Kit and method for detecting NTRK gene fusion
阅读说明：本技术 用于检测ntrk基因融合的试剂盒和方法 (Kit and method for detecting NTRK gene fusion ) 是由 吴诗扬 许嘉森 彭璨璨 刘志明 刘芳 于 2019-11-14 设计创作，主要内容包括：本发明提供了一种用于检测NTRK基因融合的试剂盒和方法。该试剂盒包括有4管试剂中的至少一管,所述4管试剂中,含有分别针对NTRK基因12种融合类型设计得到特异性引物和探针,且每管检测1种NTRK1融合、1种NTRK2融合、1种NTRK3融合,可排除同一基因各引物间的干扰,从而极大地提高了反应体系的稳定性,提高检测的敏感度和特异性,降低出现假阳性的几率。(The invention provides a kit and a method for detecting NTRK gene fusion. The kit comprises at least one tube of 4 tubes of reagents, wherein the 4 tubes of reagents contain specific primers and probes which are respectively designed according to 12 fusion types of NTRK genes, and each tube detects 1 NTRK1 fusion, 1 NTRK2 fusion and 1 NTRK3 fusion, so that the interference among the primers of the same gene can be eliminated, the stability of a reaction system is greatly improved, the detection sensitivity and specificity are improved, and the probability of false positive is reduced.)
1. A kit for detecting NTRK gene fusion is characterized by comprising at least one tube of 4 tubes of PCR reaction reagents,
each tube of PCR reaction reagent comprises:
one primer and one probe in four of A), B), C) and D) aiming at NTRK1 gene fusion; and/or
E) for NTRK2 gene fusion, F), G) and H); and/or
One primer and one probe in four types of I), J), K) and L) aiming at NTRK3 gene fusion;
in the 4 tubes of PCR reaction reagents, primers and probes among the PCR reaction reagents of each tube are not repeated;
wherein:
A) primers and probes for NTRK gene exon E10 fusion partner exon E2 of LMNA-NTRK1 gene fusion;
B) primers and probes for NTRK gene exon E11 fusion partner exon E2 of LMNA-NTRK1 gene fusion;
C) primers and probes for NTRK gene exon E11 fusion partner exon E10 of LMNA-NTRK1 gene fusion;
D) primers and probes for NTRK gene exon E10 fusion partner exon E7 for TPM3-NTRK1 gene fusion;
E) primers and probes for NTRK gene exon E12 fusion partner exon E13 for AFAP1-NTRK2 gene fusion;
F) primers and probes for NTRK gene exon E13 fusion partner exon E4 for NACC2-NTRK2 gene fusion;
G) primers and probes for NTRK gene exon E16 fusion partner exon E6 for QKI-NTRK2 gene fusion;
H) primers and probes for NTRK gene exon E15 fusion partner exon E12 for TRIM24-NTRK2 gene fusion;
I) primers and probes for NTRK gene exon E14 fusion partner exon E4 for ETV6-NTRK3 gene fusion;
J) primers and probes for NTRK gene exon E14 fusion partner exon E5 for ETV6-NTRK3 gene fusion;
K) primers and probes for NTRK gene exon E15 fusion partner exon E4 for ETV6-NTRK3 gene fusion;
l) are primers and probes for the NTRK gene exon E15 fusion partner exon E5 of the ETV6-NTRK3 gene fusion;
the 3 'end of the probe is connected with an MGB modifying group, and the 5' end is modified with a fluorescent reporter group; wherein, the probe aiming at NTRK1 gene fusion, the probe aiming at NTRK2 gene fusion and the probe aiming at NTRK3 gene fusion are respectively marked with fluorescent reporter groups with different colors.
2. The kit for detecting NTRK gene fusion according to claim 1,
A) the primer is shown as SEQ ID NO.1 and SEQ ID NO.2, and the probe is shown as SEQ ID NO. 25;
B) the primer is shown as SEQ ID NO.3 and SEQ ID NO.4, and the probe is shown as SEQ ID NO. 26;
C) the primers are shown as SEQ ID NO.5 and SEQ ID NO.6, and the probe is shown as SEQ ID NO. 27;
D) the primers are shown as SEQ ID NO.7 and SEQ ID NO.8, and the probe is shown as SEQ ID NO. 28;
E) the primer is shown as SEQ ID NO.9 and SEQ ID NO.10, and the probe is shown as SEQ ID NO. 29;
F) the primers are shown as SEQ ID NO.11 and SEQ ID NO.12, and the probe is shown as SEQ ID NO. 30;
G) the primers are shown as SEQ ID NO.13 and SEQ ID NO.14, and the probe is shown as SEQ ID NO. 31;
H) the primers are shown as SEQ ID NO.15 and SEQ ID NO.16, and the probe is shown as SEQ ID NO. 32;
I) the primers are shown as SEQ ID NO.17 and SEQ ID NO.18, and the probe is shown as SEQ ID NO. 33;
J) the primers are shown as SEQ ID NO.19 and SEQ ID NO.20, and the probe is shown as SEQ ID NO. 34;
K) the primers are shown as SEQ ID NO.21 and SEQ ID NO.22, and the probe is shown as SEQ ID NO. 35;
l) the primer is shown as SEQ ID NO.23 and SEQ ID NO.24, and the probe is shown as SEQ ID NO. 36.
3. The kit for detecting NTRK gene fusion of claim 2, comprising at least one of 4 tubes of PCR reaction reagents, wherein:
the first tube PCR reaction reagent comprises: primers with nucleotide sequences shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.17 and SEQ ID NO.18, and probes with nucleotide sequences shown as SEQ ID NO.25, SEQ ID NO.30 and SEQ ID NO. 33; probes with nucleotide sequences shown as SEQ ID NO.25, SEQ ID NO.30 and SEQ ID NO.33 are respectively marked with fluorescent reporter groups with different colors;
the second tube PCR reaction reagent comprises: primers with nucleotide sequences shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.19 and SEQ ID NO.20, and probes with nucleotide sequences shown as SEQ ID NO.26, SEQ ID NO.29 and SEQ ID NO. 34; probes with nucleotide sequences shown as SEQ ID NO.26, SEQ ID NO.29 and SEQ ID NO.34 are respectively marked with fluorescent reporter groups with different colors;
the third tube PCR reaction reagent comprises: primers with nucleotide sequences shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.21 and SEQ ID NO.22, and probes with nucleotide sequences shown as SEQ ID NO.27, SEQ ID NO.31 and SEQ ID NO. 35; probes with nucleotide sequences shown as SEQ ID NO.27, SEQ ID NO.31 and SEQ ID NO.35 are respectively marked with fluorescent reporter groups with different colors;
the fourth tube PCR reaction reagent comprises: primers with nucleotide sequences shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.21 and SEQ ID NO.22, and probes with nucleotide sequences shown as SEQ ID NO.27, SEQ ID NO.31 and SEQ ID NO. 35; the probes with nucleotide sequences shown as SEQ ID NO.27, SEQ ID NO.31 and SEQ ID NO.35 are respectively marked with fluorescent reporter groups with different colors.
4. The kit for detecting NTRK gene fusion of claim 3, comprising 4 tubes of PCR reaction reagents, wherein:
the first tube PCR reaction reagent comprises: primers with nucleotide sequences shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.17 and SEQ ID NO.18, and probes with nucleotide sequences shown as SEQ ID NO.25, SEQ ID NO.30 and SEQ ID NO. 33; probes with nucleotide sequences shown as SEQ ID NO.25, SEQ ID NO.30 and SEQ ID NO.33 are respectively marked with fluorescent reporter groups with different colors;
the second tube PCR reaction reagent comprises: primers with nucleotide sequences shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.19 and SEQ ID NO.20, and probes with nucleotide sequences shown as SEQ ID NO.26, SEQ ID NO.29 and SEQ ID NO. 34; probes with nucleotide sequences shown as SEQ ID NO.26, SEQ ID NO.29 and SEQ ID NO.34 are respectively marked with fluorescent reporter groups with different colors;
the third tube PCR reaction reagent comprises: primers with nucleotide sequences shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.21 and SEQ ID NO.22, and probes with nucleotide sequences shown as SEQ ID NO.27, SEQ ID NO.31 and SEQ ID NO. 35; probes with nucleotide sequences shown as SEQ ID NO.27, SEQ ID NO.31 and SEQ ID NO.35 are respectively marked with fluorescent reporter groups with different colors;
the fourth tube PCR reaction reagent comprises: primers with nucleotide sequences shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.21 and SEQ ID NO.22, and probes with nucleotide sequences shown as SEQ ID NO.27, SEQ ID NO.31 and SEQ ID NO. 35; the probes with nucleotide sequences shown as SEQ ID NO.27, SEQ ID NO.31 and SEQ ID NO.35 are respectively marked with fluorescent reporter groups with different colors.
5. The kit for detecting NTRK gene fusion of any one of claims 1 to 4, further comprising positive quality controls comprising at least one of 13 plasmid DNAs, wherein the 13 plasmid DNAs comprise:
LMNA-E2-NTRK1-E10 gene fusion sequence, LMNA-E2-NTRK1-E11 gene fusion sequence, LMNA-E10-NTRK1-E11 gene fusion sequence, TPM3-E7-NTRK1-E1 gene fusion sequence, AFAP 1-E1-NTRK 1-E1 gene fusion sequence, NACC 1-E1-NTRK 1-E1 gene fusion sequence, QKI-E1-NTRK 1-E1 gene fusion sequence, TRIM 1-E1-NTRK 1-E1 gene fusion sequence, ETV 1-E1-NTRK 1 gene fusion sequence, ETV 1-E1-NTRK 1-E1 gene fusion sequence and ETV 1-NTRK 1-E1 gene fusion sequence.
6. The kit for detecting NTRK gene fusion of any one of claims 1-4, further comprising PCR reaction solutions I, II, III and IV, wherein the PCR reaction solutions I, II, III and IV comprise PCR reaction buffer, DNA polymerase, MgCl, and MgCl2dNTP, purified waterAt least one of the two types of the mixed solution is premixed and distributed into eight-joint pipes.
7. A sequence for detecting NTRK gene fusion, comprising at least one set of primers and probes:
primers and probes aiming at NTRK gene exon E10 fusion partner exon E2 fused with LMNA-NTRK1 gene, wherein the primers are shown as SEQ ID NO.1 and SEQ ID NO.2, and the probes are shown as SEQ ID NO. 25;
primers and probes aiming at NTRK gene exon E11 fusion partner exon E2 fused with LMNA-NTRK1 gene, wherein the primers are shown as SEQ ID NO.3 and SEQ ID NO.4, and the probes are shown as SEQ ID NO. 26;
primers and probes aiming at NTRK gene exon E11 fusion partner exon E10 fused with LMNA-NTRK1 gene, wherein the primers are shown as SEQ ID NO.5 and SEQ ID NO.6, and the probes are shown as SEQ ID NO. 27;
primers and probes aiming at an NTRK gene exon E10 fusion partner exon E7 fused with TPM3-NTRK1 gene, wherein the primers are shown as SEQ ID NO.7 and SEQ ID NO.8, and the probes are shown as SEQ ID NO. 28;
primers and probes aiming at an NTRK gene exon E12 fusion partner exon E13 fused with AFAP1-NTRK2 gene, wherein the primers are shown as SEQ ID NO.9 and SEQ ID NO.10, and the probes are shown as SEQ ID NO. 29;
primers and probes aiming at NTRK gene exon E13 fusion partner exon E4 fused with NACC2-NTRK2 gene, wherein the primers are shown as SEQ ID NO.11 and SEQ ID NO.12, and the probes are shown as SEQ ID NO. 30;
primers and probes aiming at an exon E16 fusion partner E6 of an NTRK gene fused with a QKI-NTRK2 gene, wherein the primers are shown as SEQ ID NO.13 and SEQ ID NO.14, and the probes are shown as SEQ ID NO. 31;
primers and probes aiming at NTRK gene exon E15 fusion partner exon E12 fused with TRIM24-NTRK2 gene, wherein the primers are shown as SEQ ID NO.15 and SEQ ID NO.16, and the probes are shown as SEQ ID NO. 32;
primers and probes aiming at an NTRK gene exon E14 fusion partner exon E4 fused with an ETV6-NTRK3 gene, wherein the primers are shown as SEQ ID NO.17 and SEQ ID NO.18, and the probes are shown as SEQ ID NO. 33;
primers and probes aiming at an NTRK gene exon E14 fusion partner exon E5 fused with an ETV6-NTRK3 gene, wherein the primers are shown as SEQ ID NO.19 and SEQ ID NO.20, and the probes are shown as SEQ ID NO. 34;
primers and probes aiming at an NTRK gene exon E15 fusion partner exon E4 fused with an ETV6-NTRK3 gene, wherein the primers are shown as SEQ ID NO.21 and SEQ ID NO.22, and the probes are shown as SEQ ID NO. 35;
the primers and the probes are directed against an NTRK gene exon E15 fusion partner exon E5 fused with an ETV6-NTRK3 gene, the primers are shown as SEQ ID NO.23 and SEQ ID NO.24, and the probes are shown as SEQ ID NO. 36.
8. A method for detecting NTRK gene fusion for non-diagnostic purposes, which is characterized by comprising the following steps:
(1) preparing a fluorescent PCR reaction system, wherein the fluorescent PCR reaction system comprises a PCR reaction reagent and a sample; the PCR reaction reagent is as follows: the PCR reaction reagent of the kit for detecting NTRK gene fusion of any one of claims 1-4;
the PCR reaction reagent also comprises an internal standard primer and an internal standard probe, and the nucleotide sequence of the internal standard primer is shown as SEQID NO.37 and 38; the nucleotide sequence of the internal standard probe is shown as SEQ ID NO. 39; the 3 'end of the internal standard probe is connected with an MGB modifying group, and the 5' end is marked with a fluorescence reporting group; the color of the fluorescent reporter group of the internal standard probe is different from the fluorescent reporter group of the probe fused aiming at the NTRK1 gene, the fluorescent reporter group of the probe fused aiming at the NTRK2 gene and the fluorescent reporter group of the probe fused aiming at the NTRK3 gene;
the PCR reaction reagent also comprises PCR reaction liquids I, II, III and IV, wherein the PCR reaction liquids I, II, III and IV comprise at least one of PCR reaction buffer solution, DNA polymerase, MgCl2, dNTP and purified water, and are premixed and subpackaged into eight-union pipes;
(2) and (3) carrying out fluorescent quantitative PCR reaction and detecting a fluorescent signal.
9. The method for detecting NTRK gene fusion of non-diagnostic purposes according to claim 8, wherein the fluorescent reporter of the internal standard probe, the fluorescent reporter of the probe for NTRK1 gene fusion, the fluorescent reporter of the probe for NTRK2 gene fusion and the fluorescent reporter of the probe for NTRK3 gene fusion are respectively selected from FAM, HEX, ROX and CY5 and are different from each other.
10. The method for detecting NTRK gene fusion of non-diagnostic purposes according to claim 8 or 9, wherein the detection result is determined according to the Ct value of the detected fluorescence signal:
if the Ct value of the fluorescence signal is 0 or more than or equal to 36, the detection result is NTRK gene fusion negative;
if the fluorescence signal meets the condition that the Ct value is more than 0 and less than 36, the detection result is positive NTRK gene fusion.
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a kit and a method for detecting NTRK gene fusion.
Background
The neurotrophic factor receptor tyrosine kinase (NTRK) family includes the TRKA, TRKB and TRKC proteins of the Tropomyosin Receptor Kinase (TRK) family, which are encoded by the NTRK1, NTRK2 and NTRK3 genes, respectively, and these proteins are usually expressed in neural tissues and activated by neurotrophic factors (NTs), playing an important role in the physiology of development and function of the nervous system. NTs refer to Nerve Growth Factor (NGF), a specific ligand for TRKA, brain-derived growth factor (BDGF), a specific ligand for TRKB, neurotrophic factor-4/5 (NT-4/5), and NT-3, a specific ligand for TRKC. Binding of NTs to TRK protein induces receptor dimerization, phosphorylation and activation of TRK downstream signaling cascades via PI3K, RAS/MAPK/ERK, and PLC-gamma signaling pathways. Alterations in TRK signaling pathways, including gene fusions, protein overexpression, and single nucleotide alterations, with NTRK gene fusions being most characterized in all NTRK alterations in cancer. NTRK gene fusion will result in fusion of NTRK gene family members (NTRK1, NTRK2, NTRK3) with another unrelated gene. TRK will be in continuous active state, triggering permanent signal cascade reaction, driving TRK fusion tumor to spread and grow.
There are many different fusion types for NTRK gene fusion. The currently found NTRK1 gene fusion types include LMNA-NTRK1, TPM3-NTRK1, CD74-NTRK1, MPRIP-NTRK1, NFASC-NTRK1, SQSRM1-NTRK1, TPR-NTRK1 and the like; the NTRK2 gene fusion types comprise AFAP1-NTRK2, NACC2-NTRK2, QKI-NTRK2, TRIM24-NTRK2 and the like; the NTRK3 gene fusion types include ETV6-NTRK3, EML4-NTRK3, TPM4-NTRK3, ZNF710-NTRK3 and the like. LMNA-NTRK1 and TPM3-NTRK1 are the main types of NTRK1 fusions, which occur at 31% and 21% frequency, respectively, in all NTRK1 fusions. ETV6-NTRK3 is the most common type of NTRK3 fusion, occurring at 88% frequency in all NTRK3 fusions.
The current detection method for NTRK gene fusion comprises the following steps: next Generation Sequencing (NGS) techniques, Fluorescence In Situ Hybridization (FISH) techniques, Immunohistochemistry (IHC), fluorescent PCR methods, and the like. NGS is also called high-throughput sequencing technology, is an accurate method for detecting NTRK gene fusion, and has the advantages of high throughput, high sensitivity, high specificity and the like. However, the NGS technology applied to NTRK gene fusion detection has the following disadvantages: (1) the cost is high; (2) the operation is complicated; (3) is relatively time consuming, e.g., based on Ion torrentTMThe detection process of the NGS technical platform is fastestReports were obtained only 3 days.
FISH is considered a gold standard for detection of gene fusions. The method has the advantages of high stability, high sensitivity, good specificity, short experimental period and the like. However, it can only detect one target at a time, for example, the commonly used split-type FISH probes can only detect gene fusions and cannot specifically detect the type of fusion. In addition, designing multiplex probes for detecting NTRK fusion partners is not only costly and time consuming, and therefore not suitable for practical high-throughput assays.
IHC is more economical and faster than current molecular detection methods. However, the IHC results are always subjective and the determination of weak positive results requires further verification by techniques such as FISH. In addition, the repeatability of IHC in NTRK gene fusion detection remains to be further verified.
The advantages of fluorescent PCR are: (1) the operation is simple and easy, and the detection time is short; (2) the sensitivity is high, the specificity is good, and the detection result is accurate and reliable; (3) the result judgment is clear and intuitive, and the result can be quantitatively analyzed; (4) the method has wide application range and is suitable for various detection samples. However, the application of the method in NTRK gene fusion detection is limited to detection of a certain gene fusion type; for example, human LMNA-NTRK1 gene fusion mutation detection primers, probes and detection kit (CN108660193A) are only used for detecting the fusion of the exon 11 of LMNA gene and the exon 10 of NTRK1 gene, but other fusion types of NTRK1 gene such as TPM3-NTRK1 and NTRK2 and NTRK3 gene fusion can not be detected.
Therefore, there is a need for a more optimal detection method to meet the following requirements: (1) can identify a plurality of different NTRK gene fusion types; (2) the operation process is simple and easy to implement, and the detection time is short; (3) the sensitivity is high, the specificity is good, and the detection result is accurate and reliable; (4) the result judgment is objective and reliable; (5) the method is suitable for detecting various types of samples.
Disclosure of Invention
Based on this, the present invention aims to provide a kit for detecting NTRK gene fusion.
In order to achieve the purpose, the specific technical scheme of the invention is as follows:
a kit for detecting NTRK gene fusion comprises at least one tube of 4 tubes of PCR reaction reagents, wherein each tube of PCR reaction reagent in the 4 tubes of PCR reaction reagents respectively comprises:
one primer and one probe in four of A), B), C) and D) aiming at NTRK1 gene fusion; and/or E), F), G) and H) of NTRK2 gene fusion; and/or one primer and probe in four types of I), J), K) and L) aiming at NTRK3 gene fusion; in the 4 tubes of PCR reaction reagents, primers and probes among the PCR reaction reagents of each tube are not repeated; wherein:
A) primers and probes for NTRK gene exon E10 fusion partner exon E2 of LMNA-NTRK1 gene fusion; B) primers and probes for NTRK gene exon E11 fusion partner exon E2 of LMNA-NTRK1 gene fusion; C) primers and probes for NTRK gene exon E11 fusion partner exon E10 of LMNA-NTRK1 gene fusion; D) primers and probes for NTRK gene exon E10 fusion partner exon E7 for TPM3-NTRK1 gene fusion; E) primers and probes for NTRK gene exon E12 fusion partner exon E13 for AFAP1-NTRK2 gene fusion; F) primers and probes for NTRK gene exon E13 fusion partner exon E4 for NACC2-NTRK2 gene fusion; G) primers and probes for NTRK gene exon E16 fusion partner exon E6 for QKI-NTRK2 gene fusion; H) primers and probes for NTRK gene exon E15 fusion partner exon E12 for TRIM24-NTRK2 gene fusion; I) primers and probes for NTRK gene exon E14 fusion partner exon E4 for ETV6-NTRK3 gene fusion; J) primers and probes for NTRK gene exon E14 fusion partner exon E5 for ETV6-NTRK3 gene fusion; K) primers and probes for NTRK gene exon E15 fusion partner exon E4 for ETV6-NTRK3 gene fusion; l) are primers and probes against the NTRK gene exon E15 fusion partner exon E5 of the ETV6-NTRK3 gene fusion. The 3 'end of the probe is connected with an MGB modifying group, and the 5' end is modified with a fluorescent reporter group; wherein, the probe aiming at NTRK1 gene fusion, the probe aiming at NTRK2 gene fusion and the probe aiming at NTRK3 gene fusion are respectively marked with fluorescent reporter groups with different colors.
Preferably, in the primers and probes described above, A) the primers are shown as SEQ ID NO.1 and SEQ ID NO.2, and the probe is shown as SEQ ID NO. 25; B) the primer is shown as SEQ ID NO.3 and SEQ ID NO.4, and the probe is shown as SEQ ID NO. 26; C) the primers are shown as SEQ ID NO.5 and SEQ ID NO.6, and the probe is shown as SEQ ID NO. 27; D) the primers are shown as SEQ ID NO.7 and SEQ ID NO.8, and the probe is shown as SEQ ID NO. 28; E) the primer is shown as SEQ ID NO.9 and SEQ ID NO.10, and the probe is shown as SEQ ID NO. 29; F) the primers are shown as SEQ ID NO.11 and SEQ ID NO.12, and the probe is shown as SEQ ID NO. 30; G) the primers are shown as SEQ ID NO.13 and SEQ ID NO.14, and the probe is shown as SEQ ID NO. 31; H) the primers are shown as SEQ ID NO.15 and SEQ ID NO.16, and the probe is shown as SEQ ID NO. 32; I) the primers are shown as SEQ ID NO.17 and SEQ ID NO.18, and the probe is shown as SEQ ID NO. 33; J) the primers are shown as SEQ ID NO.19 and SEQ ID NO.20, and the probe is shown as SEQ ID NO. 34; K) the primers are shown as SEQ ID NO.21 and SEQ ID NO.22, and the probe is shown as SEQ ID NO. 35; l) the primer is shown as SEQ ID NO.23 and SEQ ID NO.24, and the probe is shown as SEQ ID NO. 36.
Preferably, the kit comprises at least one tube of 4 tubes of PCR reaction reagents, wherein:
the first tube PCR reaction reagent comprises: primers with nucleotide sequences shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.17 and SEQ ID NO.18, and probes with nucleotide sequences shown as SEQ ID NO.25, SEQ ID NO.30 and SEQ ID NO. 33; the probes with nucleotide sequences shown as SEQ ID NO.25, SEQ ID NO.30 and SEQ ID NO.33 are respectively marked with fluorescent reporter groups with different colors. The second tube PCR reaction reagent comprises: primers with nucleotide sequences shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.19 and SEQ ID NO.20, and probes with nucleotide sequences shown as SEQ ID NO.26, SEQ ID NO.29 and SEQ ID NO. 34; the probes with nucleotide sequences shown as SEQ ID NO.26, SEQ ID NO.29 and SEQ ID NO.34 are respectively marked with fluorescent reporter groups with different colors. The third tube PCR reaction reagent comprises: primers with nucleotide sequences shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.21 and SEQ ID NO.22, and probes with nucleotide sequences shown as SEQ ID NO.27, SEQ ID NO.31 and SEQ ID NO. 35; the probes with nucleotide sequences shown as SEQ ID NO.27, SEQ ID NO.31 and SEQ ID NO.35 are respectively marked with fluorescent reporter groups with different colors. The fourth tube PCR reaction reagent comprises: primers with nucleotide sequences shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.21 and SEQ ID NO.22, and probes with nucleotide sequences shown as SEQ ID NO.27, SEQ ID NO.31 and SEQ ID NO. 35; the probes with nucleotide sequences shown as SEQ ID NO.27, SEQ ID NO.31 and SEQ ID NO.35 are respectively marked with fluorescent reporter groups with different colors.
More preferably, the kit comprises the 4-tube PCR reaction reagent.
Preferably, the kit further comprises an internal standard primer and an internal standard probe, wherein the nucleotide sequence of the internal standard primer is shown in SEQ ID NO.37 and 38; the nucleotide sequence of the internal standard probe is shown as SEQ ID NO. 39.
More preferably, the 3 'end of the internal standard probe is connected with an MGB modifying group, and the 5' end is marked with a fluorescent reporter group; the color of the fluorescent reporter of the internal standard probe is distinguished from the fluorescent reporter of the probe for NTRK1 gene fusion, the fluorescent reporter of the probe for NTRK2 gene fusion and the fluorescent reporter of the probe for NTRK3 gene fusion.
Preferably, the kit further comprises a positive quality control, comprising at least one of 13 plasmid DNAs, wherein the 13 plasmid DNAs comprise: LMNA-E2-NTRK1-E10 gene fusion sequence, LMNA-E2-NTRK1-E11 gene fusion sequence, LMNA-E10-NTRK1-E11 gene fusion sequence, TPM3-E7-NTRK1-E1 gene fusion sequence, AFAP 1-E1-NTRK 1-E1 gene fusion sequence, NACC 1-E1-NTRK 1-E1 gene fusion sequence, QKI-E1-NTRK 1-E1 gene fusion sequence, TRIM 1-E1-NTRK 1-E1 gene fusion sequence, ETV 1-E1-NTRK 1 gene fusion sequence, ETV 1-E1-NTRK 1-E1 gene fusion sequence and ETV 1-NTRK 1-E1 gene fusion sequence.
The invention also provides a sequence for detecting NTRK gene fusion, which comprises at least one group of primers and probes as described above.
The invention also provides a non-diagnosis-purpose NTRK gene fusion detection method, which comprises the following steps:
(1) preparing a fluorescent PCR reaction system, wherein the fluorescent PCR reaction system comprises a PCR reaction reagent and a sample; the PCR reaction reagent is as follows: the PCR reaction reagent in the kit for detecting NTRK gene fusion described in any one of the above; the PCR reaction reagent also comprises an internal standard primer and an internal standard probe, and the nucleotide sequence of the internal standard primer is shown in SEQ ID NO.37 and 38; the nucleotide sequence of the internal standard probe is shown as SEQ ID NO. 39; the 3 'end of the internal standard probe is connected with an MGB modifying group, and the 5' end is marked with a fluorescence reporting group; the color of the fluorescent reporter group of the internal standard probe is different from the fluorescent reporter group of the probe fused aiming at the NTRK1 gene, the fluorescent reporter group of the probe fused aiming at the NTRK2 gene and the fluorescent reporter group of the probe fused aiming at the NTRK3 gene; the PCR reaction reagent also comprises PCR reaction liquids I, II, III and IV, wherein the PCR reaction liquids I, II, III and IV comprise at least one of PCR reaction buffer solution, DNA polymerase, MgCl2, dNTP and purified water, and are premixed and subpackaged into eight-union pipes; (2) and (3) carrying out fluorescent quantitative PCR reaction and detecting a fluorescent signal.
In some embodiments, the PCR reagent further comprises an internal standard primer and an internal standard probe. Preferably, the nucleotide sequence of the internal standard primer is shown in SEQ ID NO.37 and 38; the nucleotide sequence of the internal standard probe is shown as SEQ ID NO. 39. More preferably, the 3 'end of the internal standard probe is connected with an MGB modifying group, and the 5' end is marked with a fluorescent reporter group. Further preferably, the color of the fluorescent reporter of the internal standard probe is different from that of the fluorescent reporter of the NTRK1 gene fusion probe, that of the fluorescent reporter of the NTRK2 gene fusion probe and that of the fluorescent reporter of the NTRK3 gene fusion probe.
And (2) carrying out fluorescent quantitative PCR reaction and detecting a fluorescent signal.
Preferably, in the detection method of NTRK gene fusion for non-diagnostic purposes, the fluorescent reporter group of the internal standard probe, the fluorescent reporter group of the probe for NTRK1 gene fusion, the fluorescent reporter group of the probe for NTRK2 gene fusion and the fluorescent reporter group of the probe for NTRK3 gene fusion are respectively selected from FAM, HEX, ROX and CY5 and are different from each other.
Further, in the fluorescent quantitative PCR reaction, FAM, HEX, ROX and CY5 channels are selected as the fluorescent channels of the fluorescent PCR amplification instrument.
Preferably, the conditions of the fluorescent quantitative PCR reaction are as follows: pre-denaturation at 95 + -1 deg.C for 5 + -0.5 min for 1 cycle; denaturation at 95 +/-1 ℃ for 30 +/-1 seconds, annealing at 60 +/-1 ℃ for 30 +/-1 seconds, extension at 72 +/-1 ℃ for 30 +/-1 seconds and 40 +/-5 cycles; extension at 72. + -. 1 ℃ for 10. + -.1 min.
In some embodiments, the detection result is determined by the Ct value of the detected fluorescence signal:
if the Ct value of the fluorescence signal is 0 or more than or equal to 36, the detection result is NTRK gene fusion negative;
if the fluorescence signal meets the condition that the Ct value is more than 0 and less than 36, the detection result is positive NTRK gene fusion.
The preparation method of the kit for detecting NTRK gene fusion comprises the following steps: (1) designing primers and probes for detecting human NTRK gene fusion; (2) the PCR tubes were packed with primers and probes, respectively, according to the 4-tube reagents described above.
Preferably, the preparation method further comprises, before the step (2), dispensing the DNA polymerase in the PCR tube, adding paraffin in a molten state, and allowing the paraffin to solidify.
More preferably, the PCR tube is an octal tube.
Based on the technical scheme, the invention has the following beneficial effects:
(1) the detection sensitivity is high, the specificity is good: the invention designs specific primers and fluorescent probes respectively aiming at 12 fusion types of NTRK genes, the specific primers can ensure accurate and effective amplification of target sequences, and the fluorescent probes with short fragments and MGB modifying groups can be better specifically combined with target sequences, thereby ensuring high detection sensitivity and good specificity of the kit; in addition, 12 different NTRK gene fusion types are divided into 4 groups of tubes for detection, and each group detects 1 NTRK1 fusion, 1 NTRK2 fusion and 1 NTRK3 fusion, so that the interference among primers of the same gene can be eliminated, the stability of a reaction system is greatly improved, and the probability of false positive in detection is reduced.
(2) The detection operation is quick and simple: the PCR reaction liquids I, II, III and IV are ready-to-use reagents which are respectively subpackaged into eight-connected tubes, and the enzymes in the PCR reaction liquids are separated from other components by using paraffin or a compound with similar characteristics at normal temperature or low temperature; when the sample template is added for PCR reaction, the paraffin is liquefied, and the enzyme can be uniformly mixed with other substances in the tube for reaction, so that the design greatly simplifies the detection operation process. The whole fluorescence PCR detection process can be completed within 2 hours, and 12 fusion types of NTRK genes can be detected at one time.
(3) The types of samples that are suitable for use are diverse: can be used for detecting fresh, frozen or paraffin-embedded tissue samples, blood samples and other conventional samples.
Drawings
FIG. 1 is a detection curve diagram of a PCR reaction solution I of the NTRK gene fusion negative sample of the invention;
FIG. 2 is a detection curve diagram of a PCR reaction solution II of the NTRK gene fusion negative sample of the invention;
FIG. 3 is a detection curve diagram of a PCR reaction solution III of the NTRK gene fusion negative sample according to the present invention;
FIG. 4 is a diagram showing the detection curve of the PCR reaction solution IV of the NTRK gene fusion negative sample according to the present invention;
FIG. 5 is a detection curve diagram of PCR reaction solution I of the NTRK gene fusion positive sample of the present invention;
FIG. 6 is a detection curve diagram of PCR reaction solution II of the NTRK gene fusion positive sample of the present invention;
FIG. 7 is a detection curve diagram of PCR reaction solution III of the NTRK gene fusion positive sample according to the present invention;
FIG. 8 is a graph showing the detection curve of the PCR reaction solution IV of the NTRK gene fusion positive sample according to the present invention;
FIG. 9 is a detection curve diagram of the PCR reaction solution I of the positive quality control material of the present invention;
FIG. 10 is a detection curve diagram of the PCR reaction solution II of the positive quality control material of the present invention;
FIG. 11 is a detection curve diagram of the PCR reaction solution III of the positive quality control material of the present invention;
FIG. 12 is a graph showing the detection curve of the PCR reaction solution IV of the positive quality control material of the present invention.
Detailed Description
In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It will be appreciated that the experimental procedures for the following examples, where specific conditions are not indicated, are generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The various reagents used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.