CN107083417B

CN107083417B - AML1/ETO fusion gene detection kit and detection method

Info

Publication number: CN107083417B
Application number: CN201610087705.0A
Authority: CN
Inventors: 朱蓉; 吴诗扬; 廖传荣
Original assignee: Surexam Bio Tech Co Ltd
Current assignee: Surexam Bio Tech Co Ltd
Priority date: 2016-02-16
Filing date: 2016-02-16
Publication date: 2020-01-31
Anticipated expiration: 2036-02-16
Also published as: CN107083417A

Abstract

The invention discloses an AML1/ETO fusion gene detection kit, which is characterized by comprising a th group of probes for AML1 gene breakage and a second group of probes for ETO gene breakage, wherein the two groups of probes are marked with dyes, the colors of the dyes are the same as those of the group of probes, and the colors of the dyes of the different groups of probes are different, the two groups of probes are amplification products obtained by respectively using human genome DNA as templates and amplifying primers.

Description

AML1/ETO fusion gene detection kit and detection method

Technical Field

The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and particularly relates to AML1/ETO fusion gene detection kits and detection methods.

Background

Acute Myeloid Leukemia (AML), which is mainly characterized by malignant proliferation of myeloid progenitor cells, has two subtypes: the undifferentiated type of granulocytic leukemia (M1) and the partially differentiated type of granulocytic leukemia (M2). The literature reports that l 2% -20% of Acute Myeloid Leukemia (AML) have t (8; 21) (q 22; q22) chromosomal translocations, especially in AML-M₂The incidence rate can reach 40-80%.

AML1 gene located in chromosome 21q22 is fused with ETO gene of 8q22 to generate AML1/ETO and ETO/AML1 fusion genes, generally considers that the expression of the ETO/AML1 fusion gene is extremely low or unstable due to degradation, so that the AML1-ETO fusion gene is mostly studied at the present stage, AML1 gene is also called RUNX1 gene, contains 12 exons, has a full length of more than 260kb, has a breaking point of 80-90% located in intron 1, and the rest occurs in intron 2. AML1/ETO fusion protein is transcription inhibitor, can inhibit normal AML1 protein-mediated function, change the self-renewal and maturation process of hematopoietic progenitor cells, and also generates a signal for starting abnormal cell proliferation, causes leukemia cell growth, t (8; 21) (q 22; q 2) is good in adolescence and children (5% -10%), is mainly related to M2 type 2, and is smaller and the occurrence of prognosis is less, and the fusion gene is a clinically significant for the patients who are diagnosed as a positive or as a patient who has a relapse of AML 638-positive or a patient who has a relapse, a clinically-positive prognosis, a patient who is likely to have a relapse rate of AML 638-positive relapse, a patient who is closely detected by a patient who has a short-positive chemotherapy-negative gene, a patient who has a relapse rate after AML-positive patient who has a relapse rate after a patient who has a relapse rate of a patient who.

The current AML1/ETO fusion gene detection method mainly comprises 3 methods of cytogenetic detection, detection based on a PCR method and FISH detection, wherein the cytogenetic detection needs to culture cells, is long in time consumption, can only detect cells in the middle period, and is low in success rate and sensitivity, PCR-based detection such as RT-PCR, Q-PCR and the like is easy to pollute, high in false positive rate, can only detect known breaking points, and can not detect AML1/ETO fusion genes which are unknown or contain a small amount of transcripts, FISH detection has extremely high sensitivity and capability of detecting hidden translocation, the false positive rate of the FISH detection is far lower than that of PCR detection, can provide more reliable molecular genetic evidence relative to karyotype analysis, can be used for monitoring long-term prognosis, medication effect, tiny lesion residue and effect after transplantation, although the current FISH detection method obtains application and improvement of , but has the following defects that (1) the FISH detection method has more time-consuming sequences in BAC template preparation, has influence on tiny lesion residue, and the detection effect, and the detection of fluorescence specificity after transplantation, can not only does not only need to be repeated detection, but also can ensure the detection of FISH detection, the fluorescence detection of a probe is suitable for the detection, and the detection of a few of FISH detection probe not only can be obtained by the detection method, but also can be obtained by the detection method, so that the detection method, the detection of the fluorescence detection of AML 1/ET.

Disclosure of Invention

The invention aims to provide AML1/ETO fusion gene detection kits with strong specificity, high sensitivity and high detection efficiency.

The technical scheme for achieving the purpose is as follows.

AML1/ETO fusion gene detection kit, comprising a group of probes aiming at AML1 gene breakage and a second group of probes aiming at ETO gene breakage, wherein the two groups of probes are marked with dyes, the colors of the dyes of the same group of probes are the same, and the colors of the dyes of the different groups of probes are different, the two groups of probes are amplification products obtained by respectively using human genome DNA as templates and amplifying primers;

the amplification primers for the set probes are at least pairs selected from the group consisting of SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, and at least pairs selected from the group consisting of SEQ ID NO.21 and SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.30, SEQ ID NO.31 and SEQ ID NO.32, SEQ ID NO.25 and SEQ ID NO.33, SEQ ID NO.36, SEQ ID NO.38 and SEQ ID NO.38, and pairs of SEQ ID NO.38, SEQ ID NO.35 and SEQ ID NO. 38;

the amplification primers for the second set of probes are at least pairs selected from SEQ ID NO.41 and SEQ ID NO.42, SEQ ID NO.43 and SEQ ID NO.44, SEQ ID NO.45 and SEQ ID NO.46, SEQ ID NO.47 and SEQ ID NO.48, SEQ ID NO.49 and SEQ ID NO.50, SEQ ID NO.51 and SEQ ID NO.52, SEQ ID NO.53 and SEQ ID NO.54, SEQ ID NO.55 and SEQ ID NO.56, SEQ ID NO.57 and SEQ ID NO.58, SEQ ID NO.59 and SEQ ID NO.60, and at least 3579 pairs selected from SEQ ID NO.61 and SEQ ID NO.62, SEQ ID NO.63 and SEQ ID NO.64, SEQ ID NO.65 and SEQ ID NO.66, SEQ ID NO.67 and SEQ ID NO.68, SEQ ID NO.69 and SEQ ID NO.70, SEQ ID NO.71 and SEQ ID NO.72, SEQ ID NO.74 and SEQ ID NO.73, SEQ ID NO.76 and SEQ ID NO.75, SEQ ID NO.76 and SEQ ID NO.76, SEQ ID NO.75 and SEQ ID NO. 80.

In of these examples, the size of the resulting amplification product was 100-500 bp.

In embodiments in which amplification primers for the set of probes are selected from at least four of the above-described pairs of primers, e.g., at least two pairs selected from the group consisting of SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, and at least two pairs selected from the group consisting of SEQ ID NO.21 and SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.30, SEQ ID NO.23 and SEQ ID NO.35, SEQ ID NO.76 and SEQ ID NO.48, SEQ ID NO.48 and SEQ ID NO.48, SEQ ID NO.48 and SEQ ID NO.48, SEQ ID NO.48 and SEQ ID NO.48, SEQ ID NO.48 and SEQ ID NO.48, SEQ ID NO.48 and SEQ ID NO.48, SEQ ID NO.48 and SEQ ID.

In of these embodiments, the amplification primers for the set of probes are selected from at least eight of the primer pairs described above and the amplification primers for the second set of probes are selected from at least eight of the primer pairs described above.

In of these examples, the fluorochrome is selected from the group consisting of FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas Red, LC RED640, Cy5, LC RED705, Alexa Fluor 488, and Alexa Fluor 750, and the fluorochromes for the different probe sets are different from each other.

Another objective of the invention is to provide AML1/ETO fusion gene detection methods.

The technical scheme for achieving the purpose is as follows.

AML1/ETO fusion gene detection method, comprising:

(4) pretreating and flaking a sample to be detected;

(5) carrying out hybridization detection by using the AML1/ETO fusion gene detection kit;

(6) the fluorescent signals were observed under a fluorescent microscope, and different fluorescent signals were observed and counted.

In cases, the hybridization assay using the AML1/ETO fusion gene assay kit described above, included the following steps:

(2.1) probe equilibration to room temperature; preparing a section sample with a hybridization region;

(2.2) Pre-denaturation: placing slices in 70%, 85% and 100% ethanol in sequence for dehydration; drying;

(2.3) hybridization: adding a probe solution into a hybridization area of the sliced sample to perform hybridization reaction;

(2.4) washing;

and (2.5) DAPI counterstaining.

In examples, the hybridization reaction conditions in step (2.3) were 42. + -. 1 ℃ for 4. + -. 0.5 h.

The main advantages of the invention are:

(1) the FISH probe is prepared by designing primer amplification according to a non-repetitive and highly conserved sequence after sequence comparison by the inventor, and has strong specificity and lower background noise.

(2) The length of the FISH probe is the optimal length obtained by comparing and statistically analyzing the experimental results through a large number of experiments, the optimal balance between the detection specificity and the hybridization time can be achieved, the result specificity and the sensitivity can be ensured, the hybridization time can be shortened, the detection can be completed within 7-8 hours, and the detection efficiency is improved.

(3) The conventional FISH probe labeling method cannot amplify a fluorescent signal, so that a longer probe is required to ensure the detection sensitivity, thereby resulting in an excessively long hybridization time. The invention adopts a new probe marking method, can amplify the fluorescent signal on the probe and greatly improve the detection sensitivity. By optimizing the probe marking method for signal amplification and the length of the probe, the invention greatly shortens the time required by the full hybridization of the probe and the target gene and simultaneously improves the detection sensitivity and the detection efficiency.

Drawings

FIG. 1 is a signal counting guideline of the invention-typical positive cell signal pattern;

FIG. 2 is a signal counting guideline of the invention-typical negative cell signal pattern.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.

The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention, the terminology used herein "and/or" includes any and all combinations of or more of the associated listed items.

EXAMPLE 1 kit composition

The AML1/ETO fusion gene detection kit of this embodiment mainly includes:

fluorescent dye-labeled probe

The probes comprise a group of probes for AML1 gene breakage and a second group of probes for ETO gene breakage, wherein the probes are obtained by taking human genome DNA as a template and utilizing 40 pairs of specific primer pairs to carry out PCR amplification reaction, and the preparation method comprises the following steps:

1. design of amplification primers

The AML1/ETO fusion gene is formed by mutual translocation of AML1 gene positioned at 21q22 and ETO gene positioned at 8q22, the preparation of the probe of the invention is respectively aimed at the upstream and downstream sequences of AML1 gene breaking point and the upstream and downstream sequences of ETO gene, in order to realize detection, 2 groups of amplification primers are respectively designed, namely th group of amplification primers aiming at the upstream and downstream gene sequences of AML1 gene breaking point and second group of amplification primers aiming at the upstream and downstream gene sequences of ETO gene, the amplification regions of the amplification primer groups are respectively non-repetitive and highly conserved fragments in upstream and downstream target detection regions of AML1 and ETO gene breaking point, the fragment length is more than 1000bp, the corresponding amplification product length of the amplification primers is between 100 bp and 500bp, a th group and a second group of probe library are respectively formed aiming at the upstream and downstream amplification products of each gene, the sequence information of the amplification primers and the corresponding amplification products are shown in Table 1 (note: 1F/1R is an pair of primers, respectively indicating forward primer and a reverse primer, and other similar primers, and the like).

TABLE 1 amplification primers and amplification products

The primer sequences are synthesized by Shanghai Biotechnology engineering services, Inc., each synthesized sequence is prepared into 100pmol/mL stock solution by using 10mmol/L Tris Buffer, and the stock solution is marked.

2. Probe library construction

The PCR amplification is carried out by using the designed primer pair and human genome DNA as a template, and corresponding amplification products respectively form th group and a second group of probe libraries.

(1) Human genome DNA extraction: according to the prior art, the procedures can be performed with reference to molecular cloning experiments, third edition (scientific Press) or according to the instructions of commercial human genome DNA extraction kit products.

(2) Preparing PCR primer working solution, dividing corresponding amplification primers (1F/1R-20F/20R) into 4 amplification primer groups aiming at groups of probe libraries, carrying out progressive amplification on 2 non-repetitive and highly-conserved regions at the upstream and 2 non-repetitive and highly-conserved regions at the downstream of AML1 gene fragmentation site, respectively taking 50ul of corresponding amplification primers (1F/1R-5F/5R, 6F/6R-10F/10R, 11F/11R-15F/15R, 16F/16R-20F/20R) stock solution and placing the 50ul in 4 centrifuge tubes of 1.5mL, using 10mmol/LTris Buffer to prepare 4 amplification primer working solution with final concentration of 10pmol/mL respectively, correspondingly marking, aiming at a second group of probe libraries, dividing corresponding amplification primers (21F/21R-40F/40R) into 4 amplification primer groups, taking 2 non-repetitive and highly-conserved regions at the upstream and downstream of the AML1 gene fragmentation site, respectively using 35 mmol/20R-25 mmol/20R, 35-25 mmol/L of primers, 35-25-10R, 35-25-mL, 35-10 mmol/L of amplification primer working solution, and amplification primer of 35-20R.

(3) Configuring a PCR amplification system: the amplification of the 8 systems is respectively carried out, and the amplification system reagent composition is as follows:

reagent	Per reaction (μ L)
		10 Xbuffer (containing Mg)²⁺)	5
10 XdNTP Mix (Biotin-dUTP containing)	5
		Taq DNA polymerase	2
PCR primer working solution	10
		DNA(10ng/μl)	1
Sterilization double distilled water	27
		Total volume	50

(4) And (3) PCR amplification: after the system is prepared, the mixture is mixed evenly and is centrifuged for 5 to 10 seconds instantly, and amplification is carried out according to the following procedures: 95 ℃ for 5min, 95 ℃ for 30s, 59 ℃ for 30s, 72 ℃ for 30s, 30 cycles from the second step, 72 ℃ for 5min, 4 ℃ hold.

(5) And (3) product identification and purification, namely uniformly mixing amplification products aiming at 2 non-repeated and highly conserved regions at the upstream and 2 non-repeated and highly conserved regions at the downstream of the AML1 gene fracture site to obtain a group of probe libraries, uniformly mixing amplification products aiming at 2 non-repeated and highly conserved regions at the upstream and 2 non-repeated and highly conserved regions at the downstream of the ETO gene fracture site to obtain a second group of probe libraries, identifying the two groups of probe libraries through 2% agarose gel electrophoresis, cutting products with the size of 100-500bp, and recovering the products to obtain a group and a second group of probe libraries, and marking correspondingly.

3. Dye-labeled probe

Cy3 (red fluorescence) labeled group probes are preferred in this example, Alexa Fluor 488 (green fluorescence) labeled second group probes are preferred, the synthetic Cy3 labeled and Alexa Fluor 488 labeled polyA sequence is synthesized, the polyA sequence comprises 1-30 bases, preferably 10-20 bases, more preferably 2-8 bases, the tail end of the polyA sequence is modified with streptavidin, the Cy3 labeled and Alexa Fluor 488 labeled polyA sequence is mixed with group and second group probe library modified with biotin (20 uL of fluorescently labeled polyA sequence is added in every 100ug of probe library), and incubated and slowly shaking for 30min at 37 ℃ to obtain the corresponding fluorescently labeled probes, the probes are purified and precipitated, dissolved in TE buffer solution to obtain group probes labeled red and green labeled second group probes, and the probes are stored at-20 ℃.

II, other components of the kit

1. SSC buffer stock (20 XSSC, pH 5.3): 88g of sodium chloride, 44g of sodium citrate and 400mL of ultrapure water were sufficiently dissolved and mixed, the pH value of the solution was adjusted to 5.3 at room temperature, the solution was made to 500mL with ultrapure water, and the solution was filtered through a 0.45 μm filter.

2. Ethanol solution (70% and 85%): absolute ethanol 700mL/850mL and ultrapure water 300mL/150mL, and thoroughly mixed.

3. Washing buffer I: 20 XSSC (pH5.3)35mL, ethylphenylpolyethylene glycol (NP-40)3mL, and ultrapure water 912mL, were thoroughly mixed, the pH of the solution was adjusted to 7.0 at room temperature, the solution was made to 1000mL with ultrapure water, and the mixture was filtered through a 0.45 μm filter.

4. Washing buffer II: 20 XSSC (pH5.3)100mL, ethylphenylpolyethylene glycol (NP-40)1mL, and ultrapure water 849mL, were thoroughly mixed, the pH of the solution was adjusted to 7.0 at room temperature, the solution was made to 1000mL with ultrapure water, and the mixture was filtered through a 0.45 μm filter.

Example 2 detection of clinical samples Using the kit of example 1

sample pretreatment

1. Collecting 1-1.5ml of peripheral blood of a patient by using a heparin sodium anticoagulation tube, and centrifuging for 5min at 2000 rpm;

2. discarding the supernatant, adding 5-10ml of 0.075M KCl solution, blowing and beating uniformly, and performing hypotonic treatment at 37 ℃ for 30 min;

3. adding 1ml fresh fixing solution (methanol/glacial acetic acid 3:1), blowing and beating uniformly, and standing for 5 min;

4. centrifuging at 2000rpm for 5min, and discarding the supernatant;

5. adding 10ml fresh fixing solution (methanol/glacial acetic acid 3:1), blowing and beating uniformly, and standing for 10 min;

6. centrifuging at 2000rpm for 5min, and discarding the supernatant;

7. adding 10ml of fresh stationary liquid (methanol/glacial acetic acid 3:1), blowing uniformly, centrifuging at 2000rpm for 5min, and discarding the supernatant;

8. repeating the step 7 twice, and finally adding 10ul of fresh fixing solution (methanol/glacial acetic acid 3:1) to resuspend the cells;

9. the prepared cell suspension was dropped onto the slide.

Second, FISH detection

The FISH detection mainly comprises four steps of pre-denaturation, hybridization and counterstaining, and the operation steps from the hybridization are carried out under the condition of avoiding light:

1. preparation work: balancing the probe to room temperature, uniformly mixing by vortex, and centrifuging in a microcentrifuge for 2-3 s; starting and preheating a hybridization instrument, immersing the wet strips into distilled water for later use (at least soaking for 2 hours), and putting the wet strips into the hybridization instrument during hybridization; the sample hybridization area was circled on the reverse side of the section using a glass knife.

2. Pre-denaturation: the slides were soaked in 2 × SSC at room temperature for 2min, and then dehydrated in 70%, 85%, 100% ethanol for 2min each. Oven drying on a baking sheet machine for 2-5 min.

3. And (3) hybridizing (in a dark place), namely adding 10ul of probe solution (the probe concentration is 15ng/ul, the corresponding detected sample area is about 20mm multiplied by 20mm per standard human number probe amount, the dosage of a sample with a larger sample area can be increased according to actual conditions), covering the glass cover to ensure that no bubbles exist under the glass cover and the probes are uniformly distributed, covering the peripheral position of the glass cover with a piece sealing glue to form a closed sealing ring, putting the slice into a hybridization instrument with a wet strip installed, and setting a hybridization reaction program that the slice is denatured at 85 ℃ for 8min and hybridized at 42 ℃ for 4 h.

4. And (3) washing after hybridization (lightproof operation), namely opening a water bath (76 +/-1 ℃), placing a washing buffer solution II in the water bath for preheating, taking the hybridized slice out of the hybridization instrument, removing the sealing glue, placing the slice in a washing buffer solution I at room temperature for 5min, washing away the cover glass, placing the slice in a washing buffer solution II at 76 +/-1 ℃ for 5min, slightly shaking the slice for 1-3s, repeating the washing step for 1 time, finally, washing with the washing buffer solution I for times, wherein the time for washing each washing buffer solution is the same as that for washing with the buffer solution at times, and vertically drying the slice after washing.

5. DAPI counterstain (dark operation): and adding 10 mu L of counterstain solution into the sample hybridization area, covering a cover glass, and observing a fluorescence signal under a microscope after keeping out of the sun for 5-10min at room temperature. The section after DAPI counterstaining can also be stored at minus 25 ℃ to minus 18 ℃ in a dark place (the storage time is not more than 72h), and the slide is placed to the room temperature for observation when the detection is needed.

Thirdly, result judgment standard

The kit of the invention has the following result judgment standards:

1. 100 cells were counted per sample, and if the number of positive cells was less than 3 (3/100 or < 3%), the sample was judged negative.

2. 100 cells were counted per sample, and if the number of positive cells was greater than 5 (5/100 or > 5%), the sample was judged positive.

3. Counting 100 cells in each sample, and if the number of positive cells is between 3 and 5 (3 to 5%), additionally counting 100 cells by people who read the slices, summarizing the number of the counted cells by two people who read the slices and the number of the positive cells, namely, the number of the positive cells is 200 cells in total, if the total number of the positive cells is less than 8 (< 4%), the sample is judged to be negative, and if the total number of the positive cells is more than or equal to 8 (> 4%), the sample is judged to be positive.

And (3) judging the positive and negative of the cells:

the microscope field was moved up and down to look for all signals present in the nuclei.

1. The following signaling patterns were determined as AML1/ETO fusion gene positive cells (see FIG. 1 for details): present in interphase nuclei are 1 red, 1 green and 2 red-green hybridization signals (1R1G 2F);

2. the following signaling patterns were judged as AML1/ETO fusion gene-negative cells (see FIG. 2 for details): present in interphase nuclei are 2 red and 2 green randomly dispersed 4 hybridization signals (2R 2G).

Note: in the figure, black represents a red dot and white represents a green dot.

Fourthly, analysis of detection results

Peripheral blood samples (numbers 1-15) of 15 patients with acute myelocytic leukemia were tested using the kit of example 1, and Kasumi cell line was used as a positive control and K562 cell line was used as a negative control, and those skilled in the art could purchase the cell lines by knowing their names. Approximately 2000 Kasumi and K562 cells (as determined by cell counter) were each taken and mixed well and the samples were each divided into 5 aliquots numbered 16-20 and 21-25. Specific detection results are shown in table 3:

TABLE 3 sample test results

According to the detection results, the kit has good sensitivity and specificity for detecting AML1/ETO fusion genes, can realize the detection of peripheral blood samples of patients with acute promyelocytic leukemia, and simultaneously all Kasumi cells are positive for AML1/ETO fusion genes, and all K562 cells are negative for AML1/ETO fusion genes. The probe designed by the kit can specifically generate hybridization reaction with a target gene sequence, the generated fluorescent signal is strong and stable, and the specificity and the accuracy of a detection result are ensured.

EXAMPLE 3 Effect of duration of hybridization on detection Effect of kit

, length of hybridization

The design of the length of the probe of the kit and the dye labeling method is an optimal scheme obtained by comparing and statistically analyzing experimental results through a large number of experiments by the inventor, so that the optimal balance between the detection specificity and the hybridization time can be achieved, the result specificity and the sensitivity can be ensured, the hybridization time can be shortened, and the detection efficiency can be improved.

To verify the effect of hybridization duration on the detection results of the present invention, 4 experimental groups with different hybridization durations were set in this example, and the detection probes and reagents used are shown in Table 4 below, which is similar to the kit in example 1.

TABLE 4 duration of hybridization

Second, sample detection

The kit prepared by the design is adopted to detect the peripheral blood samples 26-40 of the patients with acute myelocytic leukemia according to the detection process and method described in the embodiment 2, wherein the hybridization time of each experimental group is compared with that of the table 4, and the specific experimental results are as follows:

TABLE 5 influence of hybridization duration on assay results

Comparing the detection results of 4 experimental groups, it can be known that the probe designed by the kit can be fully hybridized with the target gene of the sample within 4h, so as to realize the detection of the sample and ensure the specificity and accuracy of the result, the hybridization time of 2h causes the omission of positive cells due to the insufficient hybridization of the probe and the sample, so that the false negative result is caused, and the detection result is unstable, while the hybridization time of 8h and 16h results in the complete of 4h and the fluorescence signal intensity is basically .

EXAMPLE 4 Effect of Probe Length on the detection Effect of the kit

, probe length

In order to verify the influence of the probe length on the detection result of the present invention, 4 experimental groups with different probe lengths are provided in this example, see table 6 specifically, the amplification region positions of the 4 experimental groups are the same, and the preparation method and reagents of the detection probe used are the same as those of the kit in example 1.

TABLE 6 Probe Length

Grouping	Experimental group 5	Experimental group 6	Experimental group 7	Experimental group 8
					Length of probe	20-100bp	100-500bp	500-1000bp	＞1000bp

Second, sample detection

The kit prepared by the design is adopted to detect the peripheral blood samples 41-55 of the patients with acute myelocytic leukemia according to the detection process and method described in the embodiment 2, and the specific experimental results are as follows:

TABLE 7 influence of Probe Length on the assay results

Comparing the detection results of the 4 experimental groups, it can be known that the experimental group 6 can be fully hybridized with the target gene of the sample within 4h by using the probe of 100-500bp, thereby realizing the detection of the sample and ensuring the specificity and accuracy of the result. If the length of the probe is too short (experiment group 5), the detection specificity is reduced, higher background noise is caused, and meanwhile, the generated fluorescent signal is very weak, so that the result is misjudged; too long a probe length (panels 7 and 8) can reduce the uptake capacity of the probe by the cells, and at the same time, the time required for hybridization can be prolonged, so that the probe cannot be sufficiently hybridized with the target gene within 4h, and the accuracy and stability of the detection result are affected.

Example 5 Effect of quantity of primer pairs for constructing Probe library on detection Effect of kit

selection of primer pairs

The kit provided by the invention designs two groups of probe libraries aiming at detection and typing of AML1/ETO fusion genes, wherein each group of probe libraries is obtained by amplifying 20 pairs of primers, the amplification regions are respectively non-repetitive and highly conserved segments in target detection regions on the upstream and the downstream of AML1 and ETO gene fracture sites, 4 non-repetitive and highly conserved amplification regions are preferably selected for the target detection regions on the upstream and the downstream of AML1 and ETO gene fracture sites, and 5 pairs of amplification primers are designed for each region.

In order to detect the influence of the types and the quantity of the primer pairs used for constructing the probe library on the detection result of the invention, in this embodiment, taking the construction of the th group of probes as an example, 3 experimental groups are set, specifically, see table 8, the preparation method and the reagents of the detection probes used are similar to those of the kit in example 1, and the second group of probes is subjected to library construction by using all amplification primers.

TABLE 8 selection of primer pairs

Second, sample detection

The kit prepared by the design is adopted to detect the peripheral blood samples 56-70 of the patients with acute myelocytic leukemia according to the detection process and method described in the embodiment 2, and the specific experimental results are as follows:

TABLE 9 influence of the types and amounts of the primer pairs in the Probe library on the detection results

Comparing the detection results of the 3 experimental groups, it can be seen that when 1, 2 and 5 pairs of amplification primers are selected for each amplification region of the target gene, i.e., the probe base is constructed by using 4, 8 and 20 pairs of primers, the detection can be completed, when 4 or more pairs of primers are used for construction, the detection result tends to be stable, and when 8 pairs of amplification primers are used, the specificity and stability are good, wherein, when all 20 pairs of the library-constructing primer pairs are used, the signal is stronger and more stable, the detection effect is best, the other experimental results for selecting the types and the number of the probe base and the second probe base library-constructing primer pairs are , and the specific data are omitted.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for those skilled in the art, variations and modifications can be made without departing from the spirit of the invention, and these are within the scope of the invention. Therefore, the scope of the invention/patent should be determined by the appended claims.

Claims

The kit for detecting AML1/ETO fusion gene of species is characterized by comprising a th group of probes for AML1 gene breakage and a second group of probes for ETO gene breakage, wherein the two groups of probes are marked with dyes, the colors of the dyes of the same group of probes are the same, and the colors of the dyes of the different groups of probes are different;

the amplification primers for the set probes are at least pairs selected from the group consisting of SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, and at least pairs selected from the group consisting of SEQ ID NO.21 and SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.30, SEQ ID NO.31 and SEQ ID NO.32, SEQ ID NO.33 and SEQ ID NO.34, SEQ ID NO.36, SEQ ID NO.38 and pairs of SEQ ID NO.38, SEQ ID NO.38 and SEQ ID NO. 38;

the amplification primers for the second set of probes are at least pairs selected from SEQ ID NO.41 and SEQ ID NO.42, SEQ ID NO.43 and SEQ ID NO.44, SEQ ID NO.45 and SEQ ID NO.46, SEQ ID NO.47 and SEQ ID NO.48, SEQ ID NO.49 and SEQ ID NO.50, SEQ ID NO.51 and SEQ ID NO.52, SEQ ID NO.53 and SEQ ID NO.54, SEQ ID NO.55 and SEQ ID NO.56, SEQ ID NO.57 and SEQ ID NO.58, SEQ ID NO.59 and SEQ ID NO.60, and at least 3579 pairs selected from SEQ ID NO.61 and SEQ ID NO.62, SEQ ID NO.63 and SEQ ID NO.64, SEQ ID NO.65 and SEQ ID NO.66, SEQ ID NO.67 and SEQ ID NO.68, SEQ ID NO.69 and SEQ ID NO.70, SEQ ID NO.71 and SEQ ID NO.72, SEQ ID NO.74 and SEQ ID NO.73, SEQ ID NO.76 and SEQ ID NO.75, SEQ ID NO.76 and SEQ ID NO.76, SEQ ID NO.75 and SEQ ID NO. 75.
2. The AML1/ETO fusion gene detection kit of claim 1, wherein the amplification product has a size of 100-500 bp.
3. The AML1/ETO fusion gene detection kit of claim 1, wherein the amplification primers for said set of probes are selected from at least four of the corresponding amplification primers of claim 1, and the amplification primers for said second set of probes are selected from at least four of the corresponding amplification primer pairs of claim 1.
4. The AML1/ETO fusion gene detection kit of claim 3, wherein the amplification primers for said set of probes are selected from at least eight of the corresponding said amplification primers of claim 1, and the amplification primers for said second set of probes are selected from at least eight of the corresponding said amplification primers of claim 1.
5. The AML1/ETO fusion gene detection kit according to claim 4, wherein the amplification primers for the group of probes are SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO.30, SEQ ID NO.23 and SEQ ID NO.31, SEQ ID NO.32 and SEQ ID NO.38, SEQ ID NO.32 and SEQ ID NO.35, SEQ ID NO.36 and SEQ ID NO. 38;

the amplification primers for the second set of probes were: SEQ ID NO.41 and SEQ ID NO.42, SEQ ID NO.43 and SEQ ID NO.44, SEQ ID NO.45 and SEQ ID NO.46, SEQ ID NO.47 and SEQ ID NO.48, SEQ ID NO.49 and SEQ ID NO.50, SEQ ID NO.51 and SEQ ID NO.52, SEQ ID NO.53 and SEQ ID NO.54, SEQ ID NO.55 and SEQ ID NO.56, SEQ ID NO.57 and SEQ ID NO.58, SEQ ID NO.59 and SEQ ID NO.60, SEQ ID NO.61 and SEQ ID NO.62, SEQ ID NO.63 and SEQ ID NO.64, SEQ ID NO.65 and SEQ ID NO.66, SEQ ID NO.67 and SEQ ID NO.68, SEQ ID NO.69 and SEQ ID NO.70, SEQ ID NO.71 and SEQ ID NO.72, SEQ ID NO.73 and SEQ ID NO.74, SEQ ID NO.75 and SEQ ID NO.76, SEQ ID NO.76 and SEQ ID NO.78, SEQ ID NO.80 and SEQ ID NO. 80.
6. The AML1/ETO fusion gene detection kit of of any one of claims 1-5, wherein the fluorochrome is selected from FAM, TET, JOE, HEX, Cy3, TAMRA, ROX, Texas Red, LC RED640, Cy5, LC RED705, Alexa Fluor 488 and Alexa Fluor 750, and the fluorochromes for different probe sets are different from each other.