CN113005200A - Primer composition and kit for detecting sarcoma fusion gene mutation and application of primer composition and kit - Google Patents
Primer composition and kit for detecting sarcoma fusion gene mutation and application of primer composition and kit Download PDFInfo
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Abstract
The application discloses a primer composition for detecting sarcoma fusion gene mutation, a kit and application. The primer composition comprises a specific primer pair for detecting 38 fusion mutations and a corresponding specific single-base extension primer; the forward primers of the specific primer pairs are shown as Seq ID No.1 to 38 in sequence, the reverse primers are shown as Seq ID No.40 to 77 in sequence, and the specific single-base extension primers are shown as Seq ID No.79 to 116 in sequence. The primer composition can detect 38 sarcoma fusion gene mutations of 28 sarcoma subtypes, has a large coverage range, and is suitable for initial diagnosis and auxiliary typing. The primer composition is used for detecting on the RNA layer, so that the detection accuracy is improved, and the omission of detection is avoided; and only multiple PCR is needed to be carried out in 1 reaction, the operation is simple and convenient, the detection period is short, and the clinical detection use requirements can be better met.
Description
Technical Field
The application relates to the technical field of gene detection, in particular to a primer composition for detecting sarcoma fusion gene mutation, a kit and application.
Background
Soft tissue sarcoma is a mesenchymal tumor, occurring in about 60% of the limb, 19% of the trunk, and others, such as the abdomen and neck. Soft tissue sarcoma accounts for 1% of adult tumors, but accounts for 15% of childhood malignancies, one of the most common solid tumors in children. According to WHO data, the sarcoma has more than 100 different subtypes and shows histological diversity; at the same time, the biological behavior is also variable, ranging from inert, highly invasive to distant. The predisposition to recurrence is one of the characteristics of soft tissue sarcomas.
At present, clinical diagnosis and typing of soft tissue sarcoma are comprehensive evaluations combining multiple aspects of clinic, imaging, pathology and the like. Histopathology is the golden standard for soft tissue sarcoma diagnosis, and with the intensive study on the molecular mechanism of sarcomas, some sarcoma subtypes often contain specific fusion gene mutations, such as FUS-DDIT3 fusion mutation in 90% mucinous/round cell lipoma and EWSR1-DDIT3 fusion mutation in 5%. Detection of these fusion mutations can then assist in molecular typing of sarcomas. For the detection of fusion genes, Fluorescence In Situ Hybridization (FISH) and Real-Time fluorescent quantitative PCR (Real Time PCR, RT-PCR) are mainly used at present. The FISH has the advantages of high sensitivity and good specificity as a gold standard for detecting the fusion gene; but only one type of fusion gene can be detected at a time, and the method has the advantages of complex operation flow, high cost and low flux. RT-PCR has high detection flux, but still cannot simultaneously detect multiple types of genes, and is not favorable for assisting molecular typing of sarcoma through comprehensive detection of fusion genes. In addition, related researches are carried out on the sarcoma fusion gene and the chaperone gene thereof through a high-throughput capture sequencing technology, and the method can be used for simultaneously detecting a plurality of fusion genes, but is complex in process, long in period and high in cost, and cannot meet the detection requirement of clinical samples.
Disclosure of Invention
The application aims to provide a novel primer composition for detecting sarcoma fusion gene mutation, a kit and application.
The following technical scheme is adopted in the application:
the first aspect of the application discloses a primer composition for detecting sarcoma fusion gene mutation, which comprises a specific primer pair for detecting 38 fusion mutations and corresponding specific single-base extension primers; among the 38 fusion mutations are: BCOR-CCNB, BCOR-ITD, BCOR-MAML, CIC-FOXO, EWSR-NFATC, EWSR-WT, ASPL-TFE, EWSR-ERG, EWSR-FLI, NTRK rearrangement, SERPINE-FOSB, FUS-CREB3L, YWHAE-NUTM2, EWSR-NFATC, HEY-NCOA, SS-SSX, FUS-DDIT, EWSR-PBX, EWSR-ZNF 444, EWSR-DDIT, COL 1A-PDG, FB TR-CAMTA, YAP-TFE, EWSR-CREB, ETV-NTRK, PAX-FOXO, PAX-NCOA, PAX-FOATF, WWS-FUATF, EWWR-ATF, ATIC-JA-, CIC-ZF-, SUNAZF-, SUNASH-SUAG, USP-PAC fusion; the specific primer pairs for detecting 38 fusion mutations have sequences shown in Seq ID No.1 to Seq ID No.38 in sequence as the forward primers, sequences shown in Seq ID No.40 to Seq ID No.77 in sequence as the reverse primers, and sequences shown in Seq ID No.79 to Seq ID No.116 in sequence as the corresponding specific single-base extension primers.
In the present application, the forward primers of the specific primer pairs for detecting 38 fusion mutations are shown by Seq ID No.1 to Seq ID No.38 in sequence, which means that the 38 forward primers are shown by BCOR-CCNB3, BCOR-ITD, BCOR-MAML3, CIC-FOXO4, EWSR1-NFATC1, EWSR1-WT1, ASPL-TFE 1, EWSR1-ERG, EWSR1-FLI1, NTRK1 rearrangement, SERPINE 1-FOSB, FUS-CREB3L1, YWHEAE-NUTM 21, EWSR1-NFATC1, HEY1-NCOA 1, SSSS 1-SSX 1, FUSS 1-SSX 1, EWSR-NFET 1, EWTAXT-1-NFET 1, FO 1-NFT 1, FO 1-1, FO 1-1, FO 1-1, FO, CIC-DUX4, JAZF1-SUZ12, NAB2-STAT6, MYH 9-USP 6 and PLAG1 fused forward primers; the reverse primers are shown as sequence from Seq ID No.40 to Seq ID No.77 in sequence, and the same means that the 38 reverse primers are reverse primers for detecting the 38 fusion mutations in the sequence; the corresponding specific single-base extension primers are shown as Seq ID No.79 to Seq ID No.116 in sequence, which also means that the 38 specific single-base extension primers are the specific single-base extension primers for detecting the 38 fusion mutations in the above sequence in sequence and correspond to the corresponding forward primer and reverse primer. For example, for the fusion mutation BCOR-CCNB3, the forward primer of the specific primer pair is shown as Seq ID No.1, the reverse primer is shown as Seq ID No.40, and the corresponding specific single-base extension primer is shown as Seq ID No. 79; for the fusion mutation BCOR-ITD, the forward primer is a sequence shown by Seq ID No.2, the reverse primer is a sequence shown by Seq ID No.41, and the specific single-base extension primer is a sequence shown by Seq ID No. 80; for the fusion mutation BCOR-MAML3, the forward primer is the sequence shown in Seq ID No.3, the reverse primer is the sequence shown in Seq ID No.42, and the specific single-base extension primer is the sequence shown in Seq ID No. 81; and so on.
It is also noted that, when the primer composition is used, the specific primer pair takes a reverse transcription product extracted from the total RNA of the 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; the corresponding fusion mutation type is judged by detecting the existence of signals of the single base extension products at the position with specific molecular weight. The primer composition can detect 38 sarcoma fusion gene mutations corresponding to 28 sarcoma subtypes, has a large coverage range, is suitable for auxiliary diagnosis and typing of patients with initial diagnosis of sarcoma and sarcoma diagnosis difficulty, and provides a reference basis for making a treatment scheme and evaluating prognosis of sarcoma. The primer composition disclosed by the application is used for carrying out fusion mutation detection on an RNA layer, so that the detection accuracy is obviously improved, and the missing detection caused by the difference of fusion breakpoints in the process of breaking and reconnecting DNA is avoided. The primer composition can be obtained by performing multiplex PCR amplification 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 manner 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 internal reference gene is GAPDH, the forward primer of the internal reference specific primer pair is shown as Seq ID No.39, the reverse primer is shown as Seq ID No.78, and the internal reference specific single-base extension primer is shown as Seq ID No. 117.
It should be noted that the setting of the reference gene is to verify whether the detection process or the sample has errors; it is understood that the use of other reference genes besides the GAPDH reference gene used in the present application is not excluded, as long as the specific pair of reference-specific primers and the corresponding reference-specific single-base extension primers can be used for multiplex PCR amplification or single-base extension together with the specific pair of primers and the corresponding specific single-base extension primers for detecting 38 fusion mutations in the present application.
In one implementation of the present application, the forward and reverse primers each have 8-14nt tag sequences upstream of the primer.
In one embodiment of the present application, the forward primer and the reverse primer each have 10nt of tag sequences of the sequence shown in Seq ID No.118 upstream.
In a second aspect, the present application discloses a kit for detecting a mutation in a sarcoma fusion gene, which contains the primer composition of the present application.
In one embodiment of the present application, the kit further comprises a reverse transcription primer for reverse transcription of total RNA extracted from a sarcoma tissue sample.
In one implementation of the present application, the reverse transcription primers include 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 the 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 of the kit of the present application is that it contains the primer composition of the present application; as for other components, for example, a reverse transcription primer, a reverse transcriptase, shrimp alkaline phosphatase, a cation exchange resin, etc. can be optionally added to the kit as required, or can be purchased separately by itself; and is not particularly limited herein.
In a third aspect of the present application, there is disclosed the use of a primer composition of the present application, or a kit of the present application, in the detection of a sarcoma subtype for non-diagnostic therapeutic purposes, the development of a sarcoma therapeutic agent, or the prognostic assessment.
The fourth aspect of the application discloses a method for detecting the mutation of a sarcoma fusion gene, which comprises the following steps,
reverse transcription is carried out on total RNA extracted from a sarcoma tissue sample to be detected;
amplifying the reverse transcription product using the specific primer pair for detecting 38 fusion mutations in the primer composition according to any one of claims 1 to 4;
processing the amplified product by shrimp alkaline phosphatase, digesting residual dNTPs in the amplification reaction, and removing pyrophosphate groups from the dNTPs;
performing single-base extension on a digestion product of prawn alkaline phosphatase by using the corresponding specific single-base extension primer in the primer composition of any one of claims 1-4 to obtain a single-base extension product;
purifying the single base extension product by using cation exchange resin;
and 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 a signal of the single-base extension product at a position with a specific molecular weight.
It should be noted that the detection method of the sarcoma fusion gene mutation of the present application aims at obtaining the classification result of the sarcoma subtype, and the classification result only provides an intermediate reference for the treatment scheme formulation and prognosis evaluation of the sarcoma, and cannot be directly used for the diagnosis or treatment of the disease.
The beneficial effect of this application lies in:
the primer composition for detecting the sarcoma fusion gene mutation can detect 38 sarcoma fusion gene mutations corresponding to 28 sarcoma subtypes, has a large coverage range, and is suitable for initial diagnosis and auxiliary typing. In addition, the primer composition judges the sarcoma subtype by qualitatively detecting the fusion mutation transcript of the extracted total RNA of the tissue, and remarkably improves the detection accuracy by detecting the fusion mutation at the RNA level, thereby avoiding the missed detection caused by the difference of fusion breakpoints in the process of breaking and reconnecting DNA. The primer composition can be obtained by only performing 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 results of detection of negative samples in the first example of the present application;
FIGS. 3 to 11 are graphs showing the results of the detection of 9 commercial standards and clinical specimens of known fusion mutation types in example II of the present application.
Detailed Description
The existing detection method for the mutation of the sarcoma fusion gene mainly realizes the 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 the clinical use requirement. In view of the above, the present application creatively provides a matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) for detecting fusion products of reverse transcription products of total RNA in sarcoma tissues. Specifically, 38 fusion mutation types corresponding to 28 sarcoma subtypes including adipoblastoma, alveolar soft tissue sarcoma and the like are selected, a specific primer is designed to amplify a fusion mutation transcript enriched on an RNA layer, and the fusion mutation transcript is qualitatively detected after single base extension and nucleic acid mass spectrum detection, so that which fusion mutation occurs in a sarcoma tissue sample to be detected is judged, the tissue subtype of the sarcoma is further judged, and a reference basis is provided for the formulation of a treatment scheme and the prognosis evaluation of the sarcoma.
On the basis of the research, the primer composition for detecting the mutation of the sarcoma fusion gene is creatively developed and comprises a specific primer pair for detecting 38 fusion mutations and a corresponding specific single-base extension primer; the specific primer pairs for detecting 38 fusion mutations have sequences shown in Seq ID No.1 to Seq ID No.38 in sequence as the forward primers, sequences shown in Seq ID No.40 to Seq ID No.77 in sequence as the reverse primers, and sequences shown in Seq ID No.79 to Seq ID No.116 in sequence as the corresponding specific single-base extension primers.
Further, the primer composition also comprises an internal reference specific primer pair for detecting an internal reference gene GAPDH and a corresponding internal reference specific single-base extension primer; the internal reference specific primer pair is shown as Seq ID No.39, the reverse primer is shown as Seq ID No.78, and the internal reference specific single base extension primer is shown as Seq ID No. 117.
The method can detect 40 fusion transcript products covered by single detection, and the commonly used RT-PCR technology can only cover the detection of 1-4 fusion products by single reaction, compared with the method, the method has more comprehensive coverage and obviously reduces the detection cost of a single fusion product. Compared with the RNA sequencing method based on the high-throughput sequencing technology, the PCR amplification technology based on single base extension has the advantages of simple process and low quality requirement on RNA, and can be applied to FFPE samples.
Compared with the existing sarcoma fusion gene mutation detection, the primer combination, the kit and the sarcoma fusion gene mutation detection method have the following advantages:
(a) the application aims at 38 fusion mutation types, a specific primer is designed to amplify and enrich fusion mutation transcripts on an RNA layer, then the fusion mutation transcripts are qualitatively detected through single base extension and nucleic acid mass spectrum detection, which fusion mutation happens in a sarcoma tissue sample to be detected is judged, and then the tissue subtype of the sarcoma is judged, so that a reference basis is provided for the formulation of a treatment scheme and the prognosis evaluation of the sarcoma.
(b) The application aims at detecting sarcoma tissue samples, and judges sarcoma subtypes by qualitatively detecting fusion mutation transcripts of extracted total RNA of tissues; the detection accuracy can be obviously improved by carrying out fusion mutation detection on the RNA layer, and the missing detection caused by the difference of fusion breakpoints in the process of breaking and reconnecting DNA is avoided.
(c) The application covers 38 fusion mutations corresponding to 28 sarcoma subtypes, has a large coverage range, and is suitable for auxiliary diagnosis and typing of patients with initial diagnosis of sarcoma and sarcoma diagnosis difficulty.
(d) The application designs 39 groups of 117 amplification and extension primers for 38 fusion mutations corresponding to 28 sarcoma subtypes and one reference gene, all the primers can be used for performing multiplex PCR amplification in 1 reaction hole, the operation is simple, the detection period is short, and the use requirement of clinical sample detection can be better met.
The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application. In the following description, numerous 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 instances, or may be replaced by other kits, materials, methods. In some instances, certain operations related to the present application have not been shown or described in detail in this specification in order to avoid obscuring the core of the present application from excessive description, and it is not necessary for those skilled in the art to describe these operations in detail, so that the related operations will be fully understood from the description in the specification and the general knowledge of the art. In the following examples, the reagents or instruments used are not indicated by manufacturers, but are all conventional products available on the market.
The numbers in the present application, for example, "Seq ID No.", "first aspect", "second aspect", "third aspect", "(a)", "(b)", "(c)" and the like, are used only for distinguishing the described objects, and do not have any sequential or technical meaning.
Example one
The embodiment is based on the multiple PCR technology of single base extension and combines the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS, hereinafter referred to as nucleic acid mass spectrometry) technology to realize the high-throughput detection of the sarcoma fusion gene mutation. The method selects 38 fusion mutation types corresponding to 28 sarcoma subtypes including lipoblastoma, alveolar soft tissue sarcoma and the like, designs a specific primer to amplify the fusion mutation transcript enriched on an RNA layer, and qualitatively detects 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 and further judging the tissue subtype of the sarcoma. And corresponding amplification primers and single base extension primers are designed aiming at the internal reference gene. The method comprises the following specific steps:
(1) selecting fusion mutation types with corresponding correlation with sarcoma molecular subtypes, and designing specific amplification primers and extension primers by using online design software of agena. Specific primer pairs and corresponding specific single-base extension primers for detecting 38 fusion mutations, and internal reference specific primer pairs and corresponding internal reference specific single-base extension primers for internal reference genes, as shown in Table 1. The correspondence between the sarcoma molecular subtypes and the fusion mutations is shown in Table 2. Wherein the length of the amplification product of each pair of specific primers is about 100-220 bp; the molecular weight of the single base extension product of the extension primer is 3500Kda to 9000 Kda.
TABLE 1 amplification primer and extension primer sequences
TABLE 2 correspondence between sarcoma molecular subtypes and fusion mutations
In the specific primer pair and the reference specific primer pair shown in Table 1, the forward primer and the reverse primer have 8-14nt tag sequences respectively at the upstream, and the specific tag length in this example is 10nt, and the sequence is the sequence shown in Seq ID No. 118. Seq ID No. 118: 5'-ACGTTGGATG-3' are provided.
(2) Peripheral blood of healthy people is used, total RNA is extracted, and 1 mu g of the total RNA is subjected to reverse transcription to obtain cDNA. In this example, reverse transcription was carried out using a random primer of 6 to 8nt and a poly (T) primer under conditions of 25 ℃ for 5min, 42 ℃ for 30min and 85 ℃ for 5 min.
(3) mu.L of the reverse transcription product was amplified using primers specific for SEQ ID NO.1 to SEQ ID NO.78, and the fusion mutation transcripts that may be present were amplified and enriched.
The reaction system for specific amplification (pre-PCR) is as follows:
H2o (HPLC grade) 1.8. mu.L, 10 XPCR Buffer 0.5. mu. L, MgCl2(25mM)0.4μL、dNTP mix(25mM each)0.2μL、Primer mix(500nM each)1μL、
0.1. mu.L (5U/. mu.L), 1. mu.L of a reverse transcription product, and 5. mu.L in total.
The specific amplification reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min; then 45 cycles were entered: 94 ℃ 20sec, 56 ℃ 30sec, 72 ℃ 1 min; after the circulation was completed, extension was carried out at 72 ℃ for 1 min.
(4) After completion of the specific amplification, treating the amplification product with Shrimp Alkaline Phosphatase (SAP) to digest dNTPs remaining in the amplification reaction so that the dNTPs are deprived of pyrophosphate; specifically, after amplification is completed, the amplification product reaction tube is opened, and 1.53. mu.L of H is added to each tube2O, 0.17. mu.L of 10 XSAP Buffer and 0.3. mu.L of SAP enzyme (1.7U/. mu.L) were added and pipetted well in a total volume of 7. mu.L.
The reaction conditions for SAP digestion were 40min at 37 ℃; 5min at 85 ℃; finally standby at 4 ℃.
(5) After completion of SAP digestion, extension of SEQ ID NO.79 to SEQ ID NO.117 was usedExtending the primer to perform single-base extension on the SAP digestion product to obtain a single-base extension product; specifically, after completion of SAP digestion, the reaction tubes were opened and 0.619. mu.L of H was added to each tube2O, 0.2. mu.L of iPLEX Buffer Plus (10X), 0.2. mu.L of iPLEX Termination mix, 0.94. mu.L of iPLEX extended Primer mix, 0.041. mu.L of iPLEX enzyme in a total volume of 9. mu.L, pipetted and mixed.
The single base extension reaction condition is pre-denaturation at 94 ℃ for 30 sec; then 50 cycles were entered: 94 ℃ for 5sec, 52 ℃ for 5sec, 80 ℃ for 5 min; after the circulation was completed, extension was carried out at 72 ℃ for 3 min.
(6) Purifying the single base extension product by using cation exchange resin to remove magnesium ions, manganese ions, iron ions and the like in the reaction solution; specifically, after the completion of the elongation reaction, the reaction tube was opened and 18. mu.L of H was added2And adding cation exchange resin, sealing the reaction tube after adding, placing on a mute mixer, rotating the product plate for 40-60min at room temperature along the long axis at 360 degrees. And centrifuging the product at 4000rpm for 5min after the end of the mixed rotation.
(7) The purified product can be used for nucleic acid mass spectrometric detection, and the corresponding fusion mutation type is judged by the presence or absence of signals of the single base extension product at the position with specific molecular weight; in this example, the supernatant was spotted according to the agena massarray spotting and spotting procedure.
The results are shown in FIG. 1 and FIG. 2, wherein the abscissa represents the relative molecular weight of the single-base extension product, and the unit is KDa; the ordinate is the signal intensity, the stronger the signal intensity, the higher the copy number of the target product. The results in FIGS. 1 and 2 show that the primer composition and the extension primer thereof in this example have relatively uniform signal intensities for the negative samples extracted from peripheral blood of healthy persons, and no non-specific amplification or non-specific extension products occur.
Example two
In this example, the same specific primer pair and corresponding specific single-base extension primer, and the same reference gene amplification primer and single-base extension primer in example were used to perform sarcoma fusion gene mutation detection on RNA of 9 commercial standard samples and clinical samples of known fusion mutation types, and the related information of the samples to be detected is shown in table 3.
Table 39 samples of known fusion mutation types
| Sample numbering | Fusion forms | Fusing information | Type of mutation |
| CBP20137R | EWSR1-WT1 | chr22:29683123/chr11:128675261 | Fusion |
| CBP20128R | EWSR1-FLI1 | chr11:118353210/chr9:20365742 | Fusion |
| FA20061953 | FUS-CREB3L1 | 3.1％ | Fusion |
| FA20061465 | FUS-DDIT3 | 7.5％ | Fusion |
| FA20070162 | COL1A1-PDGFB | 8.9％ | Fusion |
| FA20061162 | EWSR1-CREB1 | 14.1％ | Fusion |
| FA20053065 | PAX7-FOXO1 | 5.3％ | Fusion |
| FA20072357 | CIC-DUX4 | 5.5％ | Fusion |
| FA20062522 | MYH9–USP6 | 14.5％ | Fusion |
Reverse transcription, pre-PCR, SAP, EP, cation exchange resin, machine testing, etc. are all the same as in example one.
The machine was spotted in the same manner as in the example, and the results are shown in FIGS. 3 to 11. FIGS. 3 to 11 are the test results of samples CBP20137R, CBP20128R, FA20061953, FA20061465, FA20070162, FA20061162, FA20053065, FA20072357 and FA20062522 in this 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 pre-PCR amplification is carried out by using the primer composition of the example, the upstream specific primer of EWSR1 and the downstream specific primer of WT1 can amplify target fusion products, and other fusion primer groups cannot amplify due to lack of target templates. After the residual dNTPs are digested by SAP, an extension primer is added for single-base extension, the extension primer is single-stranded DNA with a known sequence and molecular weight matched with a pre-PCR product, as shown in figure 3, according to the base complementary principle, the extended single base is C, the molecular weight of the extension product is 5050KDa, when a product signal is found at the position with the molecular weight of 5050KDa, the signal of a target product can be judged, and then the existence of corresponding fusion mutation is judged. The same applies to the determination of other fusion mutations, and the presence of a plurality of different fusion mutations is indicated by the detection of signals at a plurality of signal positions of the target product. If a signal is generated at a place where the non-target product has a predetermined molecular weight, it is judged as nonsense non-specific amplification and not included in the result interpretation.
The results of the above examples show that the primer composition for detecting sarcoma fusion gene mutation can detect 38 sarcoma fusion gene mutations corresponding to 28 sarcoma subtypes, and by detecting fusion mutation on the RNA layer, the detection accuracy can be improved, and missing detection caused by the difference of fusion breakpoints during DNA fragmentation and reconnection can be avoided. The primer composition only needs to perform multiple PCR amplification and single base extension in 1 reaction, and finally performs nucleic acid mass spectrometry detection, so that the primer composition is simple and convenient to operate, short in detection period and capable of better meeting the use requirement of clinical sample detection.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.
SEQUENCE LISTING
<110> Shenzhen Letu Biotech Limited
Primer composition and kit for detecting sarcoma fusion gene mutation and application of primer composition and kit
<130> 21I31477
<160> 118
<170> PatentIn version 3.3
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<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 (10)
1. A primer composition for detecting mutation of sarcoma fusion gene, comprising: the primer composition comprises a specific primer pair for detecting 38 fusion mutations and a corresponding specific single-base extension primer;
the 38 fusion mutations include BCOR-CCNB, BCOR-ITD, BCOR-MAML, CIC-FOXO, EWSR-NFATC, EWSR-WT, ASPL-TFE, EWSR-ERG, EWSR-FLI, NTRK rearrangement, SERPINE-FOSB, FUS-CREB3L, YWHAE-NUTM2, EWSR-NFATC, HEY-NCOA, SS-SSX, FUS-DDIT, EWSR-PBX, EWSR-ZNF 444, EWSR-DDIT, COL 1A-FB PDG, WWTR-CAMTA, YAP-TFE, EWSR-CREB, ETV-NTRK, PAX-FOOA, PAX-FOFUX, PAS-ATF, EWSR-ATF, EW-ATF, CIATIC-ZF-, SUWSR-CREB, SUAG-PLXO, USP-SACK;
the forward primers of the specific primer pairs for detecting 38 fusion mutations are shown as Seq ID No.1 to Seq ID No.38 in sequence, the reverse primers of the specific primer pairs are shown as Seq ID No.40 to Seq ID No.77 in sequence, and the corresponding specific single-base extension primers are shown as Seq ID No.79 to Seq ID No.116 in sequence.
2. The primer composition of 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 internal reference gene is GAPDH, the forward primer of the internal reference specific primer pair is a sequence shown by Seq ID No.39, the reverse primer is a sequence shown by Seq ID No.78, and the internal reference specific single base extension primer is a sequence shown by Seq ID No. 117.
3. The primer composition of claim 1 or 2, wherein: the forward primer and the reverse primer have 8-14nt tag sequences respectively at the upstream.
4. The primer composition of claim 1 or 2, wherein: the forward primer and the reverse primer each have 10nt upstream of them a tag sequence of the sequence shown by Seq ID No. 118.
5. A kit for detecting sarcoma fusion gene mutation, which is characterized in that: the kit contains the primer composition of any one of claims 1 to 4.
6. The kit of claim 5, wherein: the kit also contains a reverse transcription primer, and the reverse transcription primer is used for carrying out reverse transcription on total RNA extracted from a sarcoma tissue sample;
preferably, the reverse transcription primers comprise a random primer of 6-8nt and a poly (T) primer;
preferably, the kit further comprises a reverse transcriptase.
7. The kit according to claim 5 or 6, characterized in that: the kit also contains shrimp alkaline phosphatase which is used for digesting dNTPs and removing pyrophosphate groups from the dNTPs.
8. The kit according to claim 5 or 6, characterized in that: the kit also contains cation exchange resin for removing metal ions in the reaction solution.
9. Use of a primer composition according to any one of claims 1-4, or a kit according to any one of claims 5-8, for detection of a sarcoma subtype for non-diagnostic therapeutic purposes, development of a sarcoma therapeutic agent, or prognostic assessment.
10. A method for detecting a mutation in a sarcoma fusion gene for non-diagnostic therapeutic purposes, comprising: comprises the following steps of (a) carrying out,
reverse transcription is carried out on total RNA extracted from a sarcoma tissue sample to be detected;
amplifying the reverse transcription product using the specific primer pair for detecting 38 fusion mutations in the primer composition according to any one of claims 1 to 4;
processing the amplified product by shrimp alkaline phosphatase, digesting residual dNTPs in the amplification reaction, and removing pyrophosphate groups from the dNTPs;
performing single-base extension on a digestion product of prawn alkaline phosphatase by using the corresponding specific single-base extension primer in the primer composition of any one of claims 1-4 to obtain a single-base extension product;
purifying the single base extension product by using cation exchange resin;
and 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 a signal of the single-base extension product at a position with a specific molecular weight.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113981101A (en) * | 2021-12-27 | 2022-01-28 | 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 |
| 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 |
| CN117402976A (en) * | 2023-12-15 | 2024-01-16 | 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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| 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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