CN110964829A - Application method of human RCN3-SSU72 gene fusion mutation detection primer combination, probe and detection kit - Google Patents
Application method of human RCN3-SSU72 gene fusion mutation detection primer combination, probe and detection kit Download PDFInfo
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Abstract
The invention discloses a method for using a human RCN3-SSU72 gene fusion mutation detection primer combination, a probe and a detection kit, and the method adopts a specific primer to identify the human RCN3-SSU72 gene fusion mutation type, collects a fluorescent signal based on a Taqman probe method, and well guarantees the specificity. The kit of the invention has the following advantages: (1) a real-time fluorescent PCR system is established, and RCN3-SSU72 fusion mutation can be detected simultaneously; (2) the operation is simple, the detection speed is high, and the whole reverse transcription PCR and fluorescence PCR detection can be finished within half a day; (3) the accuracy is high, and the UNG enzyme-dUTP anti-pollution system and the blank control system are introduced into the system, so that false positive results can be better prevented, and the accuracy and reliability of detection results are ensured.
Description
Technical Field
The invention relates to the field of biotechnology and medicine, in particular to a human RCN3-SSU72 gene fusion mutation detection primer combination, a probe and a use method of a detection kit.
Background
Fusion gene belongs to one of gene mutation, which means that all or part of sequences of two genes are fused with each other to form a new gene, and the most common three occurrence mechanisms are chromosome translocation, middle deletion and chromosome inversion. In most cases, fusion genes may lead to the production of abnormal sequences or proteins with abnormal functions, or to the deregulation of the expression of certain genes, thereby leading to or promoting tumorigenesis. The gene fusion is firstly discovered in malignant tumors of a blood system, wherein the BCR-ABL gene fusion in chronic myelocytic leukemia is the most classical gene fusion, and the action target of the drug imatinib/gleevec for treating the chronic myelocytic leukemia is the fusion gene. With the development of detection technology, scientists find a large number of fusion genes in solid tumors such as lung cancer, prostate cancer, breast cancer, ovarian cancer, ewing sarcoma, synovial sarcoma and the like, and research shows that the fusion genes are taken as tumor driving genes and have important significance in the diagnosis, prognosis, stratification, treatment and drug development of tumors.
Taking the ALK fusion gene as an example, ALK fusion is one of the key driving mechanisms of non-small cell lung cancer (NSCLC), and the occurrence probability in NSCLC patients is about 4% -7%. Both the inhibitors of ALK fusion, cricotinib and ceritinib, have enjoyed great success in treating NSCLC patients positive for ALK fusion. Therefore, the method has great significance for accurately detecting the fusion gene, namely, researching and developing medicines or precise medical treatment.
At present, the methods for clinically detecting the mutation of the fusion gene mainly comprise the traditional PCR method and a direct sequencing method. There is no accurate and efficient method for clinically detecting the human RCN3-SSU72 gene fusion mutation. The limitations of the traditional PCR method also greatly restrict the discovery of accurate results, and the RCN3-SSU72 gene fusion point cannot be systematically and comprehensively analyzed. The genome of human tumor tissue contains a large number of mutations, which can lead to failure of the PCR assay. Even if a whole genome detection technology (WGS) is used, the detection sensitivity of the RCN3-SSU72 fusion is low, the second-generation sequencing technology is limited by short sequencing read length, high analysis difficulty and the like, and false negative results are easy to occur.
Disclosure of Invention
The invention aims to provide a primer combination, a probe and a method for using a detection kit for human RCN3-SSU72 gene fusion mutation.
The technical scheme adopted by the invention is as follows:
human RCN3-SSU72 gene fusion mutation detection primer combination and probe are characterized in that: the primer combination comprises sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2, which are respectively as follows: RCN3-SSU72 Left Primer: 5'-TCTGTTGCTACTGAGGCACG-3' (SEQ ID NO: 1); RCN3-SSU72 Right Primer: 5'-GCTCGTCGATCTCGTTCTCC-3' (SEQ ID NO: 2), and the sequence of the specific probe matched with the primer combination is shown in SEQ ID NO:3, and the specific probe is as follows: RCN3-SSU 72-P: 5'-CCCGTCTGGGCTATACACAG-3' (SEQ ID NO: 3), which is modified with FAM or VIC at the 5 'end and NFQ-MGB at the 3' end.
The kit contains the primer combination of claim 1 and a specific probe.
The kit also comprises a quality control system, wherein the quality control system comprises a quality control primer and a quality control probe, and the sequences of the quality control primer are shown as SEQ ID NO. 4 and SEQ ID NO. 5, and are respectively as follows: ATP6IP1 Left Primer: 5'-AGTGGAGGCTGAGGCTATGA-3' (SEQ ID NO: 4); ATP6IP1 Right Primer: 5'-GAGGCTGGCATTGAGCTTCA-3' (SEQ ID NO: 5), and the sequence of the quality control probe is shown in SEQ ID NO:6 as follows: ATP6IP 1-P: 5'-GGCTGCCGGTGTTTTTGTC-3' (SEQ ID NO: 6), the 5 'end of the quality control probe of the kit is modified with FAM or VIC, and the 3' end is modified with NFQ-MGB.
The kit also contains a positive control solution and a blank control solution, wherein the positive control solution is plasmid DNA containing RCN3-SSU72, and the blank control is Tris buffer solution.
The kit comprises PCR buffer solution, HotStart Taq enzyme and UNG enzyme.
The kit for detecting the human RCN3-SSU72 gene fusion mutation comprises the following steps:
step 1: extracting RNA from a test sample from a fresh sample, such as fresh blood, tumor fresh sample after surgical resection/puncture/ultra-low temperature frozen sample/sample preserved in RNA stabilizing solution;
step 2: carrying out reverse transcription on the extracted RNA to obtain cDNA, and carrying out real-time fluorescence PCR amplification reaction by using the cDNA as a template;
and step 3: judging the detection result according to the Ct value displayed by the fluorescent PCR amplification instrument: monitoring the fluorescence intensity of FAM and VIC of a reaction system, taking the cycle number Ct value required when the FAM reaches a set threshold as a negative and positive judgment standard, wherein the Ct value is more than or equal to 38: negative; ct value less than 38: and (4) positive.
The kit comprises a reaction system of PCR amplification reaction, and comprises the following components: PCR buffer solution: 15 muL and a primer: 1 mu M and a probe: 0.5 μ M, Taq enzyme: 1.0U, UNG enzyme: 0.5U, reverse transcription product cDNA: 2 muL and water: balance, total amount: 25 muL.
The invention has the advantages that: the specific primers are adopted to identify the human RCN3-SSU72 gene fusion mutation type, fluorescent signals are collected based on a Taqman probe method, and specificity is well guaranteed. The kit of the invention has the following advantages: (1) a real-time fluorescent PCR system is established, and RCN3-SSU72 fusion mutation can be detected simultaneously; (2) the operation is simple, the detection speed is high, and the whole reverse transcription PCR and fluorescence PCR detection can be finished within half a day; (3) the accuracy is high, and the UNG enzyme-dUTP anti-pollution system and the blank control system are introduced into the system, so that false positive results can be better prevented, and the accuracy and reliability of detection results are ensured.
Detailed Description
The human RCN3-SSU72 gene fusion mutation detection primer combination and probe, wherein the primer combination comprises sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2, and the sequences are respectively as follows: RCN3-SSU72 Left Primer: 5'-TCTGTTGCTACTGAGGCACG-3' (SEQ ID NO: 1); RCN3-SSU72 Right Primer: 5'-GCTCGTCGATCTCGTTCTCC-3' (SEQ ID NO: 2), and the specific probe sequence used in combination with the primer is shown in SEQ ID NO:3 as follows: RCN3-SSU 72-P: 5'-CCCGTCTGGGCTATACACAG-3' (SEQ ID NO: 3), wherein the 5 'end is modified with FAM or VIC, the 3' end is modified with NFQ-MGB, the group does not generate fluorescence, thereby greatly reducing the intensity of background signal, meanwhile, the specific probe is also connected with MGB modifying group, which can increase the Tm value of the probe by about 10 ℃, therefore, the same Tm value, MGB probe can be shorter than the common Taqman probe, and the specificity is better.
The 5 'end of the quality control probe is marked with a reporter group, such as FAM or VIC, and the 3' end is marked with a non-luminous fluorescence quenching group, such as NFQ-MGB and the like. When the probe is intact, the fluorescent energy emitted by the reporter is absorbed by the quencher and no signal is detected by the instrument. As PCR proceeds, Taq enzyme encounters the template-bound probe during DNA strand extension, its 5 'to 3' exonuclease activity cleaves the probe and the reporter group is moved away from the fluorescence quenching group, generating a fluorescent signal.
The kit comprises the primer combination of claim 1 and a specific probe.
The kit also comprises a quality control system, wherein the quality control system comprises a quality control primer and a quality control probe, and the sequences of the quality control primer are shown as SEQ ID NO. 4 and SEQ ID NO. 5, and are respectively as follows: ATP6IP1 Left Primer: 5'-AGTGGAGGCTGAGGCTATGA-3' (SEQ ID NO: 4); ATP6IP1 Right Primer: 5'-GAGGCTGGCATTGAGCTTCA-3' (SEQ ID NO: 5), and the sequence of the quality control probe is shown in SEQ ID NO:6 as follows: ATP6IP 1-P: 5'-GGCTGCCGGTGTTTTTGTC-3' (SEQ ID NO: 6), the 5 'end of the quality control probe of the kit is modified with FAM or VIC, and the 3' end is modified with NFQ-MGB. .
RNA is easy to degrade, and the requirements on the processes of sample preparation and storage, RNA extraction and reverse transcription are high. The quality control system is constructed by adopting the genes with stable expression quantity, so that the sample quality can be effectively monitored, and the generation of false negative results is avoided. Therefore, the quality control system is adopted to eliminate the hidden troubles, and the accuracy of the detection result is ensured. The quality control system comprises quality control primers and quality control probes, and other quality control systems known to those skilled in the art can be used in the invention. Preferably, the quality control system comprises quality control primers and quality control probes.
The kit also contains a positive control solution and a blank control solution, wherein the positive control solution is plasmid DNA containing RCN3-SSU72, the blank control is Tris buffer solution, the positive control solution and the blank control solution can monitor normal operation of real-time quantitative PCR reaction, the positive control solution contains two kinds of plasmid DNA mixed solution, and the plasmid DNA is RCN3-SSU72 sequence and human ATP6IP1 sequence.
The kit comprises PCR buffer, HotStart Taq enzyme and UNG enzyme.
The kit for detecting the human RCN3-SSU72 gene fusion mutation comprises the following steps:
step 1: extracting RNA from a test sample from a fresh sample, such as fresh blood, tumor fresh sample after surgical resection/puncture/ultra-low temperature frozen sample/sample preserved in RNA stabilizing solution;
step 2: carrying out reverse transcription on the extracted RNA to obtain cDNA, and carrying out real-time fluorescence PCR amplification reaction by using the cDNA as a template;
and step 3: judging the detection result according to the Ct value displayed by the fluorescent PCR amplification instrument: monitoring the fluorescence intensity of FAM and VIC of a reaction system, taking the cycle number Ct value required when the FAM reaches a set threshold as a negative and positive judgment standard, wherein the Ct value is more than or equal to 38: negative; ct value less than 38: and (4) positive.
The enzyme solution contains Taq enzyme, which is necessary for PCR reaction, and UNG enzyme, which can selectively hydrolyze and break uracil glycosidic bond in double-stranded or single-stranded DNA containing dU. In the PCR reaction, non-specific PCR amplification and contamination can be prevented by using UNG enzyme.
The kit comprises a reaction system of PCR amplification reaction, and comprises the following components: PCR buffer solution: 15 muL and a primer: 1 mu M and a probe: 0.5 μ M, Taq enzyme: 1.0U, UNG enzyme: 0.5U, reverse transcription product cDNA: 2 muL and water: balance, total amount: 25 muL.
In the 25 muL reaction system, the primers comprise specific primers and 1 pair of quality control primers, and the probes comprise specific probes and quality control probes.
The fluorescent quantitative PCR reaction conditions in the step (3) are as follows: treatment at 37 ℃ for 10min, pre-denaturation at 95 ℃ for 5min, 40 cycles: 95 ℃ for 15 seconds, 55 ℃ for 35 seconds and collecting the fluorescence signal.
After the PCR reaction, the results were judged according to the following steps: and (3) judging the effectiveness of the kit according to the weak positive control and the blank control: according to the result of the quality control system, carrying out sample validity judgment, and controlling the PCR reaction solution: ct value is less than or equal to 30, sample RNA quality is good, and adding amount is moderate.
And judging the detection result of the sample, and determining whether the sample is fused or not.
If the Ct value of the sample is larger than or equal to 38 or no Ct value, the sample is non-fusion mutation or fusion mutation outside the detection range of the kit;
and if the Ct value detected by the sample is less than 38, the sample is RCN3-SSU72 fusion mutation.
The detection method can quickly and efficiently give results, and has the advantages of strong specificity and real and credible results.
The invention is further illustrated by the following specific examples.
Example 1
The RCN3-SSU72 fusion mutation detection kit prepared by the invention comprises the following steps:
step 1: primer and probe synthesis: synthesizing 1 pair of specific primers, wherein the forward primer is SEQ ID NO. 1, the reverse primer is SEQ ID NO. 2, and the specific probe is SEQ ID NO. 3. Preparing a primer and a probe into mother liquor of 100 mu M respectively for later use;
step 2: preparing a quality control system: synthesizing 1 pair of quality control primers aiming at the human ATP6IP1 gene, wherein the sequences of the primer pair are SEQ ID NO. 4 and SEQ ID NO. 5, and the sequence of the quality control probe is SEQ ID NO. 6. Respectively preparing the quality control primer and the probe into mother liquor of 100 mu M for later use;
and step 3: preparation of other reagents: PCR buffer was prepared containing 1.0mM of MgCI2, 1.0mM of dATP, dUTP, dGTP and dCTP. Preparing enzyme mixed solution, wherein the enzyme mixed solution contains 0.5 multiplied by 10^3U/ml of Taq enzyme and 0.1 multiplied by 10^3U/ml of UNG enzyme;
and 4, step 4: preparing positive control solution and blank control solution: the positive control solution contains a mixed solution of two plasmid DNAs, namely RCN3-SSU72 and ATP6IP1 sequences, the selection and design of the plasmids are well known to those skilled in the art, and a blank buffer solution is a Tris buffer solution;
and 5: preparing a PCR reaction solution: four different systems of PCR reaction solution were prepared according to the following table,
example 2
In this example, 10 samples of freshly cryopreserved tumor tissues of gastric cancer patients were collected, RNA was extracted therefrom, and after reverse transcription into cDNA, the RCN3-SSU72 fusion mutation detection kit obtained in example 1 was used to detect the RCN3-SSU72 fusion mutation status of the sample to be tested.
1. RNA extraction from fresh frozen tumor tissue samples
Its RNA was extracted using Qiagen's fresh tissue RNA extraction kit. The specific operation steps are operated according to the kit instruction.
2. Reverse transcription of RNA into cDNA
The concentration of RNA was measured by using Qubit, the quality of RNA was measured by using Nanodrop, and cDNA was synthesized using Clontech kit using 800-1000ng of RNA as a template for cDNA synthesis as follows:
the reverse transcription reaction procedure was: incubating in a hot lid thermal cycler at 72 ℃ for 3 minutes, slowly cooling to 42 ℃ at 0.1 ℃/second, incubating for 90 minutes at 42 ℃, heating at 70 ℃ for 10 minutes to terminate the reaction, and adding 90 μ L of Elution Buffer (EB) to dilute the first strand reaction product to obtain a single-stranded cDNA solution.
3. Fluorescent quantitative PCR detection
Preparing a fluorescent quantitative PCR reaction system according to the operation of the RCN3-SSU72 fusion mutation detection kit, putting the fluorescent quantitative PCR reaction system into a fluorescent quantitative PCR instrument, setting a PCR reaction program according to the following steps, and then carrying out amplification reaction:
treating at 37 deg.C for 10min, pre-denaturing at 95 deg.C for 5min,
40 cycles: 95 ℃ for 15 seconds, 55 ℃ for 35 seconds and collecting the fluorescence signal.
4. Analysis of detection results
Detecting Ct value of FAM fluorescence signal, and judging result according to Ct value displayed by fluorescence PCR amplification instrument: detecting the fluorescence intensity of FAM of a reaction system, taking the cycle number Ct value required when the FAM reaches a set threshold value as a negative and positive judgment standard, wherein the Ct value is more than or equal to 38: negative; ct value < 38: and (4) positive. The detection result shows that 1 of 10 detected gastric cancer tumor samples has RCN3-SSU72 fusion mutation, and the 10 gastric cancer tumor samples are subjected to second-generation whole transcript sequencing, the coincidence rate of the two detection methods is 100%, and the detection accuracy of the system is further proved.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the invention, which is intended to be covered by the appended claims.
The invention adopts specific primers to identify the human RCN3-SSU72 gene fusion mutation type, and collects fluorescent signals based on a Taqman probe method, so that the specificity is well guaranteed. The kit of the invention has the following advantages: (1) a real-time fluorescent PCR system is established, and RCN3-SSU72 fusion mutation can be detected simultaneously; (2) the operation is simple, the detection speed is high, and the whole reverse transcription PCR and fluorescence PCR detection can be finished within half a day; (3) the accuracy is high, and the UNG enzyme-dUTP anti-pollution system and the blank control system are introduced into the system, so that false positive results can be better prevented, and the accuracy and reliability of detection results are ensured.
Claims (7)
1. Human RCN3-SSU72 gene fusion mutation detection primer combination and probe are characterized in that: the primer combination comprises sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2, which are respectively as follows: RCN3-SSU72 Left Primer: 5'-TCTGTTGCTACTGAGGCACG-3' (SEQ ID NO: 1); RCN3-SSU72 Right Primer: 5'-GCTCGTCGATCTCGTTCTCC-3' (SEQ ID NO: 2), and the sequence of the specific probe matched with the primer combination is shown in SEQ ID NO:3, and the specific probe is as follows: RCN3-SSU 72-P: 5'-CCCGTCTGGGCTATACACAG-3' (SEQ ID NO: 3), which is modified with FAM or VIC at the 5 'end and NFQ-MGB at the 3' end.
2. The kit for detecting human RCN3-SSU72 gene fusion mutation according to claim 1, wherein: the kit contains the primer combination of claim 1 and a specific probe.
3. The kit for detecting human RCN3-SSU72 gene fusion mutation according to claim 2, wherein: the kit also comprises a quality control system, wherein the quality control system comprises a quality control primer and a quality control probe, and the sequences of the quality control primer are shown as SEQ ID NO. 4 and SEQ ID NO. 5, and are respectively as follows: ATP6IP1 Left Primer: 5'-AGTGGAGGCTGAGGCTATGA-3' (SEQ ID NO: 4); ATP6IP1 Right Primer: 5'-GAGGCTGGCATTGAGCTTCA-3' (SEQ ID NO: 5), and the sequence of the quality control probe is shown in SEQ ID NO:6 as follows: ATP6IP 1-P: 5'-GGCTGCCGGTGTTTTTGTC-3' (SEQ ID NO: 6), the 5 'end of the quality control probe of the kit is modified with FAM or VIC, and the 3' end is modified with NFQ-MGB.
4. The kit for detecting human RCN3-SSU72 gene fusion mutation according to claim 3, wherein: the kit also contains a positive control solution and a blank control solution, wherein the positive control solution is plasmid DNA containing RCN3-SSU72, and the blank control is Tris buffer solution.
5. The kit for detecting human RCN3-SSU72 gene fusion mutation according to claim 2, 3 or 4, wherein: the kit comprises PCR buffer solution, HotStart Taq enzyme and UNG enzyme.
6. The use method of the kit for detecting human RCN3-SSU72 gene fusion mutation according to claim 2 or 5, wherein the kit comprises: the method comprises the following steps:
step 1: extracting RNA from a test sample from a fresh sample, such as fresh blood, tumor fresh sample after surgical resection/puncture/ultra-low temperature frozen sample/sample preserved in RNA stabilizing solution;
step 2: carrying out reverse transcription on the extracted RNA to obtain cDNA, and carrying out real-time fluorescence PCR amplification reaction by using the cDNA as a template;
and step 3: judging the detection result according to the Ct value displayed by the fluorescent PCR amplification instrument: monitoring the fluorescence intensity of FAM and VIC of a reaction system, taking the cycle number Ct value required when the FAM reaches a set threshold as a negative and positive judgment standard, wherein the Ct value is more than or equal to 38: negative; ct value less than 38: and (4) positive.
7. The use method of the kit for detecting human RCN3-SSU72 gene fusion mutation according to claim 6, wherein the kit comprises: the kit comprises a reaction system of PCR amplification reaction, and comprises the following components: PCR buffer solution: 15 muL and a primer: 1 mu M and a probe: 0.5 μ M, Taq enzyme: 1.0U, UNG enzyme: 0.5U, reverse transcription product cDNA: 2 muL and water: balance, total amount: 25 muL.
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