Primer probe combination, detection kit and detection method for detecting MLL-AF4 fusion gene

Abstract

The invention discloses a primer probe combination, a detection kit and a detection method for detecting an MLL-AF4 fusion gene, primer probes included in the kit are a group of universal primer probes and can be used for simultaneously detecting e9e5, e10e4 and e11e5 subtype loci of MLL-AF4, so that the accuracy of a detection result is ensured. The primer probe combination and the detection method can effectively detect the low-frequency mutation of the fusion gene MLL-AF4, have the advantages of high specificity, high sensitivity and short detection period, are rigorous and reliable in detection result and high in practicability, and provide scientific basis for diagnosis and clinical monitoring of acute leukemia patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of MLL-AF4 fusion gene detection, and particularly relates to a primer probe combination for detecting MLL-AF4 fusion gene, a detection kit and a detection method. BACKGROUND

[0002] Acute leukemia is a hematological malignancy derived from hematopoietic stem cells, characterized by clonal expansion of abnormal differentiated primitive cells; it mainly manifests as abnormal clonal cell proliferation and differentiation in bone marrow, peripheral blood and other tissues, and destroys normal hematopoiesis. It includes two main types of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). ALL is the most common type of cancer in childhood, while AML is more common in adults.

[0003] Fusion genes are formed by chromosomal translocation, rearrangement, etc., and are the result of the combined action of genetic and environmental factors. They play an important role in the pathogenesis, diagnosis, efficacy evaluation, prognosis prediction and treatment guidance of leukemia, and are also an important basis for monitoring minimal residual disease (MRD).

[0004] Mixed lineage leukemia (MLL, also known as KMT2A) gene rearrangement can lead to invasive and poor prognosis acute lymphoblastic (ALL) and acute myeloid (AML) leukemia in children and adults. MLL gene abnormalities are one of the common chromosomal abnormalities in acute leukemia, which is associated with poor prognosis. The gene can be fused with various translocation partner genes (TPG), usually associated with t(4;11)(q21;q23) translocation, forming MLL-AF4 fusion gene. MLL-AF4 positive acute lymphoblastic leukemia (ALL) is the second most common chromosomal abnormality in adult acute lymphoblastic leukemia, next to Philadelphia chromosome positive ALL. MLL-AF4 positive is observed in 5% of childhood and adult ALL cases, and this subgroup accounts for 40-60% of infant and chemotherapy-induced ALL. At the molecular level, the breakpoint distribution of MLL and AF4 genes is in the intron, and the breakpoint distribution is between exon 8 and exon 12 of MLL and between exon 3 and exon 7 of AF4. ALL and young patients usually show MLL intron 11 breakage, while acute myelogenous leukemia (AML) and older patients show more MLL intron 9 breakage.

[0005] Studies at home and abroad show that minimal residual disease (MRD) detection can not only be used for efficacy evaluation and relapse warning, but also be used for guiding the selection of treatment methods and preemptive intervention. Therefore, MRD detection has become one of the key links to reduce leukemia relapse and improve efficacy. MRD monitoring adopts two types of detection technologies that are complementary to each other. Molecular diagnostics technology aims to explain the special genomic lesions of tumor clones, such as real-time quantitative polymerase chain reaction technology (RQ-PCR), digital PCR (dPCR) and next-generation sequencing technology (NGS), etc. Molecular diagnostics technology is more accurate in terms of specificity and sensitivity in detection, and can analyze the expression level of specific leukemia subgroups.

[0006] Digital PCR is the third generation of PCR technology after the first generation of ordinary PCR and the second generation of fluorescent quantitative PCR. Compared with other analysis and detection technologies, dPCR technology is a direct detection of the copy number of target sequences without relying on standard curves, and can realize absolute quantification of samples, overcoming the common weaknesses of qPCR, and having higher sensitivity, population coverage and operation convenience. Because this detection method has higher sensitivity, specificity and accuracy than traditional qPCR, dPCR can be rapidly widely used, especially in the detection of extremely small amount of nucleic acid samples, detection of rare mutations in complex background and identification of small differences in expression. Therefore, selecting appropriate detection technology to improve the detection rate, sensitivity and specificity of fusion genes is of great significance for disease diagnosis and typing, monitoring of tumor burden and efficacy evaluation. CN118240941A (application number 202410411656.6) discloses a method and kit for digital PCR quantitative detection of acute leukemia MLL-AF4 fusion gene, and the invention only detects MLL-AF4 typing e11e5, and cannot detect typing e9e5 and e10e4.

[0007] In summary, there is a need for a digital PCR detection kit for MLL-AF4 fusion gene that can simultaneously detect multiple clinical detection sites of MLL-AF4, is simple to operate, has high accuracy and high sensitivity. SUMMARY

[0008] The technical problem to be solved by the present application is to provide a digital PCR detection kit for acute leukemia related fusion gene, which can effectively detect fusion hotspot mutations of fusion gene MLL-AF4, has high specificity, high sensitivity and short detection period, and a special primer and probe combination thereof.

[0009] The technical scheme for achieving the first object of the present application is a primer and probe combination for detecting MLL-AF4 fusion gene, comprising a forward primer MLL-AF4-F for detecting fusion gene MLL-AF4 as shown in SEQ ID No. 1, a reverse primer MLL-AF4-R for detecting fusion gene MLL-AF4 as shown in SEQ ID No. 2, and a probe MLL-AF4-P for detecting fusion gene MLL-AF4 as shown in SEQ ID No. 3.

[0010] A forward primer ABL1-F for detecting reference gene ABL1 as shown in SEQ ID No. 4, a reverse primer ABL1-R for detecting reference gene ABL1 as shown in SEQ ID No. 5, and a probe ABL1-P for detecting reference gene ABL1 as shown in SEQ ID No. 6.

[0011] The 5' end of the probe is connected with a fluorescent reporter group, and the 3' end is connected with a quencher group.

[0012] Optionally, the 5' end of the probe MLL-AF4-P for detecting fusion gene MLL-AF4 is modified by a FAM fluorescent group, and the 3' end is modified by a BHQ1 quencher group; the 5' end of the probe ABL1-P for detecting reference gene ABL1 is modified by a VIC fluorescent dye, and the 3' end is modified by a BHQ2 quencher group.

[0013] The technical scheme for achieving the second object of the present application is a detection kit for detecting MLL-AF4 fusion gene, comprising a primer and probe premix, an enzyme mixture, positive quality control, and negative quality control. The primer and probe premix and the enzyme mixture are contained in a main reaction tube, and the positive quality control and the negative quality control are respectively packaged in independent tubes.

[0014] The primer and probe premix comprises a forward primer MLL-AF4-F for detecting fusion gene MLL-AF4 as shown in SEQ ID No. 1, a reverse primer MLL-AF4-R for detecting fusion gene MLL-AF4 as shown in SEQ ID No. 2, and a probe MLL-AF4-P for detecting fusion gene MLL-AF4 as shown in SEQ ID No. 3; a forward primer ABL1-F for detecting reference gene ABL1 as shown in SEQ ID No. 4, a reverse primer ABL1-R for detecting reference gene ABL1 as shown in SEQ ID No. 5, and a probe ABL1-P for detecting reference gene ABL1 as shown in SEQ ID No. 6.

[0015] As optional, the concentration of the forward primer MLL-AF4-F for detecting the fusion gene MLL-AF4 is 400-600 nM, the concentration of the reverse primer MLL-AF4-R for detecting the fusion gene MLL-AF4 is 400-600 nM, and the concentration of the probe MLL-AF4-P for detecting the fusion gene MLL-AF4 is 200-300 nM; the concentration of the forward primer ABL1-F for detecting the reference gene ABL1 is 400-600 nM, the concentration of the reverse primer ABL1-R for detecting the reference gene ABL1 is 400-600 nM, and the concentration of the probe ABL1-P for detecting the reference gene ABL1 is 200-300 nM.

[0016] Further, the positive quality control sample contains the MLL-AF4 fusion gene and the ABL1 wild-type gene; and the negative quality control sample contains the ABL1 wild-type gene but does not contain the MLL-AF4 fusion gene.

[0017] Further, the positive quality control sample contains ≥1000 copies / ul of the MLL-AF4 fusion gene and ≥1000 copies / ul of the ABL1 wild-type gene; and the negative quality control sample contains ≥1000 copies / ul of the ABL1 wild-type gene.

[0018] As optional, the enzyme mixture in the kit is Digital 5x RNA Master, including dPCR reaction buffer, Taq polymerase, AmpErase enzyme, dATP, dCTP, dGTP, dTTP, dUTP, and sodium azide.

[0019] As optional, the volume of the enzyme mixture in the kit accounts for 15%-75% of the total volume of the reagents in the kit.

[0020] The technical solution for achieving the third object of the present application is a detection method for detecting the MLL-AF4 fusion gene, comprising the following steps:

[0021] S1, extracting RNA to obtain a to-be-tested RNA template.

[0022] S2, PCR amplification.

[0023] A PCR reaction system is prepared, the to-be-tested RNA template is added into a main reaction tube containing a primer-probe pre-mixture and an enzyme mixture, water is quantitatively added to 30 uL, and after the reaction system is prepared, the mixture is fully mixed and centrifuged.

[0024] The primer probe premix includes a forward primer MLL-AF4-F for detecting the fusion gene MLL-AF4 as shown in SEQ ID No. 1, a reverse primer MLL-AF4-R for detecting the fusion gene MLL-AF4 as shown in SEQ ID No. 2, a probe MLL-AF4-P for detecting the fusion gene MLL-AF4 as shown in SEQ ID No. 3, a forward primer ABL1-F for detecting the reference gene ABL1 as shown in SEQ ID No. 4, a reverse primer ABL1-R for detecting the reference gene ABL1 as shown in SEQ ID No. 5, and a probe ABL1-P for detecting the reference gene ABL1 as shown in SEQ ID No. 6.

[0025] The reaction mixture is subjected to PCR reverse transcription amplification reaction.

[0026] S3, result analysis; the result analysis selects a 1D scatter plot, and a positive result is displayed as red and a negative result is displayed as blue.

[0027] The present application has positive effects:

[0028] (1) The kit of the present application realizes quantitative detection at the nucleic acid level in the same tube by using digital PCR technology and hydrolysis probe technology.

[0029] The detection method using the kit of the present application is based on a digital PCR platform, and the operation principle is that nucleic acid molecules are dispersed into a large number of micro-reaction units, the number of micro-droplets / micro-holes can reach 20,000-30,000, and high sensitivity and high specificity are realized at the detection level in the form of micro-droplet / micro-hole preparation. PCR amplification and fluorescence detection are carried out in each individual micro-reaction unit, the copy number is estimated by using a Poisson distribution statistical model, and the method is not dependent on a standard curve and a standard product, so that the original copy number of nucleic acid in the starting sample can be directly detected, and the influence of PCR efficiency on the detection result is avoided.

[0030] (2) The acute leukemia related fusion gene MLL-AF4 detection kit of the application designs a set of universal primers and probes, and adopts MLL-AF4 fusion gene and ABL1 internal reference gene to form single-hole double-channel detection. Since the dispersion of the sample in digital PCR follows the Poisson distribution principle, most of the units contain 0 or 1 target nucleic acid molecules on average when the sample concentration is low, different nucleic acid molecules are allocated into different micro-reaction units, which to a certain extent avoids the amplification of different amplicons in the same micro-reaction unit, avoids competition inhibition, and guarantees the accurate quantitative detection of each type of template. The single-hole can accurately quantify the detection of e9e5, e10e4 and e11e5 three MLL-AF4 fusion gene typing at the same time, has the advantages of strong specificity, high sensitivity, short detection period, rigorous and reliable detection results, and strong practicability, and provides a scientific basis for the diagnosis and clinical monitoring of acute leukemia patients. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 It is the MLL-AF4 fusion gene typing e9e5 annealing temperature and time gradient detection result graph.

[0032] Figure 2 It is the MLL-AF4 fusion gene typing e10e4 annealing temperature and time gradient detection result graph.

[0033] Figure 3 It is the MLL-AF4 fusion gene typing e11e5 annealing temperature and time gradient detection result graph.

[0034] Figure 4 It is the MLL-AF4 fusion gene typing e9e5 accuracy detection result graph.

[0035] Figure 5 It is the MLL-AF4 fusion gene typing e10e4 accuracy detection result graph.

[0036] Figure 6 It is the MLL-AF4 fusion gene typing e11e5 accuracy detection result graph.

[0037] Figure 7 It is the MLL-AF4 fusion gene specificity detection result graph.

[0038] Figure 8 It is the MLL-AF4 fusion gene typing e9e5 sensitivity detection result graph.

[0039] Figure 9 It is the MLL-AF4 fusion gene typing e10e4 sensitivity detection result graph.

[0040] Figure 10 It is the MLL-AF4 fusion gene typing e11e5 sensitivity detection result graph. DETAILED DESCRIPTION

[0041] The following description is provided so as to enable those skilled in the art to realize the present application and is provided in connection with the exemplary embodiments. Various alternatives and modifications to the present application are possible. For example, the boundaries between the functional stages of the present application are arbitrary, and particular stages can be included within other stages in a particular implementation of the present application. Therefore, the following detailed description is not intended to be limiting. Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of the present application disclosed herein. Various embodiments of the present application are described herein with reference to the accompanying drawings. It should be noted that the features illustrated in the drawings are not necessarily drawn to scale, and that the embodiments can include other features that are not illustrated.

[0042] In the following examples, unless otherwise stated, the reagents used are of analytical purity, and the reagents used can be obtained from commercial channels. The experimental methods not specified in the text are generally according to the conventional conditions, such as the conditions described in the book "Molecular Cloning Experiment Guide" published by Science Press in 2002, or according to the conditions suggested by the manufacturer. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as familiar to those skilled in the art. In addition, any method and material similar or equivalent to those described can be applied in the present application.

[0043] (Example 1, primer and probe combination)

[0044] The design principle of the primer and probe of the present application is: the forward primer located on the 9th exon of the MLL gene, the reverse primer and the probe located on the 5th exon of the AF4 gene, the detection range of the primer and probe combination includes the breakpoint of introns 9-11 of the MLL gene, which is a set of universal primers and probes that basically cover all subtypes of MLL-AF4 fusion genes, and is suitable for the detection range of young patients and old patients.

[0045] The design site range of the fusion gene MLL-AF4 primer and probe includes the 9th exon to the 11th exon of the MLL gene, and the 5th exon to the 4th exon of the AF4 gene.

[0046] The specific primers and probes of the fusion gene MLL-AF4 and the reference gene ABL1 are designed, each group containing an upstream primer, a downstream primer and a probe. The 5' end of the fusion gene MLL-AF4-P probe is labeled with FAM fluorescent dye, and the 3' end is labeled with BHQ1 quenching group modification; the 5' end of the reference gene ABL1-P probe is labeled with VIC fluorescent dye, and the 3' end is labeled with BHQ2 quenching group modification.

[0047] The sequence characteristics of each primer in this embodiment are shown in Table 1.

[0048] Table 1 Primer sequence characteristics

[0049]

[0050] In the above table: the nucleotide sequence of the forward primer for detecting the fusion gene MLL-AF4 is shown as SEQ ID No. 1, the nucleotide sequence of the reverse primer for detecting the fusion gene MLL-AF4 is shown as SEQ ID No. 2, and the nucleotide sequence of the probe for detecting the fusion gene MLL-AF4 is shown as SEQ ID No. 3.

[0051] The nucleotide sequence of the forward primer for detecting the reference gene ABL1 is shown as SEQ ID No. 4, the nucleotide sequence of the reverse primer for detecting the reference gene ABL1 is shown as SEQ ID No. 5, and the nucleotide sequence of the probe for detecting the reference gene ABL1 is shown as SEQ ID No. 6.

[0052] The 5' end of the above-mentioned probe is connected with a fluorescent reporter group, and the 3' end is connected with a quencher group; the probe for detecting the MLL-AF4 fusion gene is modified at the 5' end with a FAM fluorescent group and at the 3' end with a BHQ1 quencher group; the probe for detecting the reference gene ABL1 is modified at the 5' end with a VIC fluorescent group and at the 3' end with a BHQ2 quencher group.

[0053] (Example 2, kit)

[0054] The kit of this example comprises: primer-probe pre-mix, enzyme mixture, positive quality control, and negative quality control; the primer-probe pre-mix and the enzyme mixture are contained in a main reaction tube, and the positive quality control and the negative quality control are respectively packaged in separate tubes.

[0055] In the primer-probe pre-mix of this example, the primers and probes used include SEQ ID No. 1 to SEQ ID No. 6 listed in Table 1.

[0056] In the primer-probe pre-mix, the concentration of the forward primer MLL-AF4-F for detecting the fusion gene MLL-AF4 is 400-600 nM, the concentration of the reverse primer MLL-AF4-R for detecting the fusion gene MLL-AF4 is 400-600 nM, and the concentration of the probe MLL-AF4-P for detecting the fusion gene MLL-AF4 is 200-300 nM; the concentration of the forward primer ABL1-F for detecting the reference gene ABL1 is 400-600 nM, the concentration of the reverse primer ABL1-R for detecting the reference gene ABL1 is 400-600 nM, and the concentration of the probe ABL1-P for detecting the reference gene ABL1 is 200-300 nM.

[0057] The enzyme mixture included in the kit of this example is a Digital 5xRNAMaster. This enzyme mix is a PCR reaction mix, available from Roche (Cat. No. 08897115001), containing dPCR reaction buffer, Taq polymerase, AmpErase enzyme, dNTPs and sodium azide, for reverse transcription and PCR amplification using a thermostable DNA polymerase.

[0058] The kit of this embodiment includes a positive quality control and a negative quality control.

[0059] The positive sample is a solution containing the MLL-AF4 fusion gene and the ABL1 wild-type gene; the negative sample is a solution containing the ABL1 wild-type gene but not the MLL-AF4 fusion gene.

[0060] The positive quality control is prepared by diluting the positive sample to a concentration of ≥ 1 x 10 3 copies / ul of sample; the negative quality control is prepared by diluting the ABL1 to a concentration of ≥ 1 x 10 3 copies / ul of sample.

[0061] The PCR reaction system prepared using the kit in this embodiment is as follows:

[0062] The PCR reaction system is prepared according to Table 2 below, and the single-person reaction system is 30 uL.

[0063] Prepare N reaction systems according to the single-person reaction system.

[0064] N = number of samples to be tested + positive quality control + negative quality control + no nucleic acid water

[0065] Table 2 PCR reaction system preparation

[0066] Reaction components Amount per reaction Primer probe master mix 4 uL Enzyme mix 6 uL Template RNA to be tested 10 uL Water Add H20 to 30 uL Total volume 30 uL

[0067] After the reaction system is thoroughly mixed and centrifuged, 30 ul is loaded into the sample injection hole of the chip, and the chip is detected after being treated with a digital PCR sample machine.

[0068] (Example 3, digital PCR detection method)

[0069] The method for detecting the MLL-AF4 fusion gene provided in this embodiment uses a one-step reverse transcription digital PCR detection method to collect fluorescence signals to quantitatively detect the expression of the fusion gene MLL-AF4, including the following steps:

[0070] S1, sample preparation.

[0071] The suitable sample type is a peripheral blood sample.

[0072] Collect blood samples, and extract RNA according to the instructions of the Kangwei Blood RNA Extraction Kit (Item No. CW0582S).

[0073] S2, PCR amplification.

[0074] Prepare the PCR reaction system according to Table 2. After the reaction system is prepared, mix thoroughly and centrifuge.

[0075] In the primer probe premix of one embodiment, the concentration of the forward primer MLL-AF4-F for detecting the fusion gene MLL-AF4 is 500 nM, the concentration of the reverse primer MLL-AF4-R for detecting the fusion gene MLL-AF4 is 500 nM, and the concentration of the probe MLL-AF4-P for detecting the fusion gene MLL-AF4 is 250 nM; the concentration of the forward primer ABL1-F for detecting the reference gene ABL1 is 500 nM, the concentration of the reverse primer ABL1-R for detecting the reference gene ABL1 is 500 nM, and the concentration of the probe ABL1-P for detecting the reference gene ABL1 is 250 nM.

[0076] Transfer the reaction mixture to the bottom of the chip sample well for passive filling. Use a universal chip (Digital Universal Nanopore Plate, Model No. 09033696001), and the sample volume for each microwell is 30 ul. After the transfer of all samples is completed, place the chip in a digital PCR sample machine for sealing liquid treatment. After the sealing liquid treatment is completed, place the chip in a digital PCR amplifier (Digital Digital Analyzer, Model No. Digital Digital System) for PCR reverse transcription amplification reaction. The reaction program is shown in Table 3.

[0077] Note 1: During the pipetting process, gently touch the bottom of the sample well at an angle towards the channel. Avoid generating bubbles during pipetting.

[0078] Note 2: For optimal results, the reaction mixture can be stored stably for a maximum of 15 minutes after being added to the microwell plate and before being dispensed.

[0079] Note 3: The analyzer running process should be started within 90 minutes after the microwell plate is dispensed.

[0080] Table 3 Reaction program

[0081]

[0082] S3, result analysis.

[0083] Result interpretation steps:

[0084] 1) Sample setup: After setting the sample name and other information according to the sample position and reaction well, select the analysis mode as "absolute quantification".

[0085] 2) Image analysis: Select 1D scatter plot for result analysis. Positive results are shown in red and negative results are shown in blue; manually set the threshold line in the range between the positive and negative clusters and close to 1 / 3 of the negative cluster.

[0086] (Test Example 1, annealing temperature and time gradient optimization experiment)

[0087] In this test example, the annealing temperature and annealing time of MLL-AF4 fusion gene typing e9e5, e10e4 and e11e5 were optimized respectively. The method of Example 3 was used for digital PCR fluorescence detection, and the annealing temperature gradient was set to 55°C, 58°C, 60°C and 62°C; the annealing time gradient was set to 30s, 60s, 90s and 120s. The detection results of MLL-AF4 fusion gene typing e9e5, e10e4 and e11e5 are shown in Table 1. Figures 1-3 The results of digital PCR experiment showed that when the reaction condition was 58°C, 120s, the MLL-AF4 universal primer probe combination was best for detecting the fluorescence signal of typing e9e5, e10e4 and e11e5.

[0088] (Test Example 2, accuracy)

[0089] The MLL-AF4-e9e5 positive sample, MLL-AF4-e10e4 positive sample, MLL-AF4-e11e5 positive sample and ABL1 negative sample were mixed in equal volume, and gradient dilution was performed by nuclease-free water, with 3 replicates for each type and each concentration. The method of Example 3 was used for digital PCR fluorescence detection, and the gradient fluorescence amplification chart is shown in Figures 4-6 , and the detection results are shown in Tables 4-6.

[0090] From the data analysis in Tables 4-6, it can be seen that the three types of positive samples can be effectively detected at different concentrations, the detection results are positive, the CV value is less than 10%, and the positive detection rate is 100%.

[0091] Table 4 Positive sample MLL-AF4-e9e5

[0092]

[0093]

[0094] Table 5 Positive sample MLL-AF4-e10e4

[0095]

[0096] Table 6 Positive sample MLL-AF4-e11e5

[0097]

[0098] (Test Example 3, specificity)

[0099] In this test example, the negative sample ABL1 was diluted with water to high and low concentrations, with 3 replicates for each concentration. Digital PCR fluorescence detection was performed using the method of Example 3, and the fluorescence amplification chart is shown in Figure 7 , and the detection results are shown in Table 7. From the data analysis in Table 7, the MLL-AF4 copy number of the negative sample at high and low concentrations was 0, the detection result was negative, and the negative detection rate was 100%.

[0100] Table 7

[0101]

[0102] (Test Example 4, sensitivity)

[0103] The positive sample MLL-AF4-e9e5 and the negative sample ABL1 were diluted by gradient dilution with nuclease-free water. The template input volume was set to 10ul, and each gradient was repeated 3 times. Digital PCR fluorescence detection was performed using the method of Example 3, and the gradient fluorescence amplification chart results are shown in Figures 8-10 . From the data analysis in Tables 8-10, the three types of positive samples can be effectively detected at low concentrations after gradient dilution, with a detection copy number as low as ≤100 copies, the detection result is positive, and the positive detection rate is 100%.

[0104] From the above data, it can be shown that this kit can reliably and accurately detect the three types of MLL-AF4 fusion genes, avoiding the risk of missed detection, and providing a diagnostic reference for clinical practice.

[0105] Table 8 Positive sample MLL-AF4-e9e5

[0106]

[0107] Table 9 Positive sample MLL-AF4-e10e4

[0108]

[0109] Table 10 Positive sample MLL-AF4-e11e5

[0110]

Claims

1. A primer and probe combination for detecting the MLL-AF4 fusion gene, characterized in that: a forward primer MLL-AF4-F for detecting the fusion gene MLL-AF4 as shown in SEQ ID No. 1, a reverse primer MLL-AF4-R for detecting the fusion gene MLL-AF4 as shown in SEQ ID No. 2, and a probe MLL-AF4-P for detecting the fusion gene MLL-AF4 as shown in SEQ ID No. 3; a forward primer ABL1-F for detecting the reference gene ABL1 as shown in SEQ ID No. 4, a reverse primer ABL1-R for detecting the reference gene ABL1 as shown in SEQ ID No. 5, and a probe ABL1-P for detecting the reference gene ABL1 as shown in SEQ ID No.

6.

2. The primer, probe combination for detecting MLL-AF4 fusion gene according to claim 1, characterized in that: The 5' end of the probe is connected with a fluorescent reporter group, and the 3' end is connected with a quencher group.

3. The primer, probe combination for detecting MLL-AF4 fusion gene according to claim 2, characterized in that: The 5' end of the probe MLL-AF4-P for detecting the fusion gene MLL-AF4 is modified with a FAM fluorescent group, and the 3' end is modified with a BHQ1 quencher group; the 5' end of the probe ABL1-P for detecting the reference gene ABL1 is modified with a VIC fluorescent dye, and the 3' end is modified with a BHQ2 quencher group.

4. A detection kit for detecting MLL-AF4 fusion gene, characterized in that: The kit comprises a primer-probe premix, an enzyme mixture, positive quality control and negative quality control; The primer-probe premix and the enzyme mixture are packaged in a main reaction tube, and the positive quality control and the negative quality control are packaged in separate tubes. The primer-probe premix comprises a forward primer MLL-AF4-F for detecting the fusion gene MLL-AF4 as shown in SEQ ID No. 1, a reverse primer MLL-AF4-R for detecting the fusion gene MLL-AF4 as shown in SEQ ID No. 2, and a probe MLL-AF4-P for detecting the fusion gene MLL-AF4 as shown in SEQ ID No. 3; a forward primer ABL1-F for detecting the reference gene ABL1 as shown in SEQ ID No. 4, a reverse primer ABL1-R for detecting the reference gene ABL1 as shown in SEQ ID No. 5, and a probe ABL1-P for detecting the reference gene ABL1 as shown in SEQ ID No.

6.

5. The test kit for detecting MLL-AF4 fusion gene according to claim 4, characterized by: In the primer-probe premix, the concentration of the forward primer MLL-AF4-F for detecting the fusion gene MLL-AF4 is 400-600 nM, the concentration of the reverse primer MLL-AF4-R for detecting the fusion gene MLL-AF4 is 400-600 nM, and the concentration of the probe MLL-AF4-P for detecting the fusion gene MLL-AF4 is 200-300 nM; In the primer-probe premix, the concentration of the forward primer MLL-AF4-F for detecting the fusion gene MLL-AF4 is 400-600 nM, the concentration of the reverse primer MLL-AF4-R for detecting the fusion gene MLL-AF4 is 400-600 nM, and the concentration of the probe MLL-AF4-P for detecting the fusion gene MLL-AF4 is 200-300 nM; 6. The test kit for detecting MLL-AF4 fusion gene according to claim 4, characterized by: The positive quality control is a sample containing MLL-AF4 fusion gene and ABL1 wild type gene; the negative quality control is a sample containing ABL1 wild type gene but not containing MLL-AF4 fusion gene.

7. The test kit for detecting MLL-AF4 fusion gene according to claim 6, characterized by: The positive quality control contains ≥1000 copies / ul of MLL-AF4 fusion gene and ≥1000 copies / ul of ABL1 wild type gene; the negative quality control contains ≥1000 copies / ul of ABL1 wild type gene.

8. The test kit for detecting MLL-AF4 fusion gene according to claim 4, characterized by: The enzyme mix in the kit is Digital 5X RNAMaster, including dPCR reaction buffer, Taq polymerase, AmpErase enzyme, dATP, dCTP, dGTP, dTTP, dUTP, sodium azide.

9. The test kit for detecting MLL-AF4 fusion gene according to claim 8, characterized by: The volume of the enzyme mixture in the kit accounts for 15% to 75% of the total volume of the reagents in the kit.

10. A detection method for detecting an MLL-AF4 fusion gene, characterized by The kit comprises the following steps: S1, extracting RNA to obtain a to-be-tested RNA template; S2, PCR amplification; A PCR reaction system is prepared, the to-be-tested RNA template is added into a main reaction tube containing a primer probe pre-mixture and an enzyme mixture, water is quantitatively added to 30 uL, and after the reaction system is prepared, the mixture is fully mixed and centrifuged; The primer probe pre-mixture comprises a forward primer MLL-AF4-F for detecting the fusion gene MLL-AF4 as shown in SEQ ID No. 1, a reverse primer MLL-AF4-R for detecting the fusion gene MLL-AF4 as shown in SEQ ID No. 2, a probe MLL-AF4-P for detecting the fusion gene MLL-AF4 as shown in SEQ ID No. 3, a forward primer ABL1-F for detecting the reference gene ABL1 as shown in SEQ ID No. 4, a reverse primer ABL1-R for detecting the reference gene ABL1 as shown in SEQ ID No. 5, and a probe ABL1-P for detecting the reference gene ABL1 as shown in SEQ ID No. 6; The reaction mixture is subjected to PCR reverse transcription amplification reaction; S3, result analysis; the result analysis selects a 1D scatter plot, and a positive result is displayed as red and a negative result is displayed as blue.

Images