CN117512112A - Method, primer and probe for screening NAB2-STAT6 fusion condition and kit - Google Patents

Abstract

The invention relates to a primer, a probe, a composition and a method for screening and identifying NAB2-STAT6 fusion conditions by using a fluorescent PCR technology, wherein the screened and identified related fusion genes comprise 4 fusion modes of NAB2E4-STAT6E2, NAB2E4-STAT6E3, NAB2E6-STAT6E16 and NAB2E6-STAT6E 17. The primer, the probe, the detection combination mode and the detection method are convenient, economical and quick, have good specificity, high sensitivity and large flux, are suitable for comprehensively evaluating the occurrence and the development of diseases by combining a large number of clinical samples with the results of tissue examination such as immunohistochemistry, FISH and the like, and predict the prognosis and the recurrence risk of isolated fibrotic tumor (SFT).

Description

Method, primer and probe for screening NAB2-STAT6 fusion condition and kit

Technical Field

The invention belongs to the field of life science and biotechnology, and particularly relates to a primer, a probe, a kit and a method for screening NAB2-STAT6 fusion conditions by adopting a multiplex fluorescence PCR technology.

Technical Field

Isolated fibrotic tumor (solitary fibrous tumors, SFT) is a rare tumor of mesenchymal origin found and proposed in pleural tumors. It originates from CD34 dendritic mesenchymal cells, occurs widely, is reported almost everywhere, it occurs mainly in three parts of pleura, meninges and extrathoracic soft tissue, and pleura is the most common part, accounting for about 30%. In addition, it has been reported in recent years that it can occur in the head and neck, four limbs, liver, kidney, soft tissue and the like. SFT is well developed in middle-aged people and is mostly benign, with only about 10% of cases showing invasiveness. At first, at the time of diagnosing SFT, judgment is made on most SFT patients through histological features and CD34 expression, but diagnosis is possibly difficult due to the diversity of SFT histology, and then more and more researches show that most SFT has a specific NAB2-STAT6 fusion gene which is fused by nerve growth factor-induced gene A binding protein 2 (nerve growth factor-reduced gene) NGFI-A-binding protein 2, NAB 2-signal transduction and transcription activator 6 (signal transducer and activator of transcription, STAT 6), has a high positive detection rate in SFT patients in clinic, is a SFT specific molecular marker and participates in the generation and development process of SFT.

NAB2 and STAT6 genes are located in the 12q13 chromosome region, and NAB2 expression product NAB2 protein includes one N-terminal early growth reaction gene 1 (early growth response gene, EGR 1) combining region, one NAB2 conserved domain and one C-terminal transcription inhibiting region. STAT6 is an important member of the STAT family, consisting of approximately 840 amino acids, and has a relative molecular mass of approximately 94kD, and its expression product includes a DNA binding region, a tyrosine kinase homolog 2 region, and a C-terminal transcriptional activation region. It was found that inversion of the 12q13 chromosome fused the 3 'end of the NAB2 gene to the 5' end of the STAT6 gene, resulting in the production of NAB2-STAT6 fusion proteins that promote transcription of early growth response factors.

NAB2-STAT6 fusion does not always occur at the same site, NAB2 has multiple chromosomal cleavage sites, and upon cleavage from the chromosome, fragments of spliced exons of unequal length are formed. It has now been found that a number of variant subsets of NAB2-STAT6, fusion can be made by splicing the 2, 4, 6, 7 exons on NAB2 with the 2, 3, 5, 6, 16, 17, 18 exons on STAT6 (NAB 2ex2/4/6/7-STAT6ex 2/3/5/6/16/17/18). Among them, NAB2 exon 4 fused with STAT6 exon 2 (NAB 2ex4-STAT6ex 2/3), NAB2 exon 6 fused with STAT6 exon 16/17 (NAB 2ex6-STAT6ex 16/17), NAB2ex4-STAT6ex2/3 showed chest SFT of non-invasive phenotype, and NAB2ex6-STAT6ex16/18 showed invasive SFT, and was easy to relapse.

SFT is a neoplastic disease caused by gene mutation to a great extent, and is easy to recur and worsen after operation, and besides surgical excision, no effective auxiliary treatment means exists at present. The current technology for detecting NAB2-STAT6 fusion genes has various technologies, and how to apply more sensitive and accurate technologies to improve the detection of SFT patients with NAB2-STAT6 fusion genes is one of the challenges. The research is based on a real-time fluorescence quantitative PCR method, and further carries out deep research on NAB2-STAT6 fusion genes and SFT, so that important basis can be provided for improving clinical diagnosis and further revealing root causes of tumorigenesis and development, more new tumor markers and specific targets are searched for treating SFT patients, and corresponding auxiliary treatment means such as targeted drugs and the like are further developed, so that the life quality of the patients is improved.

At present, NAB2-STAT6 fusion genes are mostly detected by a fluorescence quantitative PCR method, while an immunohistochemical method is simple in principle, but the test process is complicated, and the required reagents are various, so that the application of the method is limited to a certain extent; the Fluorescence In Situ Hybridization (FISH) detection result needs an experienced expert to interpret, the result interpretation has large subjectivity, and FISH can only be qualitative and cannot be used for detecting tiny residues; compared with the secondary sequencing later-stage data processing, the real-time fluorescence quantitative PCR detection method has the characteristics of visual detection result, easiness in breaking, simplicity in operation, low cost and the like.

Disclosure of Invention

In view of the fact that the current methodology for screening fusion genes cannot meet the requirements of economy, high efficiency, accurate results and accurate diagnosis, the invention designs a set of primers and probes for detecting NAB2-STAT6 fusion genes by using a multiplex fluorescence PCR technology, and designs a screening and type identification scheme which is high in sensitivity, good in specificity, large in detection flux, quick and accurate.

The invention provides a primer and a probe for screening and identifying NAB2E4-STAT6E2, NAB2E4-STAT6E3, NAB2E6-STAT6E16 and NAB2E6-STAT6E17 fusion genes by using a multiplex fluorescence PCR technology, wherein the nucleotide sequences of the primer and the probe are as follows:

further, primers and probes for amplifying housekeeping gene ACTIN as an internal reference are also included, and the nucleotide sequences are as follows:

ACTIN-F：5’-TGAGCGAGGCTACAGCTT-3’

ACTIN-R：5’-TCCTTGATGTCGCGCACGATTT-3’

ACTIN-P:5’-FAM-ACCACCACGGCCGAGCGG-BHQ2-3’

the invention also provides a method for screening and identifying 4 fusion genes of NAB2E4-STAT6E2, NAB2E4-STAT6E3, NAB2E6-STAT6E16 and NAB2E6-STAT6E17, which comprises the following steps:

the method comprises the steps of dissolving paraffin in a paraffin sheet or a paraffin roll by using a tissue transparent liquid so as to better extract nucleic acid of paraffin embedded tissues;

extracting total RNA of the tissue by using a Qiagen kit;

taking RNA in the step (2) as a template, and carrying out primary screening of 4 fusion genes by adopting a one-step method, wherein the reaction conditions are as follows: pre-denaturation at 95℃for 1min;95 ℃ for 10s and 58 ℃ for 50s, 40 cycles are total; 58 DEG C

Collecting fluorescence;

fourthly, judging whether NAB2E4 fusion or NAB2E6 is positive according to the detection result in the step 3 and the line lifting condition of different probes; under the condition that the amplification of the internal reference ACTIN gene is normal (CT value is less than or equal to 32), each fusion gene CT value is less than 36 and is judged to be positive; if NAB2E4 fusion is positive, identifying which of NAB2E4-STAT6E2 and NAB2E4-STAT6E3 fusion genes is positive; if NAB2E6 is positive, it is necessary to identify which of the NAB2E6-STAT6E16, NAB2E6-STAT6E17 fusion genes is positive.

The invention also provides a kit for detecting 4NAB2E4-STAT 6E2, NAB2E4-STAT6E3, NAB2E6-STAT6E16 and NAB2E6-STAT6E17 fused genes, which comprises a group of primers and probes, wherein the nucleotide sequences of the kit are as follows:

further, the kit further comprises primers and probes for amplifying housekeeping gene ACTIN as an internal reference, and the nucleotide sequence of the primers and probes is as follows:

ACTIN-F：5’-TGAGCGAGGCTACAGCTT-3’

ACTIN-R：5’-TCCTTGATGTCGCGCACGATTT-3’

ACTIN-P:5’-FAM-ACCACCACGGCCGAGCGG-BHQ2-3’。

the invention has the beneficial effects that:

1. when NAB2 gene is fused in isolated fibrotumor (solitary fibmus tumors, SFT), the cleavage sites are mainly concentrated on introns 4 and 6, accounting for more than 80%, so that NAB2 exons 4 and 6 are fused with STAT6 gene; and the most common fusion genes are NAB2E4-STAT6E2, NAB2E4-STAT6E3, NAB2E6-STAT6E16, NAB2E6-STAT6E 17. Since exon 4 is very short, NAB2ex3-F primer is designed on exon 3, which can well meet the detection Tm requirement of fluorescent PCR technology on one hand and enhance the specificity of the primer on the other hand. The design scheme of the primer probe of the fusion gene is shown in figure 1.

2. Cost is considered in designing primers and probes, as long as the probes and primers for the same exon are designed as one, for example NAB2E4-STAT6E2 and NAB2E4-STAT6E3 all have NAB2E4, so the upstream primer and probe are designed on exon 3 of NAB2 and the two fusion types are shared, saving cost.

3. In the invention, 3 probes are designed in total, and for convenience and simplicity, the luminous groups of 5' of the 3 probes are different, so that all primer probes can be placed in a tube for reaction to simultaneously find which group is fused, for example, HEX marked probes react, NAB2E4 participates in fusion, and at least one of NAB2E4-STAT6E2 and NAB2E4-STAT6E3 is fused.

4. The invention uses RNA as a template, adopts a one-step method without reversing the RNA into cDNA, and reduces the reaction procedure and pollution.

On the combined scheme of NAB2-STAT6 fusion gene screening, the invention considers the interference factors such as primer dimer, hairpin structure and the like, and the reaction is carried out in one tube, thereby saving the cost, increasing the detection flux and providing convenience for screening large samples. Meanwhile, the combination mode is combined with a fluorescent PCR technology, so that the uncapping reaction of the common PCR is avoided, the accuracy of the result is improved, and pollution is avoided; and the easiness in judging the result is improved, so that the detection result is more objective and easy to read.

Drawings

FIG. 1 is a schematic diagram of the design area of a primer and probe according to the present invention, wherein the numbers represent exons and the arrows indicate the positions of the primer probe design: the first figure represents NAB2E4-STAT6E 2; the second plot represents NAB2E6-STAT6E 16.

FIG. 2 is a sample screen of 5 cases of pathology initially judged as SFT, and one example of which is found to have a response to HEX signal, which represents NAB2ex4-P based on probe design, so that NAB2E4-STAT6E2 and NAB2E4-STAT6E3 were determined to be likely fusion positive.

FIGS. 3 and 4 are graphs confirming that screening positive samples belonged to that fusion, and that NAB2E4-STAT6E2 was present but NAB2E4-STAT6E3 was absent.

FIG. 5 shows a primer probe specificity experiment, in which 5 samples containing different fusion genes and primer probes of various fusion genes of NAB2-STAT6 react, no other reaction is normally found in internal reference, and the primer probe specificity is good.

Detailed Description

Example 1: sample detection program

(1) Preparing paraffin sample RNA: the tissue is cut or paraffin slide samples are scraped into a 1.5ml centrifuge tube, 1ml of tissue transparent liquid is added, and after shaking and mixing, the mixture is centrifuged at 13000rpm for 1min. The supernatant was removed, 500ml of tissue transparency was added, and after shaking and mixing, the mixture was centrifuged at 13000rpm for 1min. The supernatant was removed, 1ml of absolute ethanol was added thereto, and after shaking and mixing, the mixture was centrifuged at 13000rpm for 1min. The supernatant was removed and placed in a 37 ℃ metal bath until dry (uncapping). 150ul PKD buffer (from RNeasy FFPE KIT) was added and mixed by shaking. 10ul PK (from RNeasy FFPE KIT) was added and mixed upside down. The metal bath was carried out at 56℃for 15min and then at 80℃for 15min. After 3min on ice, centrifugation was performed at 13200rpm for 15min, the supernatant was aspirated to a new 1.5ml EP tube, and then 16ul of DNase Booster buffer (from RNeasy FFPE KIT) and 10ul of DNase1 (from RNeasy FFPE KIT) were added, followed by shaking and mixing, low-speed centrifugation, and standing at room temperature for 15min. Adding 320ul RBC buffer (from RNeasy FFPE KIT), mixing, and adding 720ul absolute ethanol. 700ul of the previous step liquid was added to the column (from RNeasy FFPE KIT), 10000rpm was used for 15s, the liquid in the tube was discarded, the remaining liquid was added to the column, 10000rpm was used for 15s, and the liquid in the tube was discarded. 500ul of RPE buffer (from RNeasy FFPE KIT) was added to the column at 10000rpm for 15s, and the waste liquid was discarded. 500ul RPE buffer,10000rpm 2min is added into the chromatographic column, the waste liquid is discarded, the cover is opened, and the solution is centrifuged at full speed for 5min. The column was placed in a fresh 1.5ml EP tube, 30ul of DEPC water was added to the column, and after 2min of standing, the column was centrifuged at full speed for 1min. The concentration and purity of RNA were measured using a NanoDrop 2000 and stored at-30℃until use.

(2) Reagent configuration: a total volume of 25. Mu.l of the system configuration was determined for a single portion according to the following table

(3) Sample adding: adding 5 mu L of total RNA into the PCR reaction liquid of the detection system; the positive control and the negative control are directly added with 5 mu L of positive control and negative control; the blank was added with 5. Mu.L of physiological saline.

(5) And (3) detection: detection is performed on a real-time fluorescent PCR instrument, which may include ABI7300, 7500 (company of America Applied Biosystems), and the like. Reaction conditions: pre-denaturation at 95℃for 1min; fluorescent signals were collected at 58℃for 35sec at 95℃for 15s at 58℃for 40 cycles.

(6) And (3) judging results: and adjusting the threshold line to be above the background signal and the negative amplification line, and automatically outputting a CT value according to the standard by the system.

Under the condition that the amplification of the internal reference ACTIN gene is normal (CT value is less than or equal to 32), each fusion gene CT value is less than 36 and is judged to be positive; if HEX marked NAB2ex4-P signal is linear and CT value is less than 36, identifying which one of NAB2E4-STAT6E2 and NAB2E4-STAT6E3 fusion genes is positive; if the CY 5-labeled NAB2ex6-P signal is on line and the CT value is less than 36, it is necessary to identify which of the NAB2E6-STAT6E16 and NAB2E6-STAT6E17 fusion genes is positive.

Example 2: sample screening

5 paraffin-embedded tissue samples with primary pathology diagnosis as SFT were taken, total RNA and configuration reagents were extracted as in example 1, and each sample was subjected to single tube screening. Meanwhile, a negative reaction tube and a positive reaction tube are arranged, the screening result is shown in figure 2, HEX signals of one sample are hatched, and the CT value is smaller than 36, so that at least one of NAB2E4-STAT6E2 and NAB2E4-STAT6E3 fusion genes is predicted to be positive.

Example 4: identification of specific fusion type of positive sample

According to example 2, a positive sample was found, so that it was necessary to determine whether NAB2E4-STAT6E2 or NAB2E4-STAT6E3 was used. The total volume of 25. Mu.l of the reaction system was divided into two tubes and the following table was prepared:

the results are shown in FIGS. 3 and 4, with FIG. 3NAB 2E4-STAT6E2 being lined and CT number < 36, and FIG. 4NAB2E4-STAT 6E3 being unlined. Thus, the positive samples of this example were positive for NAB2E4-STAT6E2 fusion gene.

Example 5: primer and Probe specificity experiments

To confirm that the primers and probes of the invention only reacted with the NAB2-STAT6 fusion gene without amplifying samples in which other fusion genes were present. 5 fusion gene positive RNA samples were selected, the fusion modes were BCR-ABL P210, PML-RARA and AML1-ETO, respectively, 2 fusion modes were SYT (SS 18) -SSX1, and detection was performed according to the procedure of example 1, the detection results are shown in FIG. 5, and no other fusion samples were reacted in the case of internal reference reaction.

Claims (6)

1. Screening and identifying primers and probes of NAB2E4-STAT6E2, NAB2E4-STAT6E3, NAB2E6-STAT6E16 and NAB2E6-STAT6E17 total of 4NAB 2-STAT6 fusion genes by using a multiplex fluorescence PCR technology, wherein the nucleotide sequences of the primers and probes are as follows:

NAB2ex3-F 5'-GAGCACCTACTTGTCCTCCTTGA-3' NAB2ex5-R 5'-TGGGGGTACATAGGACTCAGG-3' NAB2ex4-P1 5'-HEX-TTCACCCTGAAGAACTGGGAGGCCC-BHQ2-3' STAT6ex2-R 5'-TGGAGACCAGACCCCACAGA-3' STAT6ex3-R 5'-CTGGACAGTGTCTGAAAGTAGGG-3' NAB2ex6-F 5'-GAGCCGACACATCCTGCA-3' NAB2ex6-P 5'-CY5-AGACACTGATGGACGAGGGGCTGC-BHQ2-3' STAT6ex16-R 5'-GCAGAGAATGGCTGGATGTT-3' STAT6ex17-R 5'-CCTGCCATCCTTACCCATCT-3'

2. the primer and probe according to claim 1, further comprising a primer and probe for amplifying housekeeping gene ACTIN as an internal reference, the nucleotide sequence of which is as follows:

ACTIN-F：5’-TGAGCGAGGCTACAGCTT-3’

ACTIN-R：5’-TCCTTGATGTCGCGCACGATTT-3’

ACTIN-P:5’-FAM-ACCACCACGGCCGAGCGG-BHQ2-3’。

3. use of the primers and probes of any one of claims 1-2 for the preparation of a kit for detecting 4NAB 2-STAT6 fusion genes.

4. A method for screening and identifying 4 fusion genes of NAB2E4-STAT6E2, NAB2E4-STAT6E3, NAB2E6-STAT6E16, NAB2E6-STAT6E17 comprising the steps of:

the method comprises the steps of dissolving paraffin in a paraffin sheet or a paraffin roll by using a tissue transparent liquid so as to better extract nucleic acid of paraffin embedded tissues;

extracting total RNA of the tissue by using a Qiagen kit;

taking RNA in the step (2) as a template, and carrying out primary screening of 4 fusion genes by adopting a one-step method, wherein the reaction conditions of the primary screening are as follows: pre-denaturation at 95℃for 1min;95 ℃ for 10s and 58 ℃ for 50s, 40 cycles are total; collecting fluorescence at 58 ℃;

fourthly, judging whether NAB2E4 fusion or NAB2E6 is positive according to the detection result in the step 3 and the line lifting condition of different probes; under the condition that the amplification of the internal reference ACTIN gene is normal (CT value is less than or equal to 32) and the NAB2 gene is expressed (CT value is less than or equal to 32), each fusion gene CT value is less than 36 and is judged to be positive; if NAB2E4 fusion is positive, identifying which of NAB2E4-STAT6E2 and NAB2E4-STAT6E3 fusion genes is positive; if NAB2E6 is positive, it is necessary to identify which of the NAB2E6-STAT6E16, NAB2E6-STAT6E17 fusion genes is positive.

5. A kit for detecting 4NAB2E4-STAT 6E2, NAB2E4-STAT6E3, NAB2E6-STAT6E16, NAB2E6-STAT6E17 total of 4NAB 2-STAT6 fusion genes, characterized in that the kit comprises a set of primers and probes, the nucleotide sequences of which are as follows:

NAB2ex3-F 5'-GAGCACCTACTTGTCCTCCTTGA-3' NAB2ex5-R 5'-TGGGGGTACATAGGACTCAGG-3' NAB2ex4-P1 5'-HEX-TTCACCCTGAAGAACTGGGAGGCCC-BHQ2-3' STAT6ex2-R 5'-TGGAGACCAGACCCCACAGA-3' STAT6ex3-R 5'-CTGGACAGTGTCTGAAAGTAGGG-3' NAB2ex6-F 5'-GAGCCGACACATCCTGCA-3' NAB2ex6-P 5'-CY5-AGACACTGATGGACGAGGGGCTGC-BHQ2-3' STAT6ex16-R 5'-GCAGAGAATGGCTGGATGTT-3' STAT6ex17-R 5'-CCTGCCATCCTTACCCATCT-3'

6. the kit according to claim 5, further comprising primers and probes for amplifying housekeeping gene ACTIN as an internal reference, the nucleotide sequences of which are as follows:

ACTIN-F：5’-TGAGCGAGGCTACAGCTT-3’

ACTIN-R：5’-TCCTTGATGTCGCGCACGATTT-3’

ACTIN-P:5’-FAM-ACCACCACGGCCGAGCGG-BHQ2-3’。