CN115058515A - RARG-HNRNPM fusion gene detection and clinical application kit - Google Patents

RARG-HNRNPM fusion gene detection and clinical application kit Download PDF

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CN115058515A
CN115058515A CN202210417036.4A CN202210417036A CN115058515A CN 115058515 A CN115058515 A CN 115058515A CN 202210417036 A CN202210417036 A CN 202210417036A CN 115058515 A CN115058515 A CN 115058515A
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rarg
hnrnpm
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秘营昌
侯降雪
宋洋
刘凯奇
王建祥
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Institute of Hematology and Blood Diseases Hospital of CAMS and PUMC
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Abstract

The invention relates to a RARG-HNRNPM fusion gene detection and clinical application kit, which is characterized by comprising a reagent for diagnosing the mutant acute promyelocytic leukemia based on a Taqman probe and/or absolutely quantifying the RARG-HNRNPM fusion gene by using the detection kit.

Description

RARG-HNRNPM fusion gene detection and clinical application kit
Technical Field
The invention relates to the technical field of molecular biology, in particular to a RARG-HNRNPM fusion gene detection and clinical application kit.
Background
Acute Promyelocytic Leukemia (APL) is a special type of acute myeloid leukemia, typically characterized by a t (15; 17) (q 22; q21) chromosomal translocation and formation of a PML-RARA fusion gene. Its clinical features include severe coagulopathy and bleeding tendency, and sensitivity to arsenous Acid (ATO), all-trans retinoic acid (ATRA), and anthracycline treatments; is considered to be a "curable type leukemia". Variant APL, on the other hand, refers to the rare cases with clinical manifestations, morphological and immunophenotypic characteristics identical to APL, but lacking the PML-RARA fusion gene and generally insensitive to ATO and/or ATRA. The variant APL fusion genes reported so far include RARA, RARB and RARG-related fusion genes, and these patients all exhibit clinical, morphological and immunophenotypic characteristics similar to APL. 12 RARG rearrangement cases searched in literature mainly relate to CPSF6-RARG, NUP98-RARG, NPM1-RARG-NPM1, PML-RARG and HNRNPC-RARG. According to the above reports, RARG rearrangement patients are resistant to ATRA, but are partially sensitive to anthracycline-based chemotherapy, the treatment regimen is different from typical APL, and the prognosis is to be further observed.
In order to evaluate the efficacy of typical APL and predict the risk of relapse, the current monitoring methods used clinically include flow-induced Minimal Residual Disease (MRD), quantification of PML-RARA fusion gene, etc., wherein the quantitative monitoring of PML-RARA fusion gene is more sensitive to disease state prediction. However, for RARG-HNRNPM positive variant APL, a molecular index capable of more accurately evaluating a disease state is lacking at present.
Disclosure of Invention
According to the technical problems, the invention provides an absolute quantitative reagent for detecting the RARG-HNRNPM fusion gene by using a Taqman probe-based mutant acute promyelocytic leukemia diagnosis and/or detection kit.
The kit comprises an upstream primer, a downstream primer, a Taqman probe, an internal reference, a PCR reaction mixed solution and DEPC-H 2 O。
Upstream primer of RARG-HNRNPM ATGAAAATCACCGACCTCCG
RARG-HNRNPM downstream primer: CCTTGACTTTCCTTCAGCGT
AGCACTAAGGTTGGTGAGGTAAC RARG-HNRNPM probe
Internal reference GAPDH: hs02786624_ g1 introduced ordered (applied biosystems)
PCR buffer Taqman TM Gene Expression Master Mix 4369016(Applied Biosystems) including dNTP, Ca required for the reaction 2+ ,Mg 2+ And the like.
Positive control: target plasmid standard containing fusion gene with RARG-HNRNPM
Negative control: cDNA of APL cell line NB4
FAM group is marked at the 5 'end of RARG-HNRNPM fusion gene Taqman probe, and TAM group is marked at the 3' end; FAM group is marked at the 5 'end of Taqman probe of internal reference gene GAPDH, and NFQ group is marked at the 3' end. FAM is a fluorescent reporter group and NFQ is a quencher group. When the target DNA molecules exist in the sample to be detected, the probe is combined with the target DNA molecules, and the probe is degraded to release the fluorescent reporter group to emit fluorescence. When the target DNA molecule does not exist in the sample to be detected, the fluorescence reporter group is quenched and does not emit fluorescence.
The specific operation steps of the kit are as follows:
firstly, collecting and separating a sample to obtain mononuclear cells and extracting mRNA of the mononuclear cells
Second, mRNA of mononuclear cell is reversely transcribed into cDNA as sample to be detected
Thirdly, configuring a reaction system, and carrying out on-machine detection on a real-time fluorescent PCR instrument, wherein the reaction conditions are as follows: preheating at 50 deg.C for 2 min; pre-denaturation at 95 ℃ for 10 min; the temperature is 95 ℃ for 15s, the temperature is 60 ℃ for 1min, and the reaction is circulated for 40 cycles; 95 ℃ for 15s, 60 ℃ for 1min and 95 ℃ for 1 s.
And (4) analyzing results: the results obtained were obtained using QuantStudio TM Design&Analysis Software Analysis. The discrimination results of the kit are as follows:
when the internal reference, the positive sample control and the negative sample control are normal, the sample to be detected has no target gene amplification, and the signal curve is judged to be negative;
when the internal reference, the positive sample control and the negative sample control are normal, purposeful gene amplification is carried out on the sample to be detected, and the signal curve is judged to be positive;
and thirdly, when the internal reference and (or) the positive sample contrast and (or) the negative sample contrast are abnormal, the target gene amplification signal curve of the sample to be detected is meaningless and needs to be amplified again after being adjusted.
The beneficial effects of the invention are as follows:
1. the invention is used for detecting the variant acute promyelocytic leukemia, discovers and verifies the special fusion gene RARG-HNRNPM in the variant APL for the first time, can be used for detecting the expression level of the fusion gene in a patient specimen, and can be used for disease diagnosis and MRD monitoring.
2. The kit detects RARG-HNRNPM positive mutant APL samples, has high sensitivity, strong specificity and convenient operation, and can quickly detect the RARG-HNRNPM fusion gene expression level.
For variant APL, currently, diagnosis mostly depends on transcriptome sequencing to identify fusion genes of the APL, and a detection means for subsequently monitoring the disease development condition is lacked. The RARG-HNRNPM fusion gene detected by the invention can be used as a molecular marker of a patient for clinical diagnosis (and determination of treatment scheme selection) and monitoring the MRD level in the treatment process. The real-time quantitative fluorescence PCR method based on the Taqman probe can amplify low-abundance fusion gene signals from a patient sample, and is an important means for monitoring residual diseases, guiding subsequent treatment of patients and predicting relapse.
The kit for detecting the target gene RARG-HNRNPM of the mutant APL is designed by selecting a Taqman probe method and taking a wild type NB4 cell line as negative control and a target plasmid containing the RARG-HNRNPM fusion gene as positive control, and simultaneously, the preliminary diagnosis, morphological relief and molecular relief of a patient containing the RARG-HNRNPM fusion gene and the quantitative conditions of the RARG-HNRNPM fusion gene in three disease states are determined. The invention discovers that the expression of the fusion gene is higher in the initial diagnosis state of a patient during research; when the patient receives chemotherapy to achieve basic morphological remission, the expression of the fusion gene RARG-HNRNPM is reduced (but can still be detected); when the bone marrow remission quality of the patient is further improved (complete molecular remission is achieved), the fusion gene RARG-HNRNPM can not be detected. The detection trend of the fusion gene is consistent with the disease state, and the results show that RARG-HNRNPM is a molecular marker capable of indicating the disease state, and the accuracy and reliability of the kit are also proved.
The fusion gene probe provided by the invention is used for detecting the quantification of the fusion gene in the bone marrow sample of the patient in the initial state, the morphological remission state and the molecular remission state, so that the high expression of the fusion gene in the diagnosis process, the reduction of the expression amount in the morphological remission state and the conversion of the fusion gene into negative (undetectable) after the complete molecular remission are observed. The results show that the RARG-HNRNPM can be used as a novel MRD monitoring molecular marker for the patient with the variant APL; and the kit is used for detecting the expression of the RARG-HNRNPM fusion gene in a sample, has accurate and reliable result, and can quickly and accurately reflect the disease state of a patient.
Drawings
FIG. 1 is a diagram showing the structure of the RARG-HNRNPM fusion gene breakpoint and fusion fragment;
FIG. 2 is a PCR agarose gel electrophoresis of fusion sites in a patient's preliminary sample;
FIG. 3 is a graph of fusion site Sanger sequencing validation in a patient preliminary diagnosis sample;
FIG. 4 is a positive control plasmid map;
FIG. 5 is a diagram showing the results of detecting RARG-HNRNPM fusion gene according to the present invention;
FIG. 6 is a graph showing the change in mRNA levels of RARG-HNRNPM in patients who were RARG-HNRNPM positive over time during treatment.
Detailed Description
Example 1
Application of bioinformatics to detection of RARG-HNRNPM fusion gene in 1 patient with variant acute promyelocytic leukemia
25-year-old women are admitted to the hospital at 11/5 of 2020, and have similar immunophenotype and bone marrow cell morphology to APL, and no PML-RARA fusion gene is detected by conventional fusion gene screening and fluorescence in situ hybridization (FISH, RARA). In order to determine the disease type, the RNA of the bone marrow mononuclear cell is extracted, the cDNA is reversely recorded, a library is constructed, and transcriptome sequencing analysis is carried out on the basis of an illumina sequencing platform. An RARG-HNRNPM FUSION gene which is not reported in the past is screened out by using a bioinformatics technology through alignment of STAR-FUSION software based on a human genome (GRch37/hg19) (figure 1), and reverse transcription-polymerase chain reaction (RT-PCR) and Sanger sequencing verification are applied (figure 2 and figure 3).
The patient was treated with 2020.11.10 plus DA regimen (DNR 100mg d1-2,80mg d 3; Ara-c 200mg d 1-7). Bone marrow was shown to achieve morphological remission (CR) by a follow-up of bone marrow at 11/25/2020, and was treated with HAA regimen chemotherapy (HHT 3mg d 1-7; Ara-c 100mg d 1-7; Acla 20mg d3-7) at 2020.12.1. The patient was asked to review the bone puncture 24 months after 2 months in 2021, which indicated that the bone marrow was in complete molecular remission.
RARG-HNRNPM fusion gene sequence obtained by the Sanger sequencing method is applied to construct RARG-HNRNPM-MSCV-IRES-GFP plasmid as a positive control standard (figure 3),
example 2
Detection of HNRNPM-RARG fusion gene by Taqman probe method
1. Extraction of mononuclear cells from a patient's bone marrow specimen
2. Extracting total RNA of cells: extraction is carried out in a designated RNA pollution-free area in a laboratory, and the extraction of RNA is carried out by using an EP tube and a gun tip after being treated by DEPC water, so that the whole extraction process is ensured to be free of RNA pollution.
1) Taking a specimen treated by fresh trizol or frozen at-80 deg.C, 1 × 10 6 Treating cells/EP tube with trizol, repeatedly blowing with RNase-free gun tip, mixing, standing at room temperature for 5min
2) Adding 200 μ L chloroform, shaking vigorously for 15s, standing at room temperature for 10min
3) Centrifuging at 12000rpm and 4 deg.C for 15min, separating the liquid into upper and lower layers, and carefully sucking the supernatant to obtain a new 1.5ml EP tube.
4) Adding isopropanol with the same volume, and shaking up for tens of times by turning upside down. Standing on ice for 10 min.
5) After precipitation, centrifugation was carried out at 12000rpm at 4 ℃ for 15min to remove the supernatant liquid.
6) 1ml of ethanol in 75% DEPC water was added for washing the precipitate.
7) Centrifuging at 10000rpm and 4 deg.C for 5min, removing ethanol, air drying for 3-5min, and changing the precipitate from white to transparent.
3. Reverse cDNA RNA was inverted to cDNA according to the instructions of TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit from TransGen Biotech.
Taqman probe method fluorescent probe quantitative PCR
The experimental principle is as follows: a fluorescence labeling probe containing a fluorescence reporter group FAM and a quenching group TAMRA is added into a traditional PCR reaction system, and a target gene is quantitatively detected more accurately. For the purpose of detecting the target gene expression of the patient under initial diagnosis, partial remission and complete morphological remission, GAPDH of the patient is set as a reference gene, 3 biological repetitions are carried out for each gene detection, and the average value is calculated as the Ct value of the gene. And (5) calculating (average Ct of the target gene-average Ct of the reference gene GAPDH) by adopting a delta-delta Ct method to obtain a corresponding delta Ct value.
(1) Early preparation
According to the NCBI database of the national center for biotechnology information, various gene sequences are searched and primers are designed.
The corresponding probes and primers of the HNRNPM gene sequence (Ensembl ID: ENSG00000099783), RARG gene sequence (Ensembl ID: ENSG00000172819) and GAPDH gene sequence (Ensembl ID: ENSG00000111640) and the reference gene are designed as follows:
RARG-HNRNPM-F:ATGAAAATCACCGACCTCCG
RARG-HNRNPM-R：CCTTGACTTTCCTTCAGCGT
RARG-HNRNPM-Probe:AGCACTAAGGTTGGTGAGGTAAC
GAPDH is supplied by TaqMan: hs02786624_ g1 introduced ordered (applied biosystems) positive control: contains plasmid standard with RARG-HNRNPM fusion gene (FIG. 3). We diluted 10-fold the plasmid standard and verified the sensitivity of the designed target gene probe primer by detecting the absolute copy number of the target gene in the plasmid (FIG. 4).
The negative control sample is APL cell line NB4, RNA is extracted and reversed to cDNA
(2) Reaction system: reference Taqman TM In the specification of Gene Expression Master Mix, the kit adopts a 20 mu L system
1) PCR reaction solution system: each well system is 19.5 muL, including 10 muL of TaqMan Gene Expression Master Mix (2X), 0.8 muL of forward primer of Gene to be detected, 0.8 muL of backward primer of Gene to be detected, and 6.9 muL of RNAase-free Water.
Preparing a reaction reagent system: according to the parts of a person to be detected, preparing a detection system PCR reaction solution x 0.5 mu L ═ 19.5 mu LPCR reaction solution x (N parts of specimens, 1 part of positive control, 1 part of negative control and 1 part of blank control), and subpackaging 19.5 mu L of each person.
(3) Sample adding instruction: adding 0.5 mu L of cDNA of a sample to be detected into the PCR reaction solution; directly adding 0.5 mu L of positive control substance and negative control substance into the positive control substance and the negative control substance; the blank was added with 0.5. mu.L of physiological saline.
(4) And (3) computer detection: in a real-time fluorescent PCR instrument (ABI 7500), reaction conditions: preheating at 50 deg.C for 2 min; pre-denaturation at 95 ℃ for 10 min; the temperature is 95 ℃ for 15s, the temperature is 60 ℃ for 1min, and the reaction is circulated for 40 cycles; 95 ℃ for 15s, 60 ℃ for 1min and 95 ℃ for 1 s.
(5) And (4) analyzing results: the results obtained were obtained using QuantStudio TM Design&Analysis Software Analysis. The discrimination results of the kit are as follows:
when the internal reference, the positive sample control and the negative sample control are normal, the sample to be detected has no target gene amplification signal, and the curve is judged to be negative;
when the internal reference, the positive sample control and the negative sample control are normal, the target gene amplification signal of the sample to be detected is obtained, and the result is judged to be positive by the curve;
when the internal reference and (or) positive sample contrast and (or) negative sample contrast are abnormal, the target gene amplification signal of the sample to be detected has no meaning, and the target gene amplification signal needs to be amplified again after being adjusted.
The basic principles and the main features of the present invention and the advantages of the present invention have been shown and described above. While the invention has been described in terms of the preferred embodiments, it will be understood by those skilled in the art that the invention is not limited to the disclosed embodiments, which are provided in a manner that will serve to explain the principles of the invention and to enable various changes and modifications within the spirit and scope of the invention as hereinafter claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> hematological disease Hospital of Chinese medical science (institute of hematology of Chinese medical science)
<120> RARG-HNRNPM fusion gene detection and clinical application kit
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3087
<212> DNA
<213> fusion gene (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 1
atggccacca ataaggagcg actctttgcg gctggtgccc tggggcctgg atctggctac 60
ccaggggcag gtttcccctt cgccttccca ggggcactca gggggtctcc gcctttcgag 120
atgctgagcc ctagcttccg gggcctgggc cagcctgacc tccccaagga gatggcctct 180
ctgtcggtgg agacacagag caccagctca gaggagatgg tgcccagctc gccctcgccc 240
cctccgcctc ctcgggtcta caagccatgc ttcgtgtgca atgacaagtc ctctggctac 300
cactatgggg tcagctcttg tgaaggctgc aagggcttct ttcgccgaag catccagaag 360
aacatggtgt acacgtgtca ccgcgacaaa aactgtatca tcaacaaggt gaccaggaat 420
cgctgccagt actgccggct acagaagtgc ttcgaagtgg gcatgtccaa ggaagctgtg 480
cgaaatgacc ggaacaagaa gaagaaagag gtgaaggaag aagggtcacc tgacagctat 540
gagctgagcc ctcagttaga agagctcatc accaaggtca gcaaagccca tcaggagact 600
ttcccctcgc tctgccagct gggcaagtat accacgaact ccagtgcaga ccaccgcgtg 660
cagctggatc tggggctgtg ggacaagttc agtgagctgg ctaccaagtg catcatcaag 720
atcgtggagt ttgccaagcg gttgcctggc tttacagggc tcagcattgc tgaccagatc 780
actctgctca aagctgcctg cctagatatc ctgatgctgc gtatctgcac aaggtacacc 840
ccagagcagg acaccatgac cttctccgac gggctgaccc tgaaccggac ccagatgcac 900
aatgccggct tcgggcccct cacagacctt gtctttgcct ttgctgggca gctcctgccc 960
ctggagatgg atgacaccga gacagggctg ctcagcgcca tctgcctcat ctgcggagac 1020
cgcatggacc tggaggagcc cgaaaaagtg gacaagctgc aggagccact gctggaagcc 1080
ctgaggctgt acgcccggcg ccggcggccc agccagccct acatgttccc aaggatgcta 1140
atgaaaatca ccgacctccg gggcatcagc actaaggttg gtgaggtaac atacgtggag 1200
ctcttaatgg acgctgaagg aaagtcaagg gtaagtgttg ttgttgaatt caagatggaa 1260
gagagcatga aaaaagctgc ggaagtccta aacaagcata gtctgagcgg aagaccactg 1320
aaagtcaaag aagatcctga tggtgaacat gccaggagag caatgcaaaa ggtgatggct 1380
acgactggtg ggatgggtat gggaccaggt ggcccaggaa tgattactat cccacccagt 1440
atcctaaata atcccaacat cccaaatgag attatccatg cattacaggc tggaagactt 1500
ggaagcacag tatttgtagc aaatctggat tataaagttg gctggaagaa actgaaggaa 1560
gtatttagta tggctggtgt ggtggtccga gcagacattc ttgaagataa agatggaaaa 1620
agtcgtggaa taggcactgt tacttttgaa cagtccattg aagctgtgca agctatatct 1680
atgttcaatg gccagctgct atttgataga ccaatgcacg tcaagatgga tgagagggcc 1740
ttaccaaaag gagatttctt ccctcctgag cgtccacaac aacttcccca tggccttggt 1800
ggtattggca tggggttagg accaggaggg caacccattg atgccaatca cctgaataaa 1860
ggcatcggaa tgggaaacat aggtcccgca ggaatgggaa tggaaggcat aggatttgga 1920
ataaataaaa tgggaggaat ggaggggccc tttggtggtg gtatggaaaa catgggtcga 1980
tttggatctg ggatgaacat gggcaggata aatgaaatcc taagtaatgc actgaagaga 2040
ggagagatca ttgcaaagca gggaggaggt ggaggtggag gaagcgtccc tgggatcgag 2100
aggatgggtc ctggcattga ccgcctcggg ggtgccggca tggagcgcat gggcgcgggc 2160
ctgggccacg gcatggatcg cgtgggctcc gagatcgagc gcatgggcct ggtcatggac 2220
cgcatgggct ccgtggagcg catgggctcc ggcattgagc gcatgggccc gctgggcctc 2280
gaccacatgg cctccagcat tgagcgcatg ggccagacca tggagcgcat tggctctggc 2340
gtggagcgca tgggtgccgg catgggcttc ggccttgagc gcatggccgc tcccatcgac 2400
cgtgtgggcc agaccattga gcgcatgggc tctggcgtgg agcgcatggg ccctgccatc 2460
gagcgcatgg gcctgagcat ggagcgcatg gtgcccgcag gtatgggagc tggcctggag 2520
cgcatgggcc ccgtgatgga tcgcatggcc accggcctgg agcgcatggg cgccaacaat 2580
ctggagcgga tgggcctgga gcgcatgggc gccaacagcc tcgagcgcat gggcctggag 2640
cgcatgggtg ccaacagcct cgagcgcatg ggccctgcca tgggcccggc cctgggcgct 2700
ggcattgagc gcatgggcct ggccatgggt ggcggtggcg gtgccagctt tgaccgtgcc 2760
atcgagatgg agcgtggcaa cttcggagga agcttcgcag gttcctttgg tggagctgga 2820
ggccatgctc ctggggtggc caggaaggcc tgccagatat ttgtgagaaa tctgccattc 2880
gatttcacat ggaagatgct aaaggacaaa ttcaacgagt gcggccacgt gctgtacgcc 2940
gacatcaaga tggagaatgg gaagtccaag gggtgtggcg tggttaagtt cgagtcgcca 3000
gaggtggccg agagagcctg ccggatgatg aatggcatga agctgagtgg ccgagagatt 3060
gacgttcgaa ttgatagaaa cgcttaa 3087

Claims (6)

1. A RARG-HNRNPM fusion gene detection and clinical application kit is characterized in that the kit comprises a reagent for diagnosing the mutant acute promyelocytic leukemia based on a Taqman probe and/or detecting the absolute quantification of the RARG-HNRNPM fusion gene by the detection kit,
the kit comprises an upstream primer, a downstream primer, a Taqman probe, an internal reference, PCR reaction mixed liquor and DEPC-H 2 O。
2. The RARG-HNRNPM fusion gene detection and clinical application kit according to claim 1, characterized in that the upstream primer is RARG-HNRNPM upstream primer;
the downstream primer is RARG-HNRNPM downstream primer;
the probe is RARG-HNRNPM probe: a Taqman probe of RARG-HNRNPM fusion gene;
the internal reference is internal reference GAPDH;
the PCR reaction mixed solution is PCR reaction buffer solution.
3. The RARG-HNRNPM fusion gene detection and clinical application kit according to claim 2, characterized in that
The RARG-HNRNPM upstream primer is ATGAAAATCACCGACCTCCG;
the RARG-HNRNPM downstream primer is CCTTGACTTTCCTTCAGCGT;
the RARG-HNRNPM probe is AGCACTAAGGTTGGTGAGGTAAC;
the internal reference GAPDH is Hs02786624_ g1 introduced (applied biosystems);
the PCR buffer solution is Taqman TM Gene Expression Master Mix 4369016(Applied Biosystems) including dNTP, Ca required for the reaction 2+ ,Mg 2+ Etc.;
the positive control is a target plasmid standard product containing an RARG-HNRNPM fusion gene;
the negative control was cDNA of APL cell line NB 4.
4. The RARG-HNRNPM fusion gene detection and clinical application kit according to claim 1, characterized in that
FAM group is marked at the 5 'end of the RARG-HNRNPM fusion gene Taqman probe, and TAM group is marked at the 3' end of the Taqman probe;
the 5 'end of the Taqman probe of the internal reference gene GAPDH is marked with FAM group, and the 3' end is marked with NFQ group.
5. The RARG-HNRNPM fusion gene detection and clinical application kit according to claim 4, characterized in that the FAM group is a fluorescence reporter NFQ group which is a quenching group, when a target DNA molecule exists in a sample to be detected, the probe is combined with the target DNA molecule, the probe is degraded to release the fluorescence reporter group to emit fluorescence, when the target DNA molecule does not exist in the sample to be detected, the fluorescence reporter group is quenched and does not emit fluorescence.
6. The RARG-HNRNPM fusion gene detection and clinical application kit according to claim 1, characterized by the specific operational steps of the kit:
firstly, collecting and separating mononuclear cells from a specimen, and extracting mRNA of the mononuclear cells;
secondly, reverse transcribing mRNA of the mononuclear cell into cDNA serving as a sample to be detected;
thirdly, configuring a reaction system, and carrying out on-machine detection on a real-time fluorescent PCR instrument, wherein the reaction conditions are as follows: preheating at 50 deg.C for 2 min; pre-denaturation at 95 ℃ for 10 min; the temperature of 95 ℃ is 15s, the temperature of 60 ℃ is 1min, and the reaction is circulated for 40 times; 15s at 95 ℃, 1min at 60 ℃ and 1s at 95 ℃;
fourthly, analyzing the obtained results: the results obtained were obtained using QuantStudio TM Design&Analysis Software for Analysis,
the discrimination results of the kit are as follows:
when the internal reference, the positive sample control and the negative sample control are normal, the sample to be detected has no target gene amplification, and the signal curve is judged to be negative;
when the internal reference, the positive sample contrast and the negative sample contrast are normal, purposeful gene amplification is carried out on the sample to be detected, and the signal curve is judged to be positive;
when the internal reference and (or) the positive sample contrast and (or) the negative sample contrast are abnormal, the amplification signal curve of the target gene of the sample to be detected is meaningless and needs to be amplified again after adjustment.
CN202210417036.4A 2022-04-20 2022-04-20 RARG-HNRNPM fusion gene detection and clinical application kit Pending CN115058515A (en)

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