CN110551817A

CN110551817A - Kit for detecting human WT1 fusion gene and use method thereof

Info

Publication number: CN110551817A
Application number: CN201810549583.1A
Authority: CN
Inventors: 熊慧; 陶慧卿; 童光铨; 陈嘉铮; 徐任; 俞浩
Original assignee: Suzhou Yuntai Biological Pharmaceutical Co Ltd
Current assignee: Suzhou Yuntai Biological Pharmaceutical Co Ltd
Priority date: 2018-05-31
Filing date: 2018-05-31
Publication date: 2019-12-10

Abstract

The application relates to the field of fusion gene detection, in particular to a kit for detecting a human WT1 fusion gene. The kit adopts one-step reverse transcription, namely, a specific primer and an enzyme are adopted to perform reverse transcription on mRNA in a sample to form cDNA, the reverse-transcribed cDNA directly enters downstream fluorescent quantitative PCR to perform quantitative detection on the RNA of the WT1 gene in a clinical peripheral blood specimen, so that the clinical diagnosis of leukemia is assisted and the selection of related leukemia patients on clinical medicines is guided, and the kit has the technical advantages of high specificity, high accuracy and minimum detection amount.

Description

Kit for detecting human WT1 fusion gene and use method thereof

Technical Field

the application relates to the field of fusion gene detection, in particular to a kit for detecting a human WT1 fusion gene and a using method thereof.

Background

Leukemia is a malignant tumor of the hematopoietic system, and is characterized in that one or more blood cells in the hematopoietic system such as bone marrow and lymph nodes are malignant proliferated and infiltrate into various tissues and organs in the body, resulting in suppression of normal hematopoietic tissue cells and various symptoms. Leukemia is characterized clinically by fever, hemorrhage, anemia, enlargement of the liver, spleen and lymph nodes.

Recent clinical studies have shown that about 50% of leukemias have certain chromosomal aberrations, such as translocations, deletions, insertions, and the like. Related fusion genes are formed under most conditions when chromosome translocation is distorted, and the detection and monitoring of the fusion genes are beneficial to determining a treatment scheme and monitoring leukemia minimal residual lesions. However, about 50% of leukemias have no specific fusion gene, so the use of WT1 gene as a broad-spectrum marker for the detection of minimal residual lesions in leukemias is being accepted by more and more scholars. The WT1 gene is highly expressed in various leukemia cells, and the expression is increased along with the progress of the disease, thereby having the prompting and early warning functions on the prognosis and relapse of leukemia.

the WT1 gene (Wilms tumor gene) is a tumor gene isolated from Wilms tumor cells. The gene is mainly expressed during embryogenesis. In adults only a small amount of kidney, ovary, endometrium, testis, spleen and normal hematopoietic progenitor cells are expressed. The gene has time-phased expression in the blood system, and plays an important role in early hematopoietic cell differentiation.

The WT1 gene is highly expressed in bone marrow/peripheral blood of patients with primary acute leukemia, slightly expressed in normal bone marrow, and extremely low expressed in normal peripheral blood, even undetectable. The WT1 gene is found to be expressed at a high level in most acute myeloid leukemia subtypes, and about 70.0% of acute myeloid leukemia patients have high expression of WT 1-mRNA. In chronic granulosa patients, the expression level of WT1 gene gradually increases as the disease progresses from the chronic stage to the acute stage. Foreign scholars report that the positive rate of WT1 gene expression in acute lymphocytic leukemia is 47.0% -80.0%, and find that the expression of WT1 gene in acute lymphocytic leukemia is lower than that in acute myelocytic leukemia.

At present, the WT1 gene is considered to be closely related to the course of leukemia, wherein (1) the WT1 gene is highly expressed before treatment, the WT1 gene is turned negative after induction chemotherapy, and the WT1 gene expression is also increased although the original cells are less than 5% when the treatment is stopped, (2) the remission period of the WT1 gene is shorter and the patient is relapsed immediately, (3) the WT1 gene level of the patient is very low or undetectable after continuous complete remission, (4) the WT1 gene is rapidly increased after continuous high level or normal reduction, and the increase of the WT 10 ^-2 is more than 10, which indicates that the patient is likely to relapse, and (5) the patient is not likely to relapse after continuous low level expression.

The WT1 gene has high expression in various leukemia, the method for detecting residual leukemia by measuring WT1 gene can be used for almost all leukemia types, the quantitative detection of WT1 gene expression can evaluate the curative effect of chemotherapy or bone marrow transplantation and determine the residual quantity of leukemia cells, and has important clinical significance for early prediction of relapse and prognosis judgment.

Residual disease of leukemia (MRD), which means that a small amount of leukemia cells remain in vivo after a leukemia patient has been induced to achieve complete remission (including after bone marrow transplantation), can be detected by a more sensitive method.

The current methods for detecting MRD are diverse, and have morphology, cell culture, monoclonal antibody, metabolic enzyme assay, cytogenetics, molecular biology and the like, which are all different in accuracy, sensitivity and specificity. Recently developed faster and more applied are molecular biology methods: fluorescence In Situ Hybridization (FISH), Flow Cytometry (FCM), and Polymerase Chain Reaction (PCR) are all important in the detection of MRD.

(1) The FISH has the basic principle that single-stranded DNA (specific probe) for marking fluorescein is hybridized with the complementary DNA (specimen), and the condition of corresponding specific genes of the specimen is reflected through the quantity and the position of fluorescent signals, has accurate quantification, visual image and strong specificity, but has the highest sensitivity of 10 ^-2 -10 ^-3, can not meet the requirement of clinic on the MRD detection level, and is limited in application.

(2) FCM, using monoclonal antibody marked with fluorescein to detect the expression percentage and fluorescence intensity of different surface differentiation antigens of all cells in the specimen, expressing no or low expression on normal marrow or peripheral blood cells and expressing or high expression immunophenotype on leukemia cells to quantify MRD, FCM has wide application range, low price, large information amount, easy direct and accurate quantification, but its stable sensitivity is 10 ^-4, however, the related research shows that whether the residual leukemia cells can be reduced to less than 10 ^-4 after treatment has important meaning to the long-term remission of diseases.

(3) PCR the basic principle of PCR detection of MRD is that the target molecule mark is used as the target molecule, the target molecule is amplified by million times through polymerase chain reaction under the action of primer, and then the gel electrophoresis shows that the PCR sensitivity is 10 ^-5 -10 ^-6, the occurrence of real-time quantitative PCR well solves the problem that the common PCR is not easy to be directly and accurately quantified, the detection specificity is improved, false positive and false negative are reduced, and the PCR detection method is an important index for evaluating the MRD detection method.

in view of the above, the present application provides a kit for detecting human WT1 fusion gene by using fluorescent quantitative PCR technology.

Disclosure of Invention

The invention of the present application aims to provide a kit for detecting the human WT1 fusion gene.

in order to accomplish the purpose of the application, the technical scheme is as follows:

the application provides a kit for detecting a human WT1 fusion gene, which comprises a nucleic acid amplification reagent, wherein the nucleic acid amplification reagent comprises WT1PCR reaction liquid, internal reference PCR reaction liquid and mixed enzyme liquid;

The WT1PCR reaction solution contains a primer and a probe for detecting a WT1 fusion gene, the internal reference PCR reaction solution contains a primer and a probe for detecting an internal reference gene, and the mixed enzyme solution contains Taq enzyme, UNG enzyme, RT enzyme and Rnasin;

The primer and the probe for detecting the WT1 fusion gene comprise an upstream primer shown by SEQ ID NO. 1, a downstream primer shown by SEQ ID NO. 2 and a probe shown by SEQ ID NO. 3, wherein two ends of the probe are marked with fluorescent groups;

The internal reference PCR reaction solution is used for controlling the detection effectiveness, the primers and the probes for detecting the internal reference gene comprise an upstream primer shown by SEQ ID NO. 4, a downstream primer shown by SEQ ID NO. 5 and a probe shown by SEQ ID NO. 6, and fluorescent groups are marked at two ends of the probe.

optionally, FAM is marked at the 5 'end and TAMRA is marked at the 3' end of the nucleotide sequence shown in SEQ ID NO. 3, FAM is marked at the 5 'end and TAMRA is marked at the 3' end of the nucleotide sequence shown in SEQ ID NO. 6.

optionally, the kit further comprises a control substance, the control substance comprises a negative control and a positive control, the negative control is process water, and the positive control contains a K562 cell strain lysate.

optionally, the kit further comprises a reference substance, wherein the reference substance is used for quantifying a target gene and an internal reference gene, and the reference substance comprises the components shown in table 1:

TABLE 1

Components	The main components in the components
		WT1 reference 1: 1X 10⁶copies	WT1 plasmid DNA
WT1 reference 2: 1X 10⁵copies	WT1 plasmid DNA
		WT1 reference 3: 1X 10⁴copies	WT1 plasmid DNA
WT1 reference 4: 1X 10³copies	WT1 plasmid DNA
		Internal reference product 1: 1X 10⁶copies	Internal reference plasmid DNA
Internal reference 2: 1X 10⁵copies	Internal reference plasmid DNA
		Internal reference product 3: 1X 10⁴copies	internal reference plasmid DNA
Internal reference 4: 1X 10³copies	Internal reference plasmid DNA

Wherein, the nucleotide sequence of the WT1 plasmid DNA is shown as SEQ ID NO. 7, and the nucleotide sequence of the internal reference plasmid DNA is shown as SEQ ID NO. 8.

Optionally, the sample to which the kit is applied is selected from a peripheral blood sample.

Optionally, the mixed enzyme solution contains 0.6-0.9 volume part of Taq enzyme, 0.1-0.3 volume part of UNG enzyme, 0.25-0.45 volume part of RT enzyme and 0.2-0.3 volume part of Rnasin; the control enzyme solution contains 0.6-0.9 volume part of Taq enzyme and 0.1-0.3 volume part of UNG enzyme;

preferably, the mixed enzyme solution contains 0.8 volume part of Taq enzyme, 0.2 volume part of UNG enzyme, 0.375 volume part of RT enzyme and 0.25 volume part of Rnasin; the control enzyme solution contains 0.8 volume part of Taq enzyme and 0.2 volume part of UNG enzyme;

Wherein the unit of activity of UNG enzyme is 1U/. mu.l, the unit of activity of Taq enzyme is 5U/. mu.l, and the unit of activity of RT enzyme is 200U/. mu.l.

the application also relates to a primer and a probe for detecting the fusion gene of the human WT1, wherein the primer and the probe for detecting the fusion gene of the WT1 comprise an upstream primer shown by SEQ ID NO. 1, a downstream primer shown by SEQ ID NO. 2 and a probe shown by SEQ ID NO. 3, and two ends of the probe are marked with fluorescent groups;

preferably, the kit also comprises a primer and a probe for detecting the reference gene, wherein the primer and the probe for detecting the reference gene comprise an upstream primer shown by SEQ ID NO. 4, a downstream primer shown by SEQ ID NO. 5 and a probe shown by SEQ ID NO. 6, and fluorescent groups are marked at two ends of the probe.

Optionally, the 5 'end of the nucleotide sequence shown in SEQ ID NO. 3 is marked with FAM, and the 3' end is marked with TAMRA; the 5 'end of the nucleotide sequence shown in SEQ ID NO. 6 is marked with FAM, and the 3' end is marked with TAMRA.

The application also relates to a method for using the kit, which at least comprises the following steps:

(1) Sample treatment: extracting RNA to obtain a sample;

(2) Preparing an amplification reagent: mixing the WT1PCR reaction solution with the mixed enzyme solution to obtain a WT1PCR premix; mixing the internal reference PCR reaction solution with the mixed enzyme solution to obtain an internal reference PCR premix;

(3) Sample adding: respectively sampling from a sample, a negative control, a positive control, WT1 reference products 1-WT 1 reference products 4 and internal reference products 1-4, respectively adding into each PCR premix and each internal reference PCR premix,

(4) PCR amplification;

(5) And (6) detecting.

The technical scheme of the application has at least the following beneficial effects:

the kit adopts a reverse transcription-fluorescent quantitative PCR (RT-RQ-PCR) technology, namely mRNA in a sample is subjected to reverse transcription before the fluorescent quantitative PCR is carried out, a template of the fluorescent quantitative PCR is cDNA instead of genomic DNA, and a two-step reverse transcription method and a one-step reverse transcription method are commonly used. The kit adopts one-step reverse transcription, namely, mRNA in a sample is reversely transcribed into cDNA by adopting a specific primer and an enzyme, and the cDNA after reverse transcription directly enters downstream fluorescent quantitative PCR. The kit adopts PCR combined with real-time fluorescent probe technology to carry out quantitative detection on the RNA of WT1 gene in a clinical peripheral blood specimen, thereby assisting the clinical diagnosis of leukemia and guiding the selection of related leukemia patients to clinical drugs. Has the technical advantages of high specificity, high accuracy and minimum detection amount:

1. The kit has the advantages of high minimum detection quantity, sensitivity of 10 ² copies, wide linear range and capability of carrying out quantification in the range of 10 ² -10 ¹⁰ copies.

2. The kit has strong specificity: the fluorescent probe and the template are hybridized, and the change of a fluorescent signal in the PCR amplification process is directly detected through a photoelectric conduction system to obtain a quantitative result, so that the specificity is greatly improved compared with the traditional PCR technology.

3. the kit has good stability, because the threshold value is set in the exponential amplification stage, the concentration of each reaction component is relatively stable, and the C _T value and the logarithm of a fluorescence signal form a linear relation.

4. The reagent kit has little pollution: generally, PCR products need to be observed through agarose gel electrophoresis, ethidium bromide staining and ultraviolet light, or detected through polyacrylamide gel electrophoresis and silver staining, not only various instruments are needed, but also time and labor are wasted, and a staining agent ethidium bromide is harmful to human bodies, and the complicated experimental processes provide opportunities for pollution and false positive. The kit adopts one-step reverse transcription, only a cover needs to be opened once during sample adding, the subsequent process is completely closed tube operation, and the treatment after reverse transcription or PCR is not needed, so the operation is simple and convenient, the time is saved, the pollution is reduced, and a plurality of defects in the conventional PCR operation are avoided.

The present application is further illustrated with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application.

Detailed Description

The kit of the embodiment combines the fluorescent quantitative PCR technology and the reverse transcription technology, namely RT-RQ-PCR technology, and the expression level of the WT1 fusion gene in a sample is detected by a one-step method by applying the fluorescent quantitative PCR technology while reverse transcribing mRNA in the sample into cDNA. The kit provided by the embodiment of the application only needs to open the cover once during sample adding, the subsequent process is completely closed tube operation, and reverse transcription or PCR post-treatment is not needed, so that the operation is simple and convenient, the time is saved, the pollution is reduced, a plurality of defects in conventional PCR operation are avoided, and the kit has the technical advantages of high specificity, high accuracy and minimum detection amount.

Meanwhile, the kit of the embodiment of the present application also uses a uridine enzyme (UNG) antipollution system, which selectively degrades U-DNA by heating, to prevent contamination of the previous PCR amplification product; and the effectiveness of the whole kit detection process is controlled by adopting an internal reference gene.

In addition, the kit of the embodiment of the application also adopts two sets of reference substance standard curves to respectively quantify the target gene and the reference gene so as to achieve an accurate ratio.

Specifically, the kit of the embodiment of the present application is specifically shown in table 2:

TABLE 2

Wherein the mixed enzyme solution contains 0.6-0.9 volume part of Taq enzyme, 0.1-0.3 volume part of UNG enzyme, 0.25-0.45 volume part of RT enzyme and 0.2-0.3 volume part of Rnasin; preferably 0.8 volume part of Taq enzyme, 0.2 volume part of UNG enzyme, 0.375 volume part of RT enzyme and 0.25 volume part of Rnasin;

The reference enzyme solution contains 0.6-0.9 volume part of Taq enzyme and 0.1-0.3 volume part of UNG enzyme; preferably, the control enzyme solution contains 0.8 volume part of Taq enzyme and 0.2 volume part of UNG enzyme;

specifically, the nucleotide sequences of the primers and probes are specifically shown in table 3:

table 3:

specifically, the nucleotide sequence of the plasmid DNA is specifically shown in table 4:

TABLE 4

The sample requirements of the kit of the present application are:

Collecting samples: the kit is suitable for anticoagulation peripheral blood samples. The anticoagulation sample requirement is as follows: the anticoagulant is EDTA, sodium oxalate or sodium citrate.

Sample preservation: uniformly mixing an anticoagulated peripheral blood sample with Trizol in a ratio of 1:3, or enriching nucleated cells of broken red blood cells of a fresh sample, suspending, adding Trizol by 3 times of volume, uniformly mixing, and storing for no more than 24 hours at 4 ℃. Stored at-20 ℃ for a long time.

Sample transportation: storing in Trizol anticoagulated peripheral blood sample at 4 deg.C, and transporting at low temperature.

RNA extraction requires: extraction was performed using the Rneasy Mini kit. Or extracting by using a Trizol method. The extracted RNA is recommended to be detected immediately, otherwise, the RNA is required to be stored below 20 ℃ in a short time and at 80 ℃ in a long time, so that the RNA degradation is prevented.

Fresh peripheral blood samples were recommended for naive patients. The mononuclear cells obtained by separating the sample by lymphocyte separating medium are recommended for the patient with Minimal Residual Disease (MRD). According to the definition of the minimum residual focus (namely, the number of cancer cells in 1 ten thousand cells is detected, and if the number is less than 1, the cancer cells are judged to be negative), therefore, for a patient after treatment, in order to detect whether the minimum residual focus exists in a bone marrow specimen, more than 1ug of total RNA is recommended to be taken for an experiment, the stock solution is subjected to WT1 fusion gene detection, and after the RNA stock solution is diluted by 100 times, internal reference gene detection is carried out so as to ensure that the concentration of the corresponding detection gene is in a linear range.

the application method of the kit comprises the following steps:

1. Sample treatment (Note: 200. mu.l each of negative control and positive control was treated with the specimen):

RNA nucleic acid was extracted using Rneasy Mini kit, 400. mu.l of the specimen stored in Trizol was taken, 400. mu.l of chloroform was added thereto, and the mixture was mixed well and allowed to stand at room temperature for 5 minutes. Centrifuging at 15000rpm for 10 min, sucking colorless supernatant after centrifugation, adding equal volume of buffer RLT, and mixing uniformly, wherein the rest steps are described in the Rneasy Mini kit instruction.

Or extracting RNA by using a Trizol method, which comprises the following specific steps:

400 μ l of the specimen stored in Trizol was taken, 400 μ l of chloroform was added thereto, and the mixture was mixed well and allowed to stand at room temperature for 5 minutes. Centrifuged at 15000rpm for 10 minutes and the colorless supernatant was aspirated.

② adding equal volume of precooled isopropanol into the supernatant, reversing and mixing evenly, and then placing for 10 minutes at room temperature. Centrifuge at 15000rpm for 10 minutes and discard the supernatant.

③ to the centrifuge tube, 300. mu.l of precooled 70% ethanol was added, centrifuged at 15000rpm for 5 minutes, and the supernatant was discarded. The ethanol was evaporated at room temperature for 10 minutes.

200. mu.l DEPC-ddH ₂ O was added and dissolved to serve as a template for the relevant PCR reaction.

2. Preparation of amplification reagents:

And taking the WT1PCR reaction solution, the internal reference PCR reaction solution, the mixed enzyme solution, the positive control and the reference substance out of the kit, melting at room temperature, shaking and mixing uniformly, and centrifuging at 2000rpm for 10 s.

the reaction system is prepared as follows: WT1PCR reaction solution 8. mu.l + mixed enzyme solution 2. mu.l or reference gene PCR reaction solution 8. mu.l + mixed enzyme solution 2. mu.l (note that the number of tubes should be the sum of the sample number, 2-tube reference, 4-tube reference).

the prepared PCR premix is respectively subpackaged into each PCR tube according to the amount of 10 mu l per tube.

3. sample adding: mu.l of each of the control, reference and sample treatment solutions was added to a centrifuge tube containing the PCR premix, and the tube was closed and transferred to the detection zone.

PCR amplification:

4.1 amplification conditions

ABI series fluorescent PCR detector, Bio-Rad CFX96 fluorescent PCR detector amplification conditions: 30min at 42 ℃; 94 ℃ for 5 min; (94 ℃, 15 sec; 60 ℃, 60sec)40 cycles. The reaction system was 25. mu.l. The fluorescent signal was collected at 60 ℃ in the second step of the PCR cycle.

Stratagene Mx3000p fluorescent PCR detector amplification conditions: 30min at 42 ℃; 94 ℃ for 5 min; (94 ℃, 45 sec; 60 ℃, 80sec)40 cycles. The reaction system was 25. mu.l. The fluorescent signal was collected at 60 ℃ in the second step of the PCR cycle.

4.2 detection channel and reference fluorescence

the detection channels of the ABI 7300 fluorescent PCR detector and the ABI 7500 fluorescent PCR detector are as follows: FAM-TAMRA, reference fluorescence was set to none.

the detection channel of the Bio-Rad CFX96 fluorescent PCR detector is as follows: FAM.

The detection channels of the Stratagene Mx3000p fluorescent PCR detector are: FAM. The reference fluorescence was set to none.

5. And (3) detection:

(1) Determination of the baseline: the software defaults to baseline the mean fluorescence signal for 3-15 cycles. In the experiment, the section with smaller curve fluctuation and more stability is generally selected as the baseline, and the user can adjust the curve according to the actual situation. The starting point is to avoid the signal increase due to high temperature for the first few cycles, where the signal has dropped to background level and is stable, and the ending point is to avoid covering where the signal has started to increase significantly. According to different trends of the experimental curve, the general start value can be selected to be between 3 and 12; the stop value is selected on the basis that the interval between the starting point and the end point can be more than 8 cycles preferably so as to better meet the mathematical requirement of statistical baseline standard deviation.

(2) determination of the threshold: in the case of no amplification of the negative control, the threshold is set at the highest point of the sample without amplification curve, i.e. higher than the highest point of the growth curve without amplification (i.e. no point appears in the column "Component" of the result analysis), and the initial threshold is determined on the basis that no negative control is detected.

(3) note that: for the same sample, the analysis of the standard curves of negative and positive controls and reference substances needs to be carried out at the same time, and the reaction liquid of the target gene and the internal reference gene needs to be analyzed by using the same baseline and threshold value.

the present application is further illustrated by the following specific examples, which are all commercially available materials unless otherwise specified.

Example 1

a kit for detecting a human WT1 fusion gene, which comprises the following components shown in Table 5:

Table 5:

the packaging and contents of the components of the kit are shown in table 6:

Table 6:

Example 2: linear detection

Selecting 100 ul of WT1 plasmid and plasmid of internal reference gene with determined concentration, preparing high concentration DNA sample (L0), using the two samples as sample linear quality control L0 of the kit of example 1, then adding process water at 1: the L0 samples were serially diluted with 10 gradients to give two sets of linear quality controls L1, L2, L3 and L4, respectively. The specific compositions are shown in tables 9 and 10.

And (3) performing linear fitting by taking the logarithmic value of the concentration as Y and the CT mean value as X, and calculating the linear correlation coefficient r of the linear fitting. The specific experimental results are shown in tables 7 and 8:

TABLE 7

WT1 linear quality control article	Number of entry reaction templates (copies)
		WT1-L0	5×10⁶
WT1-L1	5×10⁵
		WT1-L2	5×10⁴
WT1-L3	5000
		WT1-L4	500
r value	≥0.980

TABLE 8

Internal reference gene linear quality control product	Number of entry reaction templates (copies)
		Internal reference of formula-L0	5×10⁶
Internal reference of formula-L1	5×10⁵
		Internal reference of formula-L2	5×10⁴
ABL-L3	5000
		internal reference of formula-L4	500
r value	≥0.980

The operation is carried out according to the detection method of the kit in the embodiment 1, the logarithmic value of the concentration of the reference substance is Y, the CT mean value is X, the linear fitting is carried out, the linear correlation coefficient r is calculated, and the r value is more than or equal to 0.980.

Example 3: accuracy detection of the kit of the present application

the kit of example 1 was used to test for accuracy references: the preparation method of the accuracy reference substance is shown in table 9:

TABLE 9

The prepared accurate reference substances were tested, and the experimental results are shown in table 10:

Watch 10

by adopting the kit for detecting the accuracy reference substance, the positive coincidence rate is 100%.

Example 4: assay-specific detection of the kits of the present application

The specific reference was tested using the kit of example 1: the preparation method of the specific reference substances is shown in table 11:

TABLE 11

The prepared assay-specific reference was tested and the experimental results are shown in table 12:

TABLE 12

When the kit is used for detecting the specific reference sample, the negative coincidence rate is 100%.

example 4: precision detection of the kit of the present application

The precision reference was tested using the kit of example 1: the preparation method of the precision reference substance is shown in table 13:

Watch 13

Detecting the prepared precision reference substance; the experimental results obtained are shown in table 14:

TABLE 14

The reagent kit can detect each precision reference product by repeating the detection in corresponding reaction liquid for 10 times, and the CV value of experimental data is less than or equal to 5 percent.

Example 5: minimum detection amount detection of kit of the application

The kit of example 1 was used to test the lowest detectable reference: the formulation of the lowest detectable amount reference is shown in table 15:

watch 15

Numbering	Entering a reaction mould (copies)	volume of each mark
			WT1-LN	100	mu.l of the linear quality control product L4 was added to 80. mu.l of water and mixed by shaking.

The lowest detected amount obtained by the preparation was detected, and the obtained experimental results are shown in table 16:

TABLE 16

Reference product with minimum detection amount	C_Tvalue of
		WT1-LN	31.86

The kit is used for detecting the reference substance with the lowest detection amount, and the lowest detection amount is 100 copies/reaction.

Although the present application has been described with reference to preferred embodiments, it is not intended to limit the scope of the claims, and many possible variations and modifications may be made by one skilled in the art without departing from the spirit of the application.

Sequence listing

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Claims

1. A kit for detecting a human WT1 fusion gene is characterized by comprising a nucleic acid amplification reagent, wherein the nucleic acid amplification reagent comprises WT1PCR reaction liquid, internal reference PCR reaction liquid and mixed enzyme liquid;

The primer and the probe for detecting the WT1 fusion gene comprise an upstream primer shown by SEQ ID NO. 1 and a downstream primer shown by SEQ ID NO. 2 and a probe shown by SEQ ID NO. 3, wherein two ends of the probe are marked with fluorescent groups;

the primer and the probe for detecting the reference gene comprise an upstream primer shown by SEQ ID NO. 4, a downstream primer shown by SEQ ID NO. 5 and a probe shown by SEQ ID NO. 6, wherein two ends of the probe are marked with fluorescent groups.

2. The kit according to claim 1, wherein the nucleotide sequence shown in SEQ ID NO. 3 is labeled with FAM at the 5 'end and TAMRA at the 3' end, and the nucleotide sequence shown in SEQ ID NO. 6 is labeled with FAM at the 5 'end and TAMRA at the 3' end.

3. The kit according to claim 1, further comprising a control, wherein the control comprises a negative control and a positive control, the negative control is process water, and the positive control contains a K562 cell line lysate.

4. The kit according to claim 1, further comprising a reference substance for quantifying the target gene and the internal reference gene, wherein the reference substance comprises:

5. The kit of claim 1, wherein the sample to which the kit is applied is selected from a peripheral blood sample.

6. The kit according to claim 1, wherein the mixed enzyme solution comprises 0.6 to 0.9 parts by volume of Taq enzyme, 0.1 to 0.3 parts by volume of UNG enzyme, 0.25 to 0.45 parts by volume of RT enzyme, and 0.2 to 0.3 parts by volume of Rnasin; the control enzyme solution contains 0.6-0.9 volume part of Taq enzyme and 0.1-0.3 volume part of UNG enzyme;

7. The primer and the probe for detecting the human WT1 fusion gene are characterized in that the primer and the probe for detecting the WT1 fusion gene comprise an upstream primer shown by SEQ ID NO. 1, a downstream primer shown by SEQ ID NO. 2 and a probe shown by SEQ ID NO. 3, wherein two ends of the probe are marked with fluorescent groups;

8. the primers and probes as claimed in claim 7, wherein the nucleotide sequence shown in SEQ ID NO. 3 is labeled with FAM at the 5 'end and TAMRA at the 3' end; the 5 'end of the nucleotide sequence shown in SEQ ID NO. 6 is marked with FAM, and the 3' end is marked with TAMRA.

9. A method of using the kit according to any one of claims 1 to 8, comprising at least the steps of:

(1) sample treatment: extracting RNA to obtain a sample;

(2) Preparing an amplification reagent:

Mixing the WT1PCR reaction solution with the mixed enzyme solution to obtain a WT1PCR premix; mixing the internal reference PCR reaction solution with the mixed enzyme solution to obtain an internal reference PCR premix;

(3) Sample adding: respectively sampling samples from a sample, a negative control, a positive control, WT1 reference products 1-WT 1 reference products 4 and internal reference products 1-4, and respectively adding the samples into each PCR premix and each internal reference PCR premix;

(4) PCR amplification;

(5) And (6) detecting.