Detection target for detecting gene expression abundance in RS genome and application

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a detection target spot for detecting gene expression abundance in an RS genome and application. According to the present invention, the human RS gene set is analyzed and tested to provide the detection region for detecting the gene expression abundance in the RS genome, and the experimental result proves that the fluorescent probe and the standard substance are designed for the detection region so as to accurately quantify the RS gene expression level. By measuring the expression level of the RS gene, the method can be used for predicting the drug sensitivity of clinical breast cancer, ovarian cancer and other malignant tumor patients to PARP inhibitor drugs and cis-platinum.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to detection targets and applications for detecting gene expression abundance in RS genomes. Background Art

[0002] The inventor team's prior patent CN202110204232.9 discloses an application of a biomarker for predicting the sensitivity of tumor patients to specific anti-tumor drugs. The biomarker is a gene set (RS gene set) consisting of 8 genes, MYBBP1A, NUP88, GEMIN4, PELP1, DHX33, C1QBP, WRAP53 and TSR1, which is used to predict the sensitivity of patients with normal HR function (HRP) to PARPi / cisplatin. It is proposed that the combined examination of this gene set and HRD status can more accurately predict the clinical response of PARPi / cisplatin. We found that PARPi / cisplatin selectively kills cells with high expression of this gene set through the pro-apoptotic ribosome stress mechanism induced by DNA damage signal (ATM), which together with the ATM-induced HR repair-promoting survival pathway balances the fate of cells under DNA damage. This model may represent an evolutionary strategy: when the accumulated DNA damage exceeds the repair capacity of the cell, the ribosome stress apoptosis induced by DNA damage signal can effectively prevent the occurrence of mutated repair. We validated this gene set as a marker for predicting PARPi / cisplatin sensitivity in HRP patients using published clinical datasets and their newly established ovarian cancer organoid library. We also collected clinical samples, information, and follow-up records from 50 patients with platinum-sensitive recurrent ovarian cancer who received PARPi monotherapy, and collected transcriptome (RNAseq) and whole genome sequencing (WGS) data. This may be the only PARPi clinical cohort to date that contains both RNAseq and WGS sequencing data. Validation results showed that this gene set predicted the response to PARPi in patients without obvious HRD with 100% accuracy, and its combined detection with HRD status can effectively screen out PARPi-sensitive patients in the cohort. Based on this, we proposed a new patient screening strategy: first, screen patients with high expression of this gene set for PARPi / cisplatin treatment; the remaining patients are further screened for HRD. Prioritizing the screening of gene sets can save a large number of patients from expensive and time-consuming HRD testing.

[0003] Based on the above-mentioned previous research results, there is an urgent need to develop detection products for detecting RS gene set expression that are suitable for wide promotion in the market, to assist clinicians in screening patients who are sensitive to PARP inhibitors and cisplatin, and to achieve personalized treatment for cancer patients.

[0004] The PCR-fluorescent probe method is a molecular biology method based on polymerase chain reaction (PCR) technology and combined with fluorescent probes for detection. Its principle is to add a specific fluorescent probe to the PCR reaction system, which can specifically bind to the target nucleic acid sequence. During the PCR amplification process, with the action of DNA polymerase, the probe is hydrolyzed and releases a fluorescent signal. The intensity of the fluorescent signal is proportional to the number of PCR products. By detecting the changes in the fluorescent signal, the progress of the PCR reaction can be monitored in real time, and the presence and quantity of the target nucleic acid can be determined. The key to the research and development of detection products based on the PCR-fluorescent probe method is to provide amplification primers and probes with the best detection effect. Among them, the primer is two short single-stranded DNA fragments that are complementary to the target nucleic acid sequence, so that the target sequence can be amplified. The probe is an oligonucleotide fragment with a fluorescent label. In the PCR-fluorescent probe method, it specifically binds to the target sequence. As the PCR reaction proceeds, when the probe is hydrolyzed by DNA polymerase, a fluorescent signal is released, thereby realizing real-time detection and quantitative analysis of the target nucleic acid. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a detection target and application for detecting gene expression abundance in RS genome.

[0006] In a first aspect of the present invention, a detection target for detecting gene expression abundance in a RS genome is provided, wherein the RS genome consists of MYBBP1A, NUP88, GEMIN4, PELP1, DHX33, C1QBP, WRAP53 and TSR1.

[0007] The detection target is a combination of nucleotide sequences, including: nucleotide sequence one, located on gene MYBBP1A, and its sequence is shown in SEQ ID NO.1; nucleotide sequence two, located on gene NUP88, and its sequence is shown in SEQ ID NO.4; nucleotide sequence three, located on gene Gemin4, and its sequence is shown in SEQ ID NO.7; nucleotide sequence four, located on gene PELP1, and its sequence is shown in SEQ ID NO.10; nucleotide sequence five, located on gene DHX33, and its sequence is shown in SEQ ID NO.13; nucleotide sequence six, located on gene C1QBP, and its sequence is shown in SEQ ID NO.16; nucleotide sequence seven, located on gene WRAP53, and its sequence is shown in SEQ ID NO.19; nucleotide sequence eight, located on gene TSR1, and its sequence is shown in SEQID NO.22.

[0008] The second aspect of the present invention provides the use of the detection target as described above for detecting gene expression abundance in the RS genome for preparing a detection product.

[0009] In the third aspect of the present invention, a primer probe combination for detecting the abundance of gene expression in the RS genome is provided, and the primer probe combination is obtained based on the detection target for detecting the abundance of gene expression in the RS genome as described above, including: primer probe group one, designed for nucleotide sequence one; primer probe group two, designed for nucleotide sequence two; primer probe group three, designed for nucleotide sequence three; nucleotide sequence four, designed for nucleotide sequence four; nucleotide sequence five, designed for nucleotide sequence five; nucleotide sequence six, designed for nucleotide sequence six; nucleotide sequence seven, designed for nucleotide sequence seven; nucleotide sequence eight, designed for nucleotide sequence eight.

[0010] Further, the primer probe group 1 includes a forward primer as shown in SEQ ID NO.25, a probe as shown in SEQ ID NO.26, and a reverse primer as shown in SEQ ID NO.27; the primer probe group 2 includes a forward primer as shown in SEQ ID NO.34, a probe as shown in SEQ ID NO.35, and a reverse primer as shown in SEQ ID NO.36; the primer probe group 3 includes a forward primer as shown in SEQ ID NO.43, a probe as shown in SEQ ID NO.44, and a reverse primer as shown in SEQ ID NO.45; the primer probe group 4 includes a forward primer as shown in SEQ ID NO.52, a probe as shown in SEQ ID NO.53, and a reverse primer as shown in SEQ ID NO.54; the primer probe group 5 includes a forward primer as shown in SEQ ID NO.61, a probe as shown in SEQ ID NO.62, and a reverse primer as shown in SEQ ID NO.63; the primer probe group 6 includes a forward primer as shown in SEQ ID NO.70, a probe as shown in SEQ ID NO.71, and a reverse primer as shown in SEQ ID NO.72. The probe shown in SEQ ID NO.71 and the reverse primer shown in SEQ ID NO.72; the primer probe group seven includes the forward primer shown in SEQ ID NO.79, the probe shown in SEQ ID NO.80, and the reverse primer shown in SEQ ID NO.81; the primer probe group eight includes the forward primer shown in SEQ ID NO.88, the probe shown in SEQ ID NO.89, and the reverse primer shown in SEQ ID NO.90.

[0011] In a fourth aspect of the present invention, a standard for detecting the abundance of gene expression in the RS genome is provided, wherein the standard is a plasmid, and the detection interval sequence in the standard is shown in SEQ ID NO.100.

[0012] According to a fifth aspect of the present invention, a kit is provided for detecting the abundance of gene expression in the RS genome, comprising the primer-probe combination as described above.

[0013] Furthermore, the kit includes the standard substance as described above.

[0014] In a sixth aspect of the present invention, a method for detecting gene expression abundance in the RS genome based on the kit as described above is provided, wherein the method is based on multiplex fluorescence detection technology for detection.

[0015] The present invention provides detection regions for detecting gene expression abundance in the RS genome by analyzing and testing the human RS gene set. Experimental results show that the expression level of the RS gene can be accurately quantified by designing fluorescent probes and standards for these detection regions. Determining the expression level of the RS gene can be used to predict the sensitivity of clinical breast cancer, ovarian cancer and other malignant tumor patients to PARP inhibitor drugs and cisplatin.

[0016] The present invention uses multiple fluorescence detection technology to simultaneously detect three fluorescence signals in one detection well, so that eight RS gene set genes and one internal reference gene can be detected by three real-time fluorescence quantitative PCRs.

[0017] The present invention clones the detection fragment of 8+1 genes to be tested into a plasmid, i.e., a standard plasmid, and the absolute value of the concentration of the standard plasmid can be determined by spectrophotometric quantification. The standard plasmid is gradiently diluted and then tested with the kit provided by the present invention to obtain a standard curve. The test results of the test sample are substituted into the standard curve to obtain the absolute quantitative data of the expression level of the RS gene set in the test sample.

[0018] The present invention conducts quantitative detection on the human RS gene set, and the detection results can be used to assist clinicians in screening patients who are suitable for PARP inhibitors and cisplatin sensitivity, so as to achieve individualized treatment for tumor patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0020] Figure 1 To detect the effect of real-time fluorescence quantitative PCR for the detection interval and the corresponding fluorescent probe;

[0021] Figure 2The real-time fluorescence quantitative PCR detection effect of primers with different concentrations;

[0022] Figure 3 The real-time fluorescence quantitative PCR detection effect of probes with different concentrations;

[0023] Figure 4 Implement quantitative PCR results for standard detection of fluorescent probes;

[0024] Figure 5 The results of real-time quantitative PCR with multiple fluorescent probes were tested for standards;

[0025] Figure 6 The result is the detection limit. DETAILED DESCRIPTION

[0026] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0027] The present invention provides a PCR-fluorescent probe method detection reagent for detecting RS gene sets based on the PCR-fluorescent probe method.

[0028] The following is the specific process of testing and detecting in the specific embodiment of the present invention:

[0029] 1. Reagent preparation: (reagent preparation area)

[0030] 1) Take out the kit from the refrigerator, equilibrate to room temperature, fully dissolve all components, and centrifuge quickly for 10 seconds.

[0031] 2) Calculate the number of reactions required for this experiment (n):

[0032] n = number of samples + blank control (1T) + positive control (1T)

[0033] 3) Dispense the PCR reaction solution from tubes 1 to 9 into PCR reaction tubes at a volume of 23 μl / well.

[0034] 4) Transfer the PCR reaction tube to the sample preparation area and store the remaining reagents back in a -20℃±5℃ refrigerator away from light.

[0035] 2. Sample preparation: (Sample preparation area)

[0036] 1) Sample RNA extraction: refer to the instructions of the purchased commercial RNA extraction kit for operation.

[0037] Reverse transcription reaction: Prepare the reverse transcription reaction system according to the table below. The amount of RNA used in 20 μl reaction system is 0.2-10 μg.

[0038] Table 1: Reverse transcription system

[0039] Reagents Usage EA Reverse Transcription Mix 4μL RNA 0.2-10 μg RNase Free dH2O Total system 20μL

[0040] 2) Reverse transcription reaction conditions: 37°C, 15 minutes; 85°C, 5 seconds.

[0041] 3) Add sample:

[0042] a) Add 2 μl of enzyme mixture to 18 μl of cDNA obtained by reverse transcription, add 2 μl of enzyme mixture to 18 μl of positive control, and add 2 μl of enzyme mixture to 18 μl of blank control, mix well, and centrifuge quickly for 10 seconds.

[0043] b) Take 2 μl of the mixed cDNA and add them to the reaction tubes containing the three PCR reaction solutions. That is, each cDNA sample to be tested is tested with the three PCR reaction solutions. The positive control and blank control are tested in the same way.

[0044] c) Take the standard curve buffer and divide it into 5 tubes with 27μl per tube. Take 3μl of the standard curve solution and add it to one tube of standard curve buffer to mix well. Then take 3μl and add it to the next tube of standard curve buffer until all the standard curve buffers are mixed. The standard curve solution is the standard curve reagent. Arrange the prepared standard curve reagents in the order of adding the standard curve solutions, and take 25μl for loading on the machine.

[0045] d) If the PCR reaction tube cannot be tested immediately after adding the template due to temporary circumstances, it is recommended to temporarily store the PCR reaction tube with the template at 2-8°C and test it on the machine as soon as possible within 24 hours.

[0046] 3. PCR amplification: (nucleic acid amplification region)

[0047] 1) Turn on the instrument and perform a self-test of its performance.

[0048] 2) Take the PCR reaction tubes prepared in the sample preparation area and place them in the corresponding positions of the instrument sample slots. And record the order of placement.

[0049] 3) Set the instrument amplification parameters according to Table 2 and start PCR amplification. After the reaction is completed, according to the amplification curve, the appropriate fluorescence threshold is defined (generally, the threshold is defined in the middle of the exponential growth period in the logarithmic form of the amplification curve)

[0050] Table 2: Instrument amplification related parameters

[0051]

[0052] Embodiment 1:

[0053] Based on the sequences of MYBBP1A, NUP88, GEMIN4, PELP1, DHX33, C1QBP, WRAP53 and TSR1, each sequence has multiple possible effective detection regions. The detection regions of each gene are screened and designed. The detection interval needs to select the region where the coding region of the target gene spans introns and the CG content is relatively balanced and uniform, and the design of subsequent primers and probes needs to be considered. The secondary structure formed between the primers and probes is analyzed, and the schemes with too many secondary structures and too stable are eliminated. Finally, 3 detection intervals are screened for each gene, as shown in Table 3.

[0054] Table 3 Gene detection interval sequence

[0055]

[0056]

[0057] For each detection interval, a corresponding detection reagent set is designed. Each detection reagent set includes three reagents: Forward (forward primer), Probe (probe), and Revers (reverse primer), as shown in Table 4.

[0058] Table 4 Detection reagent sets designed for 8 genes and 3 detection regions

[0059]

[0060]

[0061]

[0062] The efficiency and stability of the fluorescent probe method for detecting RS gene expression in the detection interval were tested in ovarian cancer cells A2780 and OV90. The results showed that the expression of RS gene can be measured relatively accurately and stably for the detection interval designed by us. It can be used to establish a real-time fluorescence quantitative PCR system for quantitative RS gene expression level ( Figure 1 We also comprehensively evaluated the PCR efficiency, detection stability, gene location of the detection interval and other conditions, and screened out the best detection interval and corresponding primers and fluorescent probes as well as primers and fluorescent probes for GAPDH (internal reference gene) (Table 5) for further subsequent detection.

[0063] Table 5 Optimal detection intervals, primers and probes for subsequent experiments after evaluation and screening

[0064]

[0065]

[0066] Furthermore, the effect of different primer concentrations on the detection effect was tested. The results showed that the primer concentration did not change the PCR peak cycle number (CT value) of the target gene, but changed the concentration of the final PCR amplification product, that is, the fluorescence value of the plateau phase ( Figure 2 ). Based on this, we determined the primer concentrations (Table 6) to make the final PCR amplification product concentrations of each gene as consistent as possible.

[0067] Table 6 Primer and probe concentrations

[0068]

[0069] Furthermore, the effects of different probe concentrations on the detection effect were tested. The results showed that increasing the probe concentration can increase the CT value of the detection to a certain extent, thereby improving the sensitivity of the detection ( Figure 3 ). To balance detection sensitivity and cost, we determined the concentration of the probe to be used (Table 6).

[0070] Furthermore, in order to absolutely quantify the test results, we cloned the detection interval sequence and recombined it into a plasmid to make a standard. The detection interval sequence in the standard is shown in SEQ ID NO.100. Figure 4 ) and multiplex fluorescent probe real-time quantitative PCR ( Figure 5 ) The quality of the standard was tested and the results showed that the standard had good linearity and could be used for absolute quantification.

[0071] Furthermore, the detection limit of each gene in the RS gene set was tested under the entity. We set the standard balance ratio to 8.81*10 4 The sample with 10 copies / μL was diluted 10 times until 8.81*10 0 The results showed that this detection system can achieve accurate quantification of 8.81*10 for all genes in the RS gene set. 1 The sample with a concentration of 88 copies / μL, that is, the gene content in each microliter of sample is greater than or equal to 88 molecules, can be absolutely quantified ( Figure 6 ).

[0072] Based on the above experimental results, it is proved that the design of fluorescent probes and standards for these detection areas can accurately quantify the expression level of RS gene. Determining the expression level of RS gene can be used to predict the sensitivity of clinical breast cancer, ovarian cancer and other malignant tumor patients to PARP inhibitor drugs and cisplatin.

[0073] The kit provided by the present invention is suitable for Stratagene MX3000P, AB 7500, and LightCycler 480 real-time fluorescence quantitative PCR instruments. And the instrument is required to use the FAM channel to collect fluorescence signals. The sample requirements of the kit provided by the present invention are as follows: 1. The quality of the RNA used for detection is very important. This kit is suitable for RNA samples extracted from paraffin-embedded pathological tissues, fresh patient tissues and CTCs. The samples should be confirmed by doctors to contain tumor tissues to obtain correct conclusions. It is recommended to select samples that are no more than 3 years old for paraffin-embedded tissues. 2. It is recommended to use QIAGEN RNeasy FFPE Kit (Cat No.73504) to extract sample RNA. The extracted RNA needs to be measured for concentration and purity using an ultraviolet spectrophotometer. The A260 / A280 of the RNA is required to be between 1.8 and 2.0. The extracted RNA should be tested immediately, otherwise it should be stored below -70°C for no more than 6 months. Avoid repeated freezing and thawing during sample storage.

[0074] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A detection target for detecting gene expression abundance in a RS genome, wherein the RS genome consists of MYBBP1A, NUP88, GEMIN4, PELP1, DHX33, C1QBP, WRAP53 and TSR1, characterized in that: The detection target is a nucleotide sequence combination, including: Nucleotide sequence 1, located on the gene MYBBP1A, and its sequence is shown in SEQ ID NO.1; Nucleotide sequence 2, located on gene NUP88, and its sequence is shown in SEQ ID NO.4; Nucleotide sequence three, located on gene Gemin4, and its sequence is shown in SEQ ID NO.7; Nucleotide sequence 4, located on gene PELP1, and its sequence is shown in SEQ ID NO.10; Nucleotide sequence five, located on gene DHX33, and its sequence is shown in SEQ ID NO.13; Nucleotide sequence six, located on gene C1QBP, and its sequence is shown in SEQ ID NO.16; Nucleotide sequence seven, located on gene WRAP53, the sequence of which is shown in SEQ ID NO.19; Nucleotide sequence eight is located on gene TSR1, and its sequence is shown in SEQ ID NO.

22.

2. Use of the detection target for detecting gene expression abundance in RS genome as claimed in claim 1 to prepare a detection product.

3. A primer-probe combination for detecting gene expression abundance in RS genome, characterized in that: The primer-probe combination is obtained based on the detection target for detecting gene expression abundance in the RS genome as claimed in claim 1, and comprises: Primer probe set 1, designed for nucleotide sequence 1; Primer probe set 2, designed for nucleotide sequence 2; Primer probe set three, designed for nucleotide sequence three; Nucleotide sequence four, designed based on nucleotide sequence four; Nucleotide sequence five, designed based on nucleotide sequence five; Nucleotide sequence six, designed based on nucleotide sequence six; Nucleotide sequence seven, designed based on nucleotide sequence seven; Nucleotide sequence eight, designed based on nucleotide sequence eight.

4. The primer-probe combination for detecting gene expression abundance in RS genome according to claim 3, characterized in that: The primer probe set 1 includes a forward primer as shown in SEQ ID NO.25, a probe as shown in SEQ ID NO.26, and a reverse primer as shown in SEQ ID NO.27; The primer probe set 2 includes a forward primer as shown in SEQ ID NO.34, a probe as shown in SEQ ID NO.35, and a reverse primer as shown in SEQ ID NO.36; The primer probe set three includes a forward primer as shown in SEQ ID NO.43, a probe as shown in SEQ ID NO.44, and a reverse primer as shown in SEQ ID NO.45; The primer probe set 4 includes a forward primer as shown in SEQ ID NO.52, a probe as shown in SEQ ID NO.53, and a reverse primer as shown in SEQ ID NO.54; The primer probe set 5 includes a forward primer as shown in SEQ ID NO.61, a probe as shown in SEQ ID NO.62, and a reverse primer as shown in SEQ ID NO.63; The primer probe set six includes a forward primer as shown in SEQ ID NO.70, a probe as shown in SEQ ID NO.71, and a reverse primer as shown in SEQ ID NO.72; The primer probe set 7 includes a forward primer as shown in SEQ ID NO.79, a probe as shown in SEQ ID NO.80, and a reverse primer as shown in SEQ ID NO.81; The primer probe set eight includes a forward primer as shown in SEQ ID NO.88, a probe as shown in SEQ ID NO.89, and a reverse primer as shown in SEQ ID NO.

90.

5. A standard for detecting gene expression abundance in RS genome, characterized in that: The standard is a plasmid, and the detection interval sequence in the standard is shown as SEQ ID NO.

100.

6. A kit for detecting gene expression abundance in RS genome, characterized in that: It comprises the primer-probe combination as claimed in claim 3 or 4.

7. The kit for detecting gene expression abundance in RS genome according to claim 6, characterized in that: It includes the standard product as claimed in claim 5.

8. A method for detecting gene expression abundance in RS genome based on the kit as claimed in claim 6, wherein the method is based on multiple fluorescence detection technology for detection.

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