CN115992228A - Primer and probe for detecting fusion gene related to acute myelogenous leukemia, application of primer and probe and kit - Google Patents
Primer and probe for detecting fusion gene related to acute myelogenous leukemia, application of primer and probe and kit Download PDFInfo
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- CN115992228A CN115992228A CN202210846391.3A CN202210846391A CN115992228A CN 115992228 A CN115992228 A CN 115992228A CN 202210846391 A CN202210846391 A CN 202210846391A CN 115992228 A CN115992228 A CN 115992228A
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- 239000000523 sample Substances 0.000 title claims abstract description 52
- 230000004927 fusion Effects 0.000 title claims abstract description 45
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 28
- 208000031261 Acute myeloid leukaemia Diseases 0.000 title claims abstract description 21
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 title claims abstract description 18
- 101710205841 Ribonuclease P protein component 3 Proteins 0.000 claims abstract description 5
- 239000002773 nucleotide Substances 0.000 claims description 14
- 125000003729 nucleotide group Chemical group 0.000 claims description 14
- 238000010791 quenching Methods 0.000 claims description 9
- 230000000171 quenching effect Effects 0.000 claims description 9
- 238000011144 upstream manufacturing Methods 0.000 claims description 6
- 238000010839 reverse transcription Methods 0.000 claims description 4
- 125000006853 reporter group Chemical group 0.000 claims description 3
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 239000008223 sterile water Substances 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 239000002299 complementary DNA Substances 0.000 abstract description 3
- 239000012634 fragment Substances 0.000 abstract description 3
- 238000001514 detection method Methods 0.000 description 15
- 238000005516 engineering process Methods 0.000 description 8
- 238000010802 RNA extraction kit Methods 0.000 description 4
- 239000013614 RNA sample Substances 0.000 description 4
- 238000007847 digital PCR Methods 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 230000003321 amplification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 150000007523 nucleic acids Chemical group 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 206010000830 Acute leukaemia Diseases 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to the technical field of biology, in particular to a primer and a probe for detecting fusion genes related to acute myelogenous leukemia, application of the primer and the probe and a kit. According to the invention, primer probes of corresponding sites are designed according to CBFB-MYH11 fusion (subtype A and subtype D) and AML1-ETO fusion sequences and cDNA fragments of endogenous internal reference RPP30 genes, and CBFB-MYH11 fusion (subtype A and subtype D) and AML1-ETO fusion in acute myeloid leukemia can be detected simultaneously, and a single chip can give qualitative and quantitative results simultaneously, so that the kit has good specificity.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a primer and a probe for detecting fusion genes related to acute myelogenous leukemia, application of the primer and the probe and a kit.
Background
Acute myeloid leukemia (acute myelogenous leukemia, AML) is the most common acute leukemia in adults. At present, the treatment of the disease is mainly chemotherapy, but about 70% of patients who obtain remission finally relapse and evolve into refractory leukemia, and the treatment fails to die.
The current research reports that the pathogenesis of AML is related to the formation of fusion genes from related genes. The rapid and accurate detection of different AML related fusion genes is performed simultaneously, which is helpful for providing a targeted treatment scheme for patients in time and improving the treatment efficiency of the diseases. The existing detection method mainly adopts a fluorescence quantitative PCR technology or a Fish technology. The fluorescent quantitative PCR technology is easier to qualitative, but involves quantitative, and a series of standard curves comprising internal reference and detection targets are often needed to be made, so that the operation is complicated. Technical traditional FISH detection has higher requirements on operation and interpretation technologies, and the detection cost is high and the time consumption is long.
The digital PCR technology is a third generation PCR technology developed on the qPCR technology, can theoretically realize the amplification detection of target nucleic acid fragments with single copy, and is the nucleic acid detection technology with highest sensitivity at present. However, there is currently no report of the application of digital PCR to the simultaneous detection of CBFB-MYH11 fusions (subtype A and subtype D) and AML1-ETO fusions.
Disclosure of Invention
In view of the above, the invention provides a primer and a probe for detecting fusion genes related to acute myelogenous leukemia, application thereof and a kit. The kit can simultaneously detect CBFB-MYH11 fusion (subtype A and subtype D) and AML1-ETO fusion in acute myeloid leukemia, and a single chip can simultaneously give qualitative and quantitative results with good specificity.
In order to achieve the above object, the present invention provides the following technical solutions:
the primer and the probe are characterized by comprising the following primer and probe for detecting CBFB-MYH11 fusion genes and AML1-ETO fusion genes;
primer and probe for detecting CBFB-MYH11 fusion gene: an upstream primer shown in SEQ ID NO.1, downstream primers shown in SEQ ID NO. 2-3 and probes shown in SEQ ID NO. 4-5;
primers and probes for detecting AML1-ETO fusion gene: an upstream primer shown in SEQ ID NO.6, a downstream primer shown in SEQ ID NO.7 and a probe shown in SEQ ID NO. 8.
In the invention, a fluorescent reporter group is marked at the 5 'end of the probe, and a fluorescence quenching group is marked at the 3' end of the probe. Wherein:
the 5 'end of the probe with the nucleotide sequence shown in SEQ ID NO.4 is marked with a fluorescence report group FAM, and the 3' end is marked with a fluorescence quenching group BHQ1;
the 5 'end of the probe with the nucleotide sequence shown in SEQ ID NO. 5 is marked with a fluorescence report group VIC, and the 3' end is marked with a fluorescence quenching group BHQ1;
the 5 'end of the probe with the nucleotide sequence shown in SEQ ID NO.8 is marked with a fluorescence report group CY5, and the 3' end is marked with a fluorescence quenching group BHQ2.
The invention also provides application of the primer and the probe in preparation of a kit for detecting fusion genes related to acute myelogenous leukemia.
The fusion genes related to the acute myeloid leukemia comprise subtype A and subtype D of CBFB-MYH11 fusion genes and AML1-ETO fusion genes.
The invention also provides a kit for detecting the fusion gene related to the acute myelogenous leukemia, which comprises the primer and the probe.
The kit provided by the invention further comprises a primer and a probe for detecting the reference gene RPP 30;
the nucleotide sequence of the upstream primer for detecting the reference gene RPP30 is shown as SEQ ID NO.9, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.10, and the nucleotide sequence of the probe is shown as SEQ ID NO. 11.
The 5 'end of the probe with the nucleotide sequence shown in SEQ ID NO.11 is marked with a fluorescent reporter group A425, and the 3' end is marked with a fluorescent quenching group BHQ1.
The kit provided by the invention further comprises sterile water, ddPCRMix and a reverse transcription reagent.
The invention also provides a method for detecting fusion genes related to acute myelogenous leukemia, and the primer probe or the kit is used for detecting a sample to be detected.
In the invention, the sample to be measured is a blood sample.
In the present invention, the reverse transcription program includes: 42 ℃ for 1h and 70 ℃ for 15min.
In the present invention, the PCR amplification procedure comprises: 98℃for 5min, (98℃for 15s,60℃for 60 s). Times.40.
According to the invention, primer probes of corresponding sites are designed according to CBFB-MYH11 fusion (subtype A and subtype D) and AML1-ETO fusion sequences and cDNA fragments of endogenous internal reference RPP30 genes, and CBFB-MYH11 fusion (subtype A and subtype D) and AML1-ETO fusion in acute myeloid leukemia can be detected simultaneously, and a single chip can give qualitative and quantitative results simultaneously, so that the kit has good specificity.
Detailed Description
The invention provides a primer and a probe for detecting fusion genes related to acute myeloid leukemia, application thereof and a kit. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1
1. Primer probe
1.1 designing specific primer sequences of corresponding sites according to CBFB-MYH11 fusion (subtype A and subtype D), AML1-ETO fusion sequences and cDNA fragments of endogenous reference RPP30 genes as shown in Table 1.
TABLE 1 primer probe combination sequences
2. Sample of
2.1 CBFB-MYH11-A subtype fusion RNA sample: RNA was extracted from clinical samples using RNA extraction kit.
2.2 CBFB-MYH11-D subtype fusion RNA sample: RNA was extracted from clinical samples using RNA extraction kit.
2.3 AML1-ETO fusion RNA samples: RNA was extracted from clinical samples using RNA extraction kit.
2.4 negative RNA samples: RNA was extracted from clinical samples using RNA extraction kit.
3. RNA reverse transcription
TABLE 2 reverse transcription system
4. Detection method
4.1 digital PCR amplification System
TABLE 3 Table 3
Example 2 specificity detection assay
The following samples were subjected to digital PCR detection according to the primer probe and detection method of example 1, and the results are shown in Table 4.
TABLE 4 Table 4
The result shows that the primer probe has good specificity, and the NTC group is detected without non-specific amplification; detecting normal RNA, and detecting A425 signals of endogenous internal references only; the detection of CBFB-MYH11-A subtype samples can detect FAM and A425 signals at the same time, and the ratio is stable; the detection of CBFB-MYH11-D subtype samples can detect VIC and A425 signals at the same time, and the ratio is stable; the detection of AML1-ETO fusion samples can detect CY5 and A425 signals simultaneously, and the ratio is stable.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. The primer and the probe are characterized by comprising the following primer and probe for detecting CBFB-MYH11 fusion genes and AML1-ETO fusion genes;
primer and probe for detecting CBFB-MYH11 fusion gene: an upstream primer shown in SEQ ID NO.1, downstream primers shown in SEQ ID NO. 2-3 and probes shown in SEQ ID NO. 4-5;
primers and probes for detecting AML1-ETO fusion gene: an upstream primer shown in SEQ ID NO.6, a downstream primer shown in SEQ ID NO.7 and a probe shown in SEQ ID NO. 8.
2. The primer and probe according to claim 1, wherein,
the 5 'end of the probe with the nucleotide sequence shown in SEQ ID NO.4 is marked with a fluorescence report group FAM, and the 3' end is marked with a fluorescence quenching group BHQ1;
the 5 'end of the probe with the nucleotide sequence shown in SEQ ID NO. 5 is marked with a fluorescence report group VIC, and the 3' end is marked with a fluorescence quenching group BHQ1;
the 5 'end of the probe with the nucleotide sequence shown in SEQ ID NO.8 is marked with a fluorescence report group CY5, and the 3' end is marked with a fluorescence quenching group BHQ2.
3. Use of the primer and probe according to claim 1 or 2 for preparing a kit for detecting fusion genes related to acute myeloid leukemia.
4. The use according to claim 3, wherein said acute myeloid leukemia related fusion genes comprise subtype a and subtype D of CBFB-MYH11 fusion gene and AML1-ETO fusion gene.
5. A kit for detecting a fusion gene associated with acute myeloid leukemia, comprising the primer and probe of claim 1 or 2.
6. The kit of claim 5, further comprising primers and probes for detecting the reference gene RPP 30;
the nucleotide sequence of the upstream primer for detecting the reference gene RPP30 is shown as SEQ ID NO.9, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.10, and the nucleotide sequence of the probe is shown as SEQ ID NO. 11.
7. The kit according to claim 6, wherein the probe of the nucleotide sequence shown in SEQ ID NO.11 is labeled with a fluorescent reporter group A425 at the 5 'end and a fluorescent quenching group BHQ1 at the 3' end.
8. The kit of claim 5, further comprising sterile water, ddPCRMix, and a reverse transcription reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210846391.3A CN115992228A (en) | 2022-07-19 | 2022-07-19 | Primer and probe for detecting fusion gene related to acute myelogenous leukemia, application of primer and probe and kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210846391.3A CN115992228A (en) | 2022-07-19 | 2022-07-19 | Primer and probe for detecting fusion gene related to acute myelogenous leukemia, application of primer and probe and kit |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101838682A (en) * | 2009-03-20 | 2010-09-22 | 江苏迈迪基因生物科技有限公司 | Leukemia fusion gene combined parallel detecting method and diagnostic reagent kit |
| CN103805698A (en) * | 2012-05-30 | 2014-05-21 | 北京艾迪康医学检验所有限公司 | Primer and method for detecting relative expression quantity of AML1-ETO (acutemyeloblastic leukemia one gene-eight twenty-one gene) fusion gene |
| CN104313160A (en) * | 2014-09-23 | 2015-01-28 | 邵棠 | Primer pair for simultaneously detecting CBFB-MYH11 and AML1-ETO fusion genes with pyrosequencing method and kit |
| CN104561331A (en) * | 2015-01-21 | 2015-04-29 | 苏州云泰生物医药科技有限公司 | Primer and probe for detecting leukemia-related fusion gene and kit of primer |
| CN106544437A (en) * | 2016-11-25 | 2017-03-29 | 徐州医科大学 | A kind of multiple fluorescence PCR test kit and method of detection leukemia fusion gene |
| CN109402259A (en) * | 2018-11-13 | 2019-03-01 | 中山大学达安基因股份有限公司 | A kind of kit detecting leukemia fusion gene and gene mutation |
| CN114592064A (en) * | 2022-04-01 | 2022-06-07 | 领航基因科技（杭州）有限公司 | Digital PCR kit for detecting multiple leukemia fusion genes |
-
2022
- 2022-07-19 CN CN202210846391.3A patent/CN115992228A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101838682A (en) * | 2009-03-20 | 2010-09-22 | 江苏迈迪基因生物科技有限公司 | Leukemia fusion gene combined parallel detecting method and diagnostic reagent kit |
| CN103805698A (en) * | 2012-05-30 | 2014-05-21 | 北京艾迪康医学检验所有限公司 | Primer and method for detecting relative expression quantity of AML1-ETO (acutemyeloblastic leukemia one gene-eight twenty-one gene) fusion gene |
| CN104313160A (en) * | 2014-09-23 | 2015-01-28 | 邵棠 | Primer pair for simultaneously detecting CBFB-MYH11 and AML1-ETO fusion genes with pyrosequencing method and kit |
| CN104561331A (en) * | 2015-01-21 | 2015-04-29 | 苏州云泰生物医药科技有限公司 | Primer and probe for detecting leukemia-related fusion gene and kit of primer |
| CN106544437A (en) * | 2016-11-25 | 2017-03-29 | 徐州医科大学 | A kind of multiple fluorescence PCR test kit and method of detection leukemia fusion gene |
| CN109402259A (en) * | 2018-11-13 | 2019-03-01 | 中山大学达安基因股份有限公司 | A kind of kit detecting leukemia fusion gene and gene mutation |
| CN114592064A (en) * | 2022-04-01 | 2022-06-07 | 领航基因科技（杭州）有限公司 | Digital PCR kit for detecting multiple leukemia fusion genes |
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