CN113005200B - Primer composition for detecting sarcoma fusion gene mutation, kit and application - Google Patents
Primer composition for detecting sarcoma fusion gene mutation, kit and application Download PDFInfo
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Abstract
The application discloses a primer composition, a kit and application for detecting sarcoma fusion gene mutation. The primer composition comprises specific primer pairs for detecting 38 fusion mutations and corresponding specific single-base extension primers; the forward primers of the specific primer pair are sequences shown by Seq ID No.1 to 38 in sequence, the reverse primers are sequences shown by Seq ID No.40 to 77 in sequence, and the specific single base extension primers are sequences shown by Seq ID No.79 to 116 in sequence. The primer composition can detect 38 sarcoma fusion gene mutations of 28 sarcoma subtypes, has large coverage, and is suitable for initial diagnosis and auxiliary typing. The primer composition detects on the RNA level, so that the detection accuracy is improved, and missed detection is avoided; in addition, the multiplex PCR is only needed to be carried out in 1 reaction, the operation is simple and convenient, the detection period is short, and the clinical detection use requirement can be better met.
Description
Technical Field
The application relates to the technical field of gene detection, in particular to a primer composition, a kit and application for detecting sarcoma fusion gene mutation.
Background
Soft tissue sarcoma is a kind of mesenchymal tumor, about 60% occurs in limb parts, 19% occurs in trunk parts, and other parts such as abdomen and neck. Soft tissue sarcoma accounts for 1% of adult tumors, but accounts for 15% of childhood malignant tumors, one of the most common solid tumors in childhood. According to WHO data, currently sarcomas have 100 different subtypes, showing histological diversity; at the same time, the biological behavior is variable, from inertia, high invasiveness to distant metastasis. Susceptibility to recurrence is one of the characteristics of soft tissue sarcomas.
At present, the clinical diagnosis and typing of soft tissue sarcomas are comprehensively evaluated in combination with various aspects such as clinic, imaging, pathology and the like. Histopathology is the gold standard for diagnosis of soft tissue sarcomas, and as the molecular mechanism of sarcomas is studied in depth, some sarcoma subtypes often contain specific fusion gene mutations, such as the fusion mutation of FUS-DDIT3 in 90% of myxoid/round cell lipomas, 5% of which are EWSR1-DDIT3 fusion mutations. Detection of these fusion mutations can then aid in molecular typing of sarcomas. For the detection of fusion genes, currently, fluorescence in situ hybridization (Fluorescence in situ hybridization, FISH) and Real-Time fluorescent quantitative PCR (Real Time PCR, RT-PCR) are mainly used. The gold standard for detecting the fusion gene by using the FISH has the advantages of high sensitivity and good specificity; however, only one type of fusion gene can be detected at a time, the operation flow is complex, the cost is high, and the flux is low. RT-PCR, although having a high detection throughput, still cannot be used for simultaneously detecting multiple types of genes, and is unfavorable for assisting the molecular typing of sarcomas through the comprehensive detection of fusion genes. In addition, related researches carry out capture sequencing on sarcoma fusion genes and chaperones thereof by a high-throughput capture sequencing technology, and the method can detect a plurality of fusion genes simultaneously, but is complex in flow, long in period and high in cost, and is difficult to meet the detection requirement of clinical samples.
Disclosure of Invention
The purpose of the application is to provide a novel primer composition for detecting sarcoma fusion gene mutation, a kit and application.
The application adopts the following technical scheme:
the first aspect of the application discloses a primer composition for detecting sarcoma fusion gene mutation, which comprises specific primer pairs for detecting 38 fusion mutations and corresponding specific single base extension primers; among them, 38 fusion mutations included: BCOR-CCNB3, BCOR-ITD, BCOR-MAML3, CIC-FOXO4, EWSR1-NFATC2, EWSR1-WT1, ASPL-TFE3, EWSR1-ERG, EWSR1-FLI1, NTRK1 rearrangement, SERPINE1-FOSB, FUS-CREB3L1, FUS-CREB3L2, YWHAE-NUTM2A, EWSR1-NFATC1, HEY1-NCOA2, SS18-SSX1, SS18-SSX4, FUS-DDIT3, EWSR1-PBX1, EWSR1-ZNF444, EWSR1-DDIT3, COL 1-PDGFB, WTR1-CAMTA1, YAP1-TFE3, EWSR1-CREB1, ETV6-NTRK 3-FO1, PAX3-NCOA1, PAX7-FOX 1, FUS 1-6-NTSC 1, CIATC 1-6-NAATZ, SUATZ 2, and UATZ-6-ZATZ fusion system 1; specific primer pairs for detecting 38 fusion mutations, the forward primers of which are shown as sequences from SEQ ID No.1 to SEQ ID No.38, the reverse primers of which are shown as sequences from SEQ ID No.40 to SEQ ID No.77, and the corresponding specific single-base extension primers of which are shown as sequences from SEQ ID No.79 to SEQ ID No. 116.
It should be noted that, in the present application, the forward primer of the specific primer pair for detecting 38 fusion mutations is the sequence shown in Seq ID No.1 to Seq ID No.38, which means that the 38 forward primers are the primers for detecting BCOR-CCNB3, BCOR-ITD, BCOR-MAML3, CIC-FOXO4, EWSR1-NFATC2, EWSR1-WT1, ASPL-TFE3, EWSR1-ERG, EWSR1-FLI1, NTRK1 rearrangement, SERPINE1-FOSB, FUS-CREB3L1, FUS-CREB3L2, YWHAE-NUTM2A, EWSR1-NFATC1, HEY1-NCOA2, SS18-SSX1, SS18-SSX4, FUS-DD3, EWSR1-ZNF444, EWSR1-DDIT3, COL 1-PDGFB 1, YA 1-ZMC 1, JAS-CREB 3L1, FUS-CREB3L2, YWWA-6, JA-6-FATS 1, XU-6, and XWATS-6, and the primers for fusing to the primers; the sequence of the reverse primers shown in Seq ID No.40 to Seq ID No.77 is the sequence of the 38 reverse primers, and the sequence of the 38 reverse primers is the reverse primer for detecting 38 fusion mutations in the sequence; the corresponding specific single base extension primers are the sequences shown in Seq ID No.79 to Seq ID No.116 in sequence, and the same means that the 38 specific single base extension primers are the specific single base extension primers for detecting 38 fusion mutations in the above sequence in sequence, and correspond to the corresponding forward primer and reverse primer. For example, for the fusion mutant BCOR-CCNB3, the forward primer of the specific primer pair is the sequence shown by Seq ID No.1, the reverse primer is the sequence shown by Seq ID No.40, and the corresponding specific single base extension primer is the sequence shown by Seq ID No. 79; for fusion mutation BCOR-ITD, the forward primer is the sequence shown by the Seq ID No.2, the reverse primer is the sequence shown by the Seq ID No.41, and the specific single-base extension primer is the sequence shown by the Seq ID No. 80; for fusion mutation BCOR-MAML3, the forward primer is the sequence shown by the Seq ID No.3, the reverse primer is the sequence shown by the Seq ID No.42, and the specific single-base extension primer is the sequence shown by the Seq ID No. 81; and so on.
The primer composition is characterized in that when in use, the specific primer pair uses a reverse transcription product of total RNA extracted from a sarcoma tissue sample to be detected as a template for PCR amplification; then, carrying out single base extension on the PCR amplification product by adopting a specific single base extension primer; and judging the corresponding fusion mutation type by detecting whether the signal of the single-base extension product at the position with the specific molecular weight is present or not. The primer composition can detect 38 sarcoma fusion gene mutations corresponding to 28 sarcoma subtypes, has large coverage, is suitable for the initial diagnosis and the auxiliary diagnosis and typing of patients with difficult sarcoma diagnosis, and provides reference basis for the treatment scheme establishment and prognosis evaluation of the sarcoma. According to the primer composition, fusion mutation detection is carried out on the RNA layer, so that the detection accuracy is remarkably improved, and missed detection caused by the difference of fusion break points in the DNA breaking reconnection process is avoided. The primer composition can be amplified by multiplex PCR in 1 reaction hole, is simple and convenient to operate, has a short detection period, and can better meet the use requirement of clinical sample detection.
In one implementation of the present application, the primer composition of the present application further comprises an internal reference specific primer pair for detecting an internal reference gene and a corresponding internal reference specific single base extension primer; the reference gene is GAPDH, the forward primer of the reference specific primer pair is the sequence shown by the Seq ID No.39, the reverse primer is the sequence shown by the Seq ID No.78, and the reference specific single-base extension primer is the sequence shown by the Seq ID No. 117.
It should be noted that the reference gene is set to verify whether the detection process or the sample has an error; it will be appreciated that other reference genes besides the GAPDH reference gene employed herein are not excluded, as long as multiplex PCR amplification or single base extension can be performed with the specific reference specific primer pairs and corresponding reference specific single base extension primers of the present application, together with the specific primer pairs and corresponding specific single base extension primers for detecting 38 fusion mutations.
In one implementation of the present application, the forward primer and the reverse primer are each upstream of 8-14nt tag sequences.
In one implementation of the present application, the forward primer and the reverse primer are each upstream of a tag sequence of the sequence shown in Seq ID No.118 of 10 nt.
In a second aspect, the present application discloses a kit for detecting mutation of sarcoma fusion gene, wherein the kit contains the primer composition of the present application.
In one implementation of the present application, the kit further comprises a reverse transcription primer for reverse transcription of total RNA extracted from the sarcoma tissue sample.
In one implementation of the present application, the reverse transcription primer includes a random primer of 6-8nt and a poly (T) primer.
In one embodiment of the present application, the kit further comprises a reverse transcriptase.
In one implementation of the present application, the kit further comprises shrimp alkaline phosphatase for digesting dNTPs to remove pyrophosphate groups from the dNTPs.
In one implementation of the present application, the kit further comprises a cation exchange resin for removing metal ions from the reaction solution.
The key point of the kit is that the kit contains the primer composition; as for other components, for example, reverse transcription primer, reverse transcriptase, shrimp alkaline phosphatase, cation exchange resin, etc., can be optionally added to the kit according to need or purchased separately; the present invention is not particularly limited herein.
The third aspect of the present application discloses the use of the primer composition of the present application, or the kit of the present application, in the detection of a subtype of sarcoma for non-diagnostic therapeutic purposes, in the development of a therapeutic drug for sarcoma, or in prognostic evaluation.
In a fourth aspect, the present application discloses a method for detecting a mutation of a sarcoma fusion gene, comprising the steps of,
performing reverse transcription on total RNA extracted from a sarcoma tissue sample to be tested;
amplifying the reverse transcription product using the specific primer pair of any one of claims 1-4 for detecting 38 fusion mutations;
treating the amplified product by using shrimp alkaline phosphatase, and digesting the dNTPs remained in the amplification reaction to remove pyrophosphoric acid groups from the dNTPs;
single base extension of the digested product of the prawn alkaline phosphatase using the corresponding specific single base extension primer of any one of claims 1-4 to obtain a single base extension product;
purifying the single base extension product using a cation exchange resin;
detecting the purified product by adopting a matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology, and judging the corresponding mutation type of the sarcoma fusion gene by using the signal of the single-base extension product at the position of the specific molecular weight.
It should be noted that, the method for detecting mutation of sarcoma fusion gene in the present application is directly aimed at obtaining classification results of sarcoma subtypes, and the classification results only provide intermediate reference basis for treatment scheme formulation and prognosis evaluation of sarcoma, and cannot be directly used for diagnosis or treatment of diseases.
The beneficial effects of this application lie in:
the primer composition for detecting the sarcoma fusion gene mutation can detect 38 sarcoma fusion gene mutations corresponding to 28 sarcoma subtypes, has large coverage, and is suitable for initial diagnosis and auxiliary typing. In addition, the primer composition judges the sarcoma subtype by carrying out qualitative detection of fusion mutation transcripts on the extracted total RNA of tissues, and the accuracy of detection is obviously improved by carrying out fusion mutation detection on the RNA layer, so that missed detection caused by the difference of fusion break points in the process of breaking and reconnecting DNA is avoided. The primer composition can be used for carrying out multiplex PCR amplification in 1 reaction, is simple and convenient to operate, has a short detection period, and can better meet the use requirement of clinical sample detection.
Drawings
FIGS. 1 and 2 are graphs showing the detection results of a negative sample in the first embodiment of the present application;
FIGS. 3 to 11 are graphs showing the detection results of 9 commercial standards and clinical samples of known fusion mutation types in the second embodiment of the present application.
Detailed Description
The existing sarcoma fusion gene mutation detection method mainly realizes simultaneous detection of a plurality of fusion genes by a high-throughput capture sequencing technology; however, the high-throughput capture sequencing process is complex, long in period and high in cost, and is difficult to meet clinical use requirements. In view of this, the present application creatively proposes that the matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) detect fusion products of reverse transcription products of total RNA in sarcoma tissue. Specifically, the application selects 38 fusion mutation types corresponding to 28 sarcoma subtypes including lipoblastoma, alveolar soft tissue sarcoma and the like, designs a fusion mutation transcript enriched on an RNA layer by specific primer amplification, and carries out qualitative detection on the fusion mutation transcript after single base extension and nucleic acid mass spectrum detection, thereby judging which fusion mutation occurs in a sarcoma tissue sample to be detected, further judging the tissue subtype of the sarcoma, and providing a reference basis for the treatment scheme formulation and prognosis evaluation of the sarcoma.
Based on the above research, the application creatively develops a primer composition for detecting sarcoma fusion gene mutation, wherein the primer composition comprises specific primer pairs for detecting 38 fusion mutations and corresponding specific single base extension primers; specific primer pairs for detecting 38 fusion mutations, the forward primers of which are shown as sequences from SEQ ID No.1 to SEQ ID No.38, the reverse primers of which are shown as sequences from SEQ ID No.40 to SEQ ID No.77, and the corresponding specific single-base extension primers of which are shown as sequences from SEQ ID No.79 to SEQ ID No. 116.
Further, the primer composition also comprises an internal reference specific primer pair for detecting the internal reference gene GAPDH and a corresponding internal reference specific single base extension primer; the reference specific primer pair has a forward primer with a sequence shown by a Seq ID No.39, a reverse primer with a sequence shown by a Seq ID No.78, and a reference specific single base extension primer with a sequence shown by a Seq ID No. 117.
The method can detect up to 40 fusion transcript products covered by a single time, and the common RT-PCR technology can only cover detection of 1-4 fusion products by a single reaction, so that the method is more comprehensive in coverage and the detection cost of a single fusion product is obviously reduced. Compared with an RNA sequencing method based on a high-throughput sequencing technology, the method is based on a single-base extension PCR amplification technology, has simple flow and low requirement on RNA quality, and is applicable to FFPE samples.
The primer combination, the kit and the sarcoma fusion gene mutation detection method have the following advantages compared with the existing sarcoma fusion gene mutation detection method:
(a) Aiming at 38 fusion mutation types, a specific primer is designed to amplify fusion mutation transcripts enriched on an RNA layer, then single-base extension and nucleic acid mass spectrum detection are carried out to qualitatively detect the fusion mutation transcripts, and then which fusion mutation occurs in a sarcoma tissue sample to be detected is judged, so that the tissue subtype of the sarcoma is judged, and a reference basis is provided for the treatment scheme formulation and prognosis evaluation of the sarcoma.
(b) The application aims at detecting a sarcoma tissue sample, and judging a sarcoma subtype by carrying out qualitative detection of fusion mutation transcripts on extracted total tissue RNA; the accuracy of detection can be remarkably improved by carrying out fusion mutation detection on the RNA layer, and missing detection caused by the difference of fusion break points in the process of DNA fracture reconnection is avoided.
(c) The application covers 38 fusion mutations corresponding to 28 sarcoma subtypes, has a large coverage range, and is suitable for the primary sarcoma and the auxiliary diagnosis typing of patients with difficult sarcoma diagnosis.
(d) The application designs 39 groups of 117 amplification and extension primers for 38 fusion mutations corresponding to 28 sarcoma subtypes and one internal reference gene, and all the primers can be used for multiplex PCR amplification in 1 reaction hole, so that the operation is simple, the detection period is short, and the use requirement of clinical sample detection can be better met.
The present application is described in further detail below by way of specific examples. The following examples are merely illustrative of the present application and should not be construed as limiting the present application. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present application. Those skilled in the art will readily recognize that some of the features may be omitted in various situations or may be replaced by other kits, materials, methods. In some instances, some operations associated with the present application have not been shown or described in the specification to avoid obscuring the core portions of the present application, and may not be necessary for a person skilled in the art to describe in detail the relevant operations based on the description herein and the general knowledge of one skilled in the art. In the following examples, the reagents or apparatus used were conventional products commercially available without reference to the manufacturer.
The numbers of the present application, for example, "Seq ID No.", "first aspect", "second aspect", "third aspect", "(a)", "(b)", "(c)", etc., are used only to distinguish the described objects, and do not have any sequential or technical meaning.
Example 1
The high-throughput detection of the sarcoma fusion gene mutation is realized by combining a single-base extension-based multiplex PCR technology with a matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology (MALDI-TOF-MS, hereinafter referred to as a nucleic acid mass spectrometry technology). In the embodiment, 38 fusion mutation types corresponding to 28 sarcoma subtypes including lipoblastoma, alveolar soft tissue sarcoma and the like are selected, a specific primer is designed to amplify fusion mutation transcripts enriched on an RNA layer, and the fusion mutation transcripts are qualitatively detected after single base extension and nucleic acid mass spectrometry detection, so that which fusion mutation occurs in a tissue sample of the sarcoma to be detected is judged, and the tissue subtype of the sarcoma is further judged. And designing corresponding amplification primers and single base extension primers aiming at the reference genes. The method comprises the following steps:
(1) Fusion mutation types with corresponding correlation to sarcoma molecule subtypes were selected and specific amplification primers and extension primers were designed using the agena online design software. Specific primer pairs for the detection of 38 fusion mutations and corresponding specific single base extension primers, and internal reference specific primer pairs for the internal reference gene and corresponding internal reference specific single base extension primers are shown in Table 1. The correspondence of the sarcoma molecular subtype to the fusion mutation is shown in Table 2. Wherein the amplified product length of each pair of specific primers is about 100-220bp; the molecular weight of the single base extension product of the extension primer is 3500Kda to 9000Kda.
TABLE 1 amplification primer and extension primer sequences
TABLE 2 correspondence of sarcoma molecular subtypes and fusion mutations
The specific primer pair and the reference specific primer pair shown in Table 1 have a tag sequence of 8-14nt upstream of each of the forward primer and the reverse primer, in this example, a tag length of 10nt, and a sequence shown in Seq ID No. 118. Seq ID No.118:5'-ACGTTGGATG-3'.
(2) Using peripheral blood of healthy persons and extracting total RNA, 1. Mu.g of total RNA was subjected to reverse transcription to obtain cDNA. In this example, 6-8nt of random primer and poly (T) primer were used for reverse transcription under conditions of 25℃for 5min, 42℃for 30min and 85℃for 5min.
(3) 1. Mu.L of the reverse transcription product was amplified using specific primers of SEQ ID NO.1 to SEQ ID NO.78, and fusion mutant transcripts that may be present were amplified and enriched.
The reaction system for specific amplification (pre-PCR) is as follows:
H 2 o (HPLC grade) 1.8. Mu.L, 10 XPCR Buffer 0.5. Mu. L, mgCl 2 (25mM)0.4μL、dNTP mix(25mM each)0.2μL、Primer mix(500nM each)1μL、
(5U/. Mu.L) 0.1. Mu.L, 1. Mu.L of reverse transcription product, and 5. Mu.L in total.
The specific amplification reaction conditions were: pre-denaturation at 94℃for 2min; then enter 45 cycles: 94 ℃ for 20sec, 56 ℃ for 30sec and 72 ℃ for 1min; after the cycle was completed, the extension was carried out at 72℃for 1min.
(4) After the specific amplification is completed, treating the amplified product with Shrimp Alkaline Phosphatase (SAP) to digest dntps remaining in the amplification reaction, such that dntps remove pyrophosphate groups; specifically, after amplification is completed, the amplification product reaction tubes are opened, and 1.53 mu L of H is added into each tube 2 O, 0.17. Mu.L of 10 XSAP Buffer and 0.3. Mu.L of SAP enzyme (1.7U/. Mu.L), were mixed by pipetting after addition and the total volume was 7. Mu.L.
The reaction conditions of SAP digestion are that the temperature is 37 ℃ for 40min;85 ℃ for 5min; and finally, standing by at 4 ℃.
(5) After the SAP digestion is completed, the extension primer from SEQ ID NO.79 to SEQ ID NO.117 is used for single base extension of the SAP digestion product to obtain single baseExtension products; specifically, after SAP digestion is completed, the reaction tube is opened, and 0.619 mu L of H is added into each tube 2 O, 0.2. Mu.L iPLEX Buffer Plus (10X), 0.2. Mu.L iPLEX Termination mix, 0.94. Mu.L iPLEX Extend Primer mix, 0.041. Mu.L iPLEX enzyme, total volume 9. Mu.L, and mix by pipetting.
The single base extension reaction conditions were 94℃for 30sec of pre-denaturation; then enter 50 cycles: 94 ℃ for 5sec, 52 ℃ for 5sec, 80 ℃ for 5min; after the cycle was completed, the extension was carried out at 72℃for 3min.
(6) Purifying the single-base extension product by using cation exchange resin, wherein the aim is to remove magnesium ions, manganese ions, iron ions and the like in the reaction solution; specifically, after the completion of the extension reaction, the reaction tube was opened and 18. Mu.L of H was added 2 O and adding cation exchange resin, placing the closed reaction tube on a mute mixer after adding, rotating the reaction tube at room temperature along the long axis by 360 degrees, and rotating the product plate for 40-60min. After the completion of the mixing, the product was centrifuged at 4000rpm for 5min.
(7) The purified product can be used for detecting nucleic acid mass spectrometry on-line, and the corresponding fusion mutation type can be judged by whether signals of the single-base extension product at the position of the specific molecular weight size exist or not; specifically, the supernatant was sampled and loaded onto the machine according to the agena massarray sample application and loading flow.
The results of the machine are shown in fig. 1 and 2, wherein the abscissa is the relative molecular weight of the single-base extension product, and the unit is kDa; the ordinate is the signal intensity, and the stronger the signal intensity, the higher the copy number of the target product is. The results of FIGS. 1 and 2 show that the primer composition of this example and its extended primer have a relatively uniform signal intensity for negative samples extracted from peripheral blood of healthy persons, and no non-specific amplification and non-specific extension products were observed.
Example two
In this example, the same specific primer pair and corresponding specific single-base extension primer, and the same internal reference gene amplification primer and single-base extension primer are used in the example, and sarcoma fusion gene mutation detection is performed on RNA of 9 commercial standard products and clinical samples with known fusion mutation types, and the relevant information of the samples to be detected is shown in Table 3.
Table 39 examples of known fusion mutation types
| Sample numbering | Fusion forms | Fusing information | Mutation type |
| CBP20137R | EWSR1-WT1 | chr22:29683123/chr11:128675261 | Fusion of |
| CBP20128R | EWSR1-FLI1 | chr11:118353210/chr9:20365742 | Fusion of |
| FA20061953 | FUS-CREB3L1 | 3.1％ | Fusion of |
| FA20061465 | FUS-DDIT3 | 7.5％ | Fusion of |
| FA20070162 | COL1A1-PDGFB | 8.9％ | Fusion of |
| FA20061162 | EWSR1-CREB1 | 14.1％ | Fusion of |
| FA20053065 | PAX7-FOXO1 | 5.3％ | Fusion of |
| FA20072357 | CIC-DUX4 | 5.5％ | Fusion of |
| FA20062522 | MYH9–USP6 | 14.5％ | Fusion of |
Reverse transcription, pre-PCR, SAP, EP, cation exchange resins, on-press testing, etc. were the same as in example one.
The sample was applied to the machine in the same manner as in example one, and the results are shown in FIGS. 3 to 11. Fig. 3 to 11 are test results of samples CBP20137R, CBP20128R, FA20061953, FA20061465, FA20070162, FA20061162, FA20053065, FA20072357 and FA20062522 in order. The results in FIG. 3 show that each sample has a product signal at the target product, indicating the presence of the corresponding fusion mutation. Taking the CBP20137R sample as an example, the sample is known to contain the EWSR1-WT1 fusion mutation, when the primer composition of the example is used for pre-PCR amplification, the upstream specific primer of the EWSR1 and the downstream specific primer of the WT1 can amplify the target fusion product, and other fusion primer sets cannot amplify due to lack of the target template. After the remaining dNTPs are digested by SAP, an extension primer is added for single-base extension, wherein the extension primer is single-stranded DNA with known sequence and molecular weight matched with the pre-PCR product, as shown in figure 3, according to the base complementation principle, the single base extended is C, the molecular weight of the extension product is 5050kDa, and when a product signal is found at the position of 5050kDa, the extension primer can be judged to be a target product signal, and further the existence of a corresponding fusion mutation is judged. The same is true for the judgment of other fusion mutations, which indicates that there are a plurality of different fusion mutations if signals are detected at a plurality of target product signal positions. If a signal is generated at a place other than the target product with a predetermined molecular weight, it is judged that nonsensical non-specific amplification is not performed, and the result interpretation is not included.
The results of the above examples show that the primer composition for detecting the mutation of the sarcoma fusion gene can detect 38 sarcoma fusion gene mutations corresponding to 28 sarcoma subtypes, and can improve detection accuracy by detecting the fusion mutation on the RNA level, thereby avoiding missed detection caused by the difference of fusion break points in the process of reconnecting DNA. The primer composition only needs to carry out multiplex PCR amplification and single base extension in 1 reaction, and finally carries out nucleic acid mass spectrum detection, so that the primer composition is simple and convenient to operate, short in detection period and capable of better meeting the use requirements of clinical sample detection.
The foregoing is a further detailed description of the present application in connection with the specific embodiments, and it is not intended that the practice of the present application be limited to such descriptions. It will be apparent to those skilled in the art to which the present application pertains that several simple deductions or substitutions may be made without departing from the spirit of the present application.
SEQUENCE LISTING
<110> Shenzhen Leshi Biotechnology Co., ltd
<120> primer composition for detecting sarcoma fusion gene mutation, kit and application
<130> 21I31477
<160> 118
<170> PatentIn version 3.3
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ccaagtcaat atagccaaca g 21
<210> 7
<211> 20
<212> DNA
<213> artificial sequence
<400> 7
<210> 8
<211> 26
<212> DNA
<213> artificial sequence
<400> 8
tcctacagcc aagctccaag ccaata 26
<210> 9
<211> 24
<212> DNA
<213> artificial sequence
<400> 9
agcagttact ctcagcagaa cacc 24
<210> 10
<211> 21
<212> DNA
<213> artificial sequence
<400> 10
ccaggtggag cagtataaga a 21
<210> 11
<211> 20
<212> DNA
<213> artificial sequence
<400> 11
<210> 12
<211> 27
<212> DNA
<213> artificial sequence
<400> 12
ctgtggctga ttacttcaag cagattg 27
<210> 13
<211> 22
<212> DNA
<213> artificial sequence
<400> 13
tagcagttct cagagcagca gc 22
<210> 14
<211> 26
<212> DNA
<213> artificial sequence
<400> 14
cacttatcat gcagttgtta cgtgat 26
<210> 15
<211> 23
<212> DNA
<213> artificial sequence
<400> 15
cccactgaga ctagtcaacc tca 23
<210> 16
<211> 23
<212> DNA
<213> artificial sequence
<400> 16
cgagatcctg cagatgaccg tgg 23
<210> 17
<211> 20
<212> DNA
<213> artificial sequence
<400> 17
<210> 18
<211> 20
<212> DNA
<213> artificial sequence
<400> 18
<210> 19
<211> 20
<212> DNA
<213> artificial sequence
<400> 19
<210> 20
<211> 22
<212> DNA
<213> artificial sequence
<400> 20
acagttatcc ccaggtacct gg 22
<210> 21
<211> 22
<212> DNA
<213> artificial sequence
<400> 21
acagttatcc ccaggtacct gg 22
<210> 22
<211> 25
<212> DNA
<213> artificial sequence
<400> 22
aatggcgtcc acggattaca gtacc 25
<210> 23
<211> 19
<212> DNA
<213> artificial sequence
<400> 23
gccgagatgg catccctgg 19
<210> 24
<211> 21
<212> DNA
<213> artificial sequence
<400> 24
atgtccgctc gcactcgtcg c 21
<210> 25
<211> 20
<212> DNA
<213> artificial sequence
<400> 25
<210> 26
<211> 22
<212> DNA
<213> artificial sequence
<400> 26
tcctacagcc aagctccaag tc 22
<210> 27
<211> 20
<212> DNA
<213> artificial sequence
<400> 27
<210> 28
<211> 20
<212> DNA
<213> artificial sequence
<400> 28
<210> 29
<211> 20
<212> DNA
<213> artificial sequence
<400> 29
<210> 30
<211> 19
<212> DNA
<213> artificial sequence
<400> 30
cctccaaccm catgaaccc 19
<210> 31
<211> 20
<212> DNA
<213> artificial sequence
<400> 31
<210> 32
<211> 25
<212> DNA
<213> artificial sequence
<400> 32
caaggattaa atgacagtgt gactc 25
<210> 33
<211> 24
<212> DNA
<213> artificial sequence
<400> 33
ccaagcttcc catcacagtg tacc 24
<210> 34
<211> 18
<212> DNA
<213> artificial sequence
<400> 34
ggctcctctc cctgtacc 18
<210> 35
<211> 20
<212> DNA
<213> artificial sequence
<400> 35
<210> 36
<211> 21
<212> DNA
<213> artificial sequence
<400> 36
cttgtcctcc ttgaagggct c 21
<210> 37
<211> 19
<212> DNA
<213> artificial sequence
<400> 37
agggcacgga aggctaagc 19
<210> 38
<211> 23
<212> DNA
<213> artificial sequence
<400> 38
ttgttggaca cttgggaact gcc 23
<210> 39
<211> 22
<212> DNA
<213> artificial sequence
<400> 39
ccacccatgg caaattccat gg 22
<210> 40
<211> 23
<212> DNA
<213> artificial sequence
<400> 40
gtggtttctc cataatgttt ggt 23
<210> 41
<211> 21
<212> DNA
<213> artificial sequence
<400> 41
gtacatggtg ggtccagctt g 21
<210> 42
<211> 22
<212> DNA
<213> artificial sequence
<400> 42
cctgaaacct tctttgccct tg 22
<210> 43
<211> 21
<212> DNA
<213> artificial sequence
<400> 43
gcttcttcac ggtttcgaga g 21
<210> 44
<211> 22
<212> DNA
<213> artificial sequence
<400> 44
gctcaatgtc ggcgtttcta ag 22
<210> 45
<211> 20
<212> DNA
<213> artificial sequence
<400> 45
<210> 46
<211> 23
<212> DNA
<213> artificial sequence
<400> 46
cgtcatcgtc tttgtagtcg gac 23
<210> 47
<211> 23
<212> DNA
<213> artificial sequence
<400> 47
cccaggtgat gcagctggag ttg 23
<210> 48
<211> 25
<212> DNA
<213> artificial sequence
<400> 48
ccaggatctg atacggatct ggctg 25
<210> 49
<211> 19
<212> DNA
<213> artificial sequence
<400> 49
gtgggttctc gatgatgtg 19
<210> 50
<211> 18
<212> DNA
<213> artificial sequence
<400> 50
gttcccggca tgtcgtag 18
<210> 51
<211> 26
<212> DNA
<213> artificial sequence
<400> 51
ccctgatgtc ccctgtaatt tgtgag 26
<210> 52
<211> 21
<212> DNA
<213> artificial sequence
<400> 52
ctggaggggc tgtgggtctg a 21
<210> 53
<211> 18
<212> DNA
<213> artificial sequence
<400> 53
gggcagagcc gtgaacac 18
<210> 54
<211> 20
<212> DNA
<213> artificial sequence
<400> 54
<210> 55
<211> 24
<212> DNA
<213> artificial sequence
<400> 55
gcaccagttg ggctttgcaa tgtg 24
<210> 56
<211> 20
<212> DNA
<213> artificial sequence
<400> 56
atgtttcccc cttttgggtc 20
<210> 57
<211> 22
<212> DNA
<213> artificial sequence
<400> 57
gtcttgttaa tcttctccaa gg 22
<210> 58
<211> 19
<212> DNA
<213> artificial sequence
<400> 58
gtggcttcaa taaatttgg 19
<210> 59
<211> 24
<212> DNA
<213> artificial sequence
<400> 59
aacactgcca gggccttctg tagg 24
<210> 60
<211> 23
<212> DNA
<213> artificial sequence
<400> 60
gaaggtcttg ccacactcgc agc 23
<210> 61
<211> 27
<212> DNA
<213> artificial sequence
<400> 61
tcatgcttaa tacagattca ccattcg 27
<210> 62
<211> 24
<212> DNA
<213> artificial sequence
<400> 62
atcaaaggag cggatcgagt ggtc 24
<210> 63
<211> 26
<212> DNA
<213> artificial sequence
<400> 63
gaactttgac cccgactgtt tcctta 26
<210> 64
<211> 21
<212> DNA
<213> artificial sequence
<400> 64
gagtgtggtg gacaggtact g 21
<210> 65
<211> 21
<212> DNA
<213> artificial sequence
<400> 65
gtaccccatc ggtaccattg t 21
<210> 66
<211> 20
<212> DNA
<213> artificial sequence
<400> 66
<210> 67
<211> 21
<212> DNA
<213> artificial sequence
<400> 67
caccctctgg attgagcatc c 21
<210> 68
<211> 20
<212> DNA
<213> artificial sequence
<400> 68
<210> 69
<211> 22
<212> DNA
<213> artificial sequence
<400> 69
ccttcattct gcacacgaat ga 22
<210> 70
<211> 21
<212> DNA
<213> artificial sequence
<400> 70
ctccatctgt gcctggactt g 21
<210> 71
<211> 20
<212> DNA
<213> artificial sequence
<400> 71
<210> 72
<211> 24
<212> DNA
<213> artificial sequence
<400> 72
gaggtcttgc cagcaaagca gtag 24
<210> 73
<211> 18
<212> DNA
<213> artificial sequence
<400> 73
caggggagtg cagaccag 18
<210> 74
<211> 23
<212> DNA
<213> artificial sequence
<400> 74
gctatgagat tccgagttcg aag 23
<210> 75
<211> 21
<212> DNA
<213> artificial sequence
<400> 75
tttttctggg ggcatcttgg a 21
<210> 76
<211> 20
<212> DNA
<213> artificial sequence
<400> 76
tgtggatgtg aactgcggtc 20
<210> 77
<211> 20
<212> DNA
<213> artificial sequence
<400> 77
<210> 78
<211> 24
<212> DNA
<213> artificial sequence
<400> 78
tctagacggc aggtcaggtc cacc 24
<210> 79
<211> 17
<212> DNA
<213> artificial sequence
<400> 79
gggctcctct gtagagt 17
<210> 80
<211> 18
<212> DNA
<213> artificial sequence
<400> 80
tagatctggt ggaattca 18
<210> 81
<211> 20
<212> DNA
<213> artificial sequence
<400> 81
<210> 82
<211> 22
<212> DNA
<213> artificial sequence
<400> 82
ctcgcccaag cgcaagatga ga 22
<210> 83
<211> 23
<212> DNA
<213> artificial sequence
<400> 83
aggagagaac cggagcatga gtg 23
<210> 84
<211> 23
<212> DNA
<213> artificial sequence
<400> 84
agcagagttc aatctgacag gac 23
<210> 85
<211> 25
<212> DNA
<213> artificial sequence
<400> 85
ctggccccga acggccggcg ccaca 25
<210> 86
<211> 25
<212> DNA
<213> artificial sequence
<400> 86
cagagttcat tccgacagga ccacc 25
<210> 87
<211> 25
<212> DNA
<213> artificial sequence
<400> 87
agagtagcta tggtcaacaa agcag 25
<210> 88
<211> 25
<212> DNA
<213> artificial sequence
<400> 88
gccaagctgg acaatgccag gcagt 25
<210> 89
<211> 26
<212> DNA
<213> artificial sequence
<400> 89
ttcctgcagc tcagcagccg ccgcca 26
<210> 90
<211> 26
<212> DNA
<213> artificial sequence
<400> 90
aaacaggaca gcccatgatt aatctg 26
<210> 91
<211> 27
<212> DNA
<213> artificial sequence
<400> 91
gggagctaca gccagcagcc tagctat 27
<210> 92
<211> 17
<212> DNA
<213> artificial sequence
<400> 92
accgggcgtg accgcga 17
<210> 93
<211> 28
<212> DNA
<213> artificial sequence
<400> 93
gttacaacca gcccagccta ggatatgg 28
<210> 94
<211> 28
<212> DNA
<213> artificial sequence
<400> 94
aggagggaaa ggttactttg acgcgcac 28
<210> 95
<211> 28
<212> DNA
<213> artificial sequence
<400> 95
tgaccaggga cagtatggaa attaccag 28
<210> 96
<211> 29
<212> DNA
<213> artificial sequence
<400> 96
tgaccaggga cagtatggaa attaccagc 29
<210> 97
<211> 30
<212> DNA
<213> artificial sequence
<400> 97
ctatggccaa gatcaatcct ccatgagtag 30
<210> 98
<211> 31
<212> DNA
<213> artificial sequence
<400> 98
ccaccatctt atcctcctac cagctactcc t 31
<210> 99
<211> 29
<212> DNA
<213> artificial sequence
<400> 99
tcttatcctc ctaccagcta ctcctctac 29
<210> 100
<211> 32
<212> DNA
<213> artificial sequence
<400> 100
aatgcataca ccgcccaacc cactcaagga ta 32
<210> 101
<211> 31
<212> DNA
<213> artificial sequence
<400> 101
cggacccccc ggaccccctg gcctcggagg a 31
<210> 102
<211> 33
<212> DNA
<213> artificial sequence
<400> 102
agccccgcgc agcagcacgc gcacctccgc cag 33
<210> 103
<211> 34
<212> DNA
<213> artificial sequence
<400> 103
cagacggtgc ccatgcggct gcggaagctg cccg 34
<210> 104
<211> 34
<212> DNA
<213> artificial sequence
<400> 104
ctacgggcag cagagttcat tccgacagga ccac 34
<210> 105
<211> 34
<212> DNA
<213> artificial sequence
<400> 105
tgaagaagat aactgtgtcc agaggacccc cagg 34
<210> 106
<211> 34
<212> DNA
<213> artificial sequence
<400> 106
gcatggattt tccagctata cagacagctt tgtg 34
<210> 107
<211> 35
<212> DNA
<213> artificial sequence
<400> 107
cccagcacca ggcatggatt ttccagctat acaga 35
<210> 108
<211> 35
<212> DNA
<213> artificial sequence
<400> 108
cccaggtgat gagcatcctg agcaacccca gcgct 35
<210> 109
<211> 32
<212> DNA
<213> artificial sequence
<400> 109
gtaactatgg ccaagatcaa tcctccatga gt 32
<210> 110
<211> 33
<212> DNA
<213> artificial sequence
<400> 110
ggttgttaag atgaacaaga gaactgggca acc 33
<210> 111
<211> 34
<212> DNA
<213> artificial sequence
<400> 111
ggaaacggca gcagccgcaa gcagcacaag cctc 34
<210> 112
<211> 35
<212> DNA
<213> artificial sequence
<400> 112
tcccagcccc gcagggggcc ctgaccccac ctcac 35
<210> 113
<211> 37
<212> DNA
<213> artificial sequence
<400> 113
tcttcgttcc gcagcagcac tccgacaggc agcgagt 37
<210> 114
<211> 37
<212> DNA
<213> artificial sequence
<400> 114
gtgcaaacct aaagagaatg ggagtgtgtc gtgtgag 37
<210> 115
<211> 38
<212> DNA
<213> artificial sequence
<400> 115
cacccggccg ccgcccgagc gctcgagaaa gtcctctc 38
<210> 116
<211> 36
<212> DNA
<213> artificial sequence
<400> 116
tccctttttg ctttctttta cgaccacatg aaactt 36
<210> 117
<211> 35
<212> DNA
<213> artificial sequence
<400> 117
cccatcacca tcttccagga gcgagatccc tccaa 35
<210> 118
<211> 10
<212> DNA
<213> artificial sequence
<400> 118
Claims (12)
1. A primer composition for detecting a mutation in a sarcoma fusion gene, characterized in that: the primer composition comprises specific primer pairs for detecting 38 fusion mutations and corresponding specific single-base extension primers;
the 38 fusion mutations include BCOR-CCNB3, BCOR-ITD, BCOR-MAML3, CIC-FOXO4, EWSR1-NFATC2, EWSR1-WT1, ASPL-TFE3, EWSR1-ERG, EWSR1-FLI1, NTRK1 rearrangement, SERPINE1-FOSB, FUS-CREB3L1, FUS-CREB3L2, YWHAE-NUTM2A, EWSR1-NFATC1, HEY1-NCOA2, SS18-SSX1, SS18-SSX4, FUS-DDIT3, EWSR1-PBX1, EWSR1-ZNF444, EWSR1-DDIT3, COL 1-PDGFB, WTR1-CAMTA1, YAP1-TFE3, ETV6-NTRK3, PAX 3-XO 1, PAX3-NCOA1, X7-PAX 1, FUS-6-FATS 1, QUATZ 1-6, and UATZ fusion 1-6;
the forward primers of the specific primer pair for detecting 38 fusion mutations are sequences shown in Seq ID No.1 to Seq ID No.38, the reverse primers of the specific primer pair are sequences shown in Seq ID No.40 to Seq ID No.77, and the corresponding specific single-base extension primers are sequences shown in Seq ID No.79 to Seq ID No. 116.
2. The primer composition according to claim 1, wherein: the primer composition also comprises an internal reference specific primer pair for detecting an internal reference gene and a corresponding internal reference specific single base extension primer;
the reference gene is GAPDH, the forward primer of the reference specific primer pair is a sequence shown by a Seq ID No.39, the reverse primer is a sequence shown by a Seq ID No.78, and the reference specific single-base extension primer is a sequence shown by a Seq ID No. 117.
3. The primer composition according to claim 1 or 2, characterized in that: upstream of the forward and reverse primers there is a tag sequence of 8-14nt each.
4. The primer composition according to claim 1 or 2, characterized in that: upstream of the forward and reverse primers there is a tag sequence of the sequence shown in Seq ID No.118 of 10nt each.
5. A kit for detecting a sarcoma fusion gene mutation, characterized in that: the kit contains the primer composition according to any one of claims 1 to 4.
6. The kit of claim 5, wherein: the kit also contains a reverse transcription primer, wherein the reverse transcription primer is used for carrying out reverse transcription on total RNA extracted from a sarcoma tissue sample.
7. The kit of claim 6, wherein: the reverse transcription primer includes a random primer of 6-8nt and a poly (T) primer.
8. The kit of claim 6, wherein: the kit also contains reverse transcriptase.
9. The kit of any one of claims 5-8, wherein: the kit also contains shrimp alkaline phosphatase for digesting dNTPs to remove pyrophosphoric acid groups from the dNTPs.
10. The kit of any one of claims 5-8, wherein: the kit also contains cation exchange resin for removing metal ions in the reaction liquid.
11. The primer composition according to any one of claims 1-4, or the kit according to any one of claims 5-10, for use in the detection of a subtype of sarcoma for non-diagnostic therapeutic purposes, development of a therapeutic drug for sarcoma or prognosis evaluation.
12. A method for detecting mutation of sarcoma fusion gene for non-diagnostic treatment purpose is characterized in that: comprises the steps of,
performing reverse transcription on total RNA extracted from a sarcoma tissue sample to be tested;
amplifying the reverse transcription product using the specific primer pair of any one of claims 1-4 for detecting 38 fusion mutations;
treating the amplified product by using shrimp alkaline phosphatase, and digesting the dNTPs remained in the amplification reaction to remove pyrophosphoric acid groups from the dNTPs;
single base extension of the digested product of the prawn alkaline phosphatase using the corresponding specific single base extension primer of any one of claims 1-4 to obtain a single base extension product;
purifying the single base extension product using a cation exchange resin;
detecting the purified product by adopting a matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology, and judging the corresponding mutation type of the sarcoma fusion gene by using the signal of the single-base extension product at the position of the specific molecular weight.
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| CN114317741A (en) * | 2021-11-24 | 2022-04-12 | 上海桐树生物科技有限公司 | Soft tissue sarcoma gene detection kit and system |
| CN113981101B (en) * | 2021-12-27 | 2022-03-15 | 首都医科大学附属北京朝阳医院 | Rhabdomyosarcoma fusion gene related circular RNA molecular marker and application thereof |
| CN117402976B (en) * | 2023-12-15 | 2024-03-12 | 首都医科大学附属北京儿童医院 | Rhabdomyosarcoma detection primer probe set, kit and application thereof |
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