CN115992228A - Primer and probe for detecting fusion gene related to acute myelogenous leukemia, application of primer and probe and kit - Google Patents

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

Primer and probe for detecting fusion gene related to acute myelogenous leukemia, application of primer and probe and kit

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.