detection results of PML-RARA fusion gene sequence PUC57 plasmids with different copy numbers;
FIG. 2 shows the result of specific detection of PML-RARA fusion gene.
Detailed Description
The technical scheme of the invention is further described below with reference to the attached drawings and specific embodiments.
Example 1
The specific sequence near the fusion site of bcr1 (the nucleotide sequence is shown as SEQ ID NO: 18), bcr2 (the nucleotide sequence is shown as SEQ ID NO: 19) and bcr3 (the nucleotide sequence is shown as SEQ ID NO: 20) is utilized to design an LAMP primer group for detecting the PML-RARA fusion gene, wherein the LAMP primer group comprises an LAMP primer group for detecting the bcr1 gene, an LAMP primer group for detecting the bcr2 gene and an LAMP primer group for detecting the bcr3 gene, 17 LAMP primers are designed in total, and the primer sequences are shown in the table 1. The primer sequence is sent to the Kirschner Co., ltd for synthesis, and diluted to 10uM or 20uM for standby.
TABLE 1 primer sequences
Example 2
A method for detecting a PML-RARA fusion gene: the loop-mediated isothermal amplification was carried out by using PML-RARA fusion genes bcr1, bcr2 and bcr3 sequence plasmids (synthesized and constructed by Jiangsu Style biological company) constructed on a PUC57 vector as templates, and by using the LAMP primer group for detecting bcr1 gene, the LAMP primer group for detecting bcr2 gene and the LAMP primer group for detecting bcr3 gene described in Table 1 of example 1, wherein the reaction system is as follows:
reaction system for detecting bcr1 gene subtype:
reaction system for detecting bcr2 gene subtype:
reaction system for detecting bcr3 gene subtype:
in the reaction system, bstDNA polymerase and a matched reaction buffer solution are prepared from the fluorescent visual detection test solution which is a product of Rongsheng biotechnology Co.
The reaction conditions are as follows: the reaction is carried out for 30min at the constant temperature of 63 ℃, and the enzyme inactivation treatment is carried out for 2min in boiling water, thus stopping the reaction.
Example 3
Sensitivity experiment:
the PML-RARA fusion genes bcr1, bcr2, bcr3 (constructed by the synthesis of Jiangsu srue biosystems) constructed on the PUC57 vector were used as templates, diluted 10-fold with ultrapure water at an initial concentration of 10copy/uL to 4 concentration gradients: 10 2 copy/uL，10 3 copy/uL，10 4 copy/uL，10 5 copy/uL, water was used as negative control instead of template, and the following steps were performed:
(1) According to the embodiment2, sequentially adding samples into the sterilized PCR tubes from top to bottom in the reaction system, wherein the template concentration of each PCR tube is 0, 10copy/uL and 10 respectively 2 copy/uL，10 3 copy/uL，10 4 copy/uL，10 5 copy/uL；
(2) Lightly flicking the bottom of the test tube for 20 times to fully mix the test tube, putting the test tube into a centrifugal machine to centrifuge for a plurality of seconds, and sinking the reaction liquid to the bottom;
(3) Placing the prepared reaction test tube into a water bath kettle, reacting for 30min at the constant temperature of 63 ℃ for 5min, flicking the bottom of the test tube by a finger, and then placing the test tube back into the water bath kettle for continuous reaction;
(4) Transferring the reaction test tube into boiling water for 2min for enzyme inactivation treatment, and stopping the reaction;
(5) And taking out the reaction tube, and placing the reaction tube under white light for visual observation, or using an ultraviolet irradiation device to irradiate the bottom of the reaction tube for color observation, wherein green fluorescence is a positive result.
FIG. 1 shows the detection results of the PML-RARA fusion gene sequence PUC57 plasmids with different copy numbers, and as can be seen from FIG. 1, when the PML-RARA fusion gene sequence PUC57 plasmid is 10copy/uL, the loop-mediated isothermal amplification reaction can still be carried out, and a clear reaction effect can be observed visually under white light.
Example 4
Specificity experiments
The plasmid of the sequence of the PML-RARA fusion genes bcr1, bcr2 and bcr3 (synthesized and constructed by Jiangsu Style biological company) constructed on the PUC57 vector is used as a template, and the concentration is 10 2 copy/uL was detected by loop-mediated isothermal amplification using the reaction system of example 2, and simultaneously, as negative controls, water, human genomic DNA (genomic DNA was extracted from human embryonic kidney upper batch 293T cell line) and PUC57 empty plasmid were used as the replacement templates, respectively, and the detection was performed in the same manner as in example 3, and the detection results are shown in fig. 2.
FIG. 2 shows the result of specific detection of PML-RARA fusion gene. As can be seen, the negative control groups, each with water, human genomic DNA and the empty plasmid of PUC57 as templates, were identical in color and at a concentration of 10 2 copy/uL PML-RARA fusion base constructed on PUC57 vectorThe experimental group with bcr1, bcr2, bcr3 sequence plasmid as template exhibited a clear color difference.
Sequence listing
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Claims (6)
1. A LAMP primer group for detecting PML-RARA fusion genes is characterized by comprising a LAMP primer group for detecting bcr1 genes, a LAMP primer group for detecting bcr2 genes and a LAMP primer group for detecting bcr3 genes,
the LAMP primer group for detecting the bcr1 gene comprises a bcr1-F3 primer, a bcr1-B3 primer, a bcr1-FIP primer, a bcr1-BIP primer, a bcr1-LF primer and a bcr1-LB primer, wherein the nucleotide sequence of the bcr1-F3 primer is shown as SEQ ID NO. 1, the nucleotide sequence of the bcr1-B3 primer is shown as SEQ ID NO. 2, the nucleotide sequence of the bcr1-FIP primer is shown as SEQ ID NO. 3, the nucleotide sequence of the bcr1-BIP primer is shown as SEQ ID NO. 4, the nucleotide sequence of the bcr1-LF primer is shown as SEQ ID NO. 5, and the nucleotide sequence of the bcr1-LB primer is shown as SEQ ID NO. 6;
the LAMP primer group for detecting the bcr2 gene comprises a bcr2-F3 primer, a bcr2-B3 primer, a bcr2-FIP primer, a bcr2-BIP primer and a bcr2-LF primer, wherein the nucleotide sequence of the bcr2-F3 primer is shown as SEQ ID NO. 7, the nucleotide sequence of the bcr2-B3 primer is shown as SEQ ID NO. 8, the nucleotide sequence of the bcr2-FIP primer is shown as SEQ ID NO. 9, the nucleotide sequence of the bcr2-BIP primer is shown as SEQ ID NO. 10, and the nucleotide sequence of the bcr2-LF primer is shown as SEQ ID NO. 11;
the LAMP primer group for detecting the bcr3 gene comprises a bcr3-F3 primer, a bcr3-B3 primer, a bcr3-FIP primer, a bcr3-BIP primer, a bcr3-LF primer and a bcr3-LB primer, wherein the nucleotide sequence of the bcr3-F3 primer is shown as SEQ ID NO. 12, the nucleotide sequence of the bcr3-B3 primer is shown as SEQ ID NO. 13, the nucleotide sequence of the bcr3-FIP primer is shown as SEQ ID NO. 14, the nucleotide sequence of the bcr3-BIP primer is shown as SEQ ID NO. 165, the nucleotide sequence of the bcr3-LF primer is shown as SEQ ID NO. 16, and the nucleotide sequence of the bcr3-LB primer is shown as SEQ ID NO. 17.
2. A kit for detecting a PML-RARA fusion gene is characterized by comprising the LAMP primer group for detecting the bcr1 gene, the LAMP primer group for detecting the bcr2 gene and the LAMP primer group for detecting the bcr3 gene according to claim 1.
3. The kit for detecting a PML-RARA fusion gene according to claim 2, wherein said kit further comprises a 2x reaction buffer, a fluorescent visual detection solution, and Bst DNA polymerase.
4. The kit for detecting the PML-RARA fusion gene according to claim 2, wherein the amplification product is obtained by loop-mediated isothermal amplification by using the sample DNA to be detected as a template and by using the LAMP primer group for detecting the bcr1 gene, the LAMP primer group for detecting the bcr2 gene and the LAMP primer group for detecting the bcr3 gene according to claim 1, and the amplification product is detected and judged according to the detection result.
5. The kit for detecting a PML-RARA fusion gene according to claim 4, wherein the reaction system for detecting the bcr1 gene is as follows:
the reaction system for detecting bcr2 gene is:
the reaction system for detecting bcr3 gene is:
6. the kit for detecting a PML-RARA fusion gene according to claim 4 or 5, wherein the reaction conditions for loop-mediated isothermal amplification are: the reaction was carried out at a constant temperature of 63℃for 30min.
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| PCT/CN2020/122786 WO2022041424A1 (en) | 2020-08-26 | 2020-10-22 | Lamp primer group, kit and detection method for detecting pml-rara fusion gene |
| LU500408A LU500408B1 (en) | 2020-08-26 | 2020-10-22 | A LAMP primer set, a kit and a detection method for detecting PML-RARA fusion gene |
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| CN104328211A (en) * | 2014-11-24 | 2015-02-04 | 山东省医学科学院基础医学研究所 | Primers and kit used in loop-mediated isothermal amplification detection method of PML-RAR alpha fusion gene |
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| CN104561331B (en) * | 2015-01-21 | 2016-08-31 | 苏州云泰生物医药科技有限公司 | A kind of primer detecting leukaemia correlation fusion gene and probe and kit thereof |
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| LU500408B1 (en) | 2022-01-10 |
| CN111850131A (en) | 2020-10-30 |
| WO2022041424A1 (en) | 2022-03-03 |
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