CN114592059A - Kit for evaluating 5-FU treatment sensitivity/drug resistance - Google Patents

Kit for evaluating 5-FU treatment sensitivity/drug resistance Download PDF

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CN114592059A
CN114592059A CN202210028785.8A CN202210028785A CN114592059A CN 114592059 A CN114592059 A CN 114592059A CN 202210028785 A CN202210028785 A CN 202210028785A CN 114592059 A CN114592059 A CN 114592059A
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赵丽瑛
马强
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Southern Hospital Southern Medical University
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Abstract

The invention discloses a kit for evaluating 5-FU treatment sensitivity/drug resistance, which is feasible for detecting Fatty Acyl-CoA Reductase 1 gene in a colorectal cancer sample. The invention can help a clinician to determine whether a colorectal cancer patient is suitable for the treatment of fluorouracil medicaments according to the expression condition of the Fatty Acyl-CoA Reductase 1 gene in the tumor tissue of the colorectal cancer patient, thereby formulating a personalized treatment scheme. The kit is simple and convenient to operate, short in detection time, capable of realizing high-throughput detection, low in price, mainly applied to detection of the Fatty Acyl-CoA Reductase 1 gene, and capable of guiding a clinician to use 5-FU for colorectal cancer patients to take medicines.

Description

Kit for evaluating 5-FU treatment sensitivity/drug resistance
Technical Field
The invention relates to the technical field of biological medicines, in particular to a kit for evaluating 5-FU treatment sensitivity/drug resistance.
Background
Colorectal cancer (CRC) is one of the third most common digestive tract malignancies with a global incidence rate, and both the incidence rate and the fatality rate keep on rising trend year by year. In recent years, more and more medicines with different action targets bring different treatment options to colorectal cancer patients and more survival benefits to the patients.
5-fluorouracil (5-FU) is the most widely used drug in colorectal cancer chemotherapy, however, the effect of chemotherapy is poor and satisfactory due to primary or secondary drug resistance of tumor cells, the overall effectiveness of 5-FU drugs is low, and the sensitivity/drug resistance of tumors to 5-FU drugs becomes one of the main factors restricting the chemotherapy and prognosis of patients. Therefore, the 5-FU drug sensitive and drug resistant related gene is urgently needed to be searched, so that a clinician can conveniently formulate a reasonable colorectal cancer personalized treatment scheme, and the clinical significance is positive.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a kit for evaluating 5-FU treatment sensitivity/drug resistance, and the invention discovers that the expression level of the Fatty Acyl-CoA Reductase 1(FAR1) gene has high correlation with the 5-FU treatment sensitivity/drug resistance, the expression level of the Fatty Acyl-CoA Reductase 1 gene is high, the colorectal cancer patient has low sensitivity and high drug resistance to 5-FU drugs, and the expression level of the Fatty Acyl-CoA Reductase 1 gene is low, the 5-FU drug sensitivity and the drug resistance are low. Therefore, the Fatty Acyl-CoA Reductase 1 gene is expected to become a new target for colorectal cancer diagnosis and medication, and the development of a kit for detecting the expression level of the Fatty Acyl-CoA Reductase 1 gene has important clinical significance.
The invention aims to provide application of a product for detecting the expression quantity of a Fatty Acyl-CoA Reductase 1 gene in preparing a kit for evaluating the treatment sensitivity/drug resistance of 5-FU.
In order to achieve the purpose, the invention is realized by the following scheme:
the application of a product for detecting the expression quantity of the Fatty Acyl-CoA Reductase 1 gene in preparing a kit for evaluating the treatment sensitivity/drug resistance of 5-FU.
Preferably, the expression level of the Fatty Acyl-CoA Reductase 1 gene is the expression level of mRNA of the Fatty Acyl-CoA Reductase 1 gene.
Preferably, the kit further comprises a reagent for detecting the expression amount of mRNA of the reference gene GAPDH.
Preferably, assessing 5-FU therapy susceptibility/resistance is assessed for colorectal cancer 5-FU therapy susceptibility/resistance.
Preferably, the product for detecting the expression level of the Fatty Acyl-CoA Reductase 1 gene is a detection kit for the expression level of mRNA of the Fatty Acyl-CoA Reductase 1 gene.
More preferably, the kit for detecting the expression level of mRNA of the Fatty Acyl-CoA Reductase 1 gene contains a primer with a nucleotide sequence shown as SEQ ID NO. 1-2 and a probe with a nucleotide sequence shown as SEQ ID NO. 3.
More preferably, the kit for detecting the expression level of mRNA of the Fatty Acyl-CoA Reductase 1 gene further comprises a primer with a nucleotide sequence shown as SEQ ID NO. 4-5 and a probe with a nucleotide sequence shown as SEQ ID NO. 6.
More preferably, the 5' end of the probe is labeled with any one of FAM, VIC, HEX, or CY5 fluorophore;
more preferably, the 3' of the probe is labeled with any one of quencher groups BHQ1 and BHQ 2.
More preferably, the kit for detecting the expression level of mRNA of the Fatty Acyl-CoA Reductase 1 gene further comprises a negative quality control substance and/or a positive quality control substance.
More preferably, the kit for detecting the expression level of mRNA of the Fatty Acyl-CoA Reductase 1 gene further comprises a FAR1 quantitative reference and/or an internal reference gene quantitative reference.
The invention further provides a kit for evaluating the treatment sensitivity/drug resistance of 5-FU, which is used for detecting the expression quantity of mRNA of Fatty Acyl-CoA Reductase 1 gene and reference gene GAPDH by a relative quantification method based on a standard curve method.
Specifically, it contains the following primers and probes:
the upstream primer of the Fatty Acyl-CoA Reductase 1 gene: 5'-TGTGCAGCTACAGTAAGGTTT-3' (SEQ ID NO.1),
downstream primer of Fatty Acyl-CoA Reductase 1 gene: 5'-GATTCTTCATTTGTTGTGCAAGG-3' (SEQ ID NO.2),
fatty Acyl-CoA Reductase 1 gene probe: 5 '-FAM-ATGTGATTGCAACGCGACAGCTT-BHQ 1-3' (SEQ ID NO.3),
internal reference gene GAPDH upstream primer: 5'-AAAACCTGCCAAATATGATGACA-3' (SEQ ID NO.4),
internal reference gene GAPDH downstream primer: 5'-TGAAGTCAGAGGAGACCACC-3' (SEQ ID NO.5),
internal reference gene GAPDH probe: 5 '-VIC-CAGTGTAGCCCAGGATGCCCTT-BHQ 1-3' (SEQ ID NO. 6);
also contains PCR buffer, reverse transcriptase, RNase inhibitor, hot start Taq enzyme, dNTPs, FAR1 quantitative reference (concentration is 1 × 10)6copies/mL、1×105copies/mL、1×104copies/mL、1×103Standards of copies/mL), reference gene quantitative reference (concentration 1X 10)6copies/mL、1×105copies/mL、1×104copies/mL、1×103A copies/mL standard), a negative quality control product and a positive quality control product.
FAR1 quantitative reference: the standard substance is diluted by a defined amount of Fatty Acyl-CoA Reductase 1 plasmid, and the concentration is 1X 106copies/mL、1×105copies/mL、1×104copies/mL、1×103The gene sequence of the Fatty Acyl-CoA Reductase 1 is shown in SEQ ID NO. 8;
quantitative reference products of internal reference genes: is a standard substance diluted with a predetermined amount of reference plasmid, and has a concentration of 1 × 106copies/mL、1×105copies/mL、1×104copies/mL、1×103The copies/mL, the reference gene quantitative reference product is that artificially constructed plasmid containing a reference target gene sequence is diluted to corresponding concentration by 1 × TE, and the reference gene target gene sequence is shown as SEQ ID NO. 7;
the negative quality control product is RNA of a clinical sample, and the quantitative value of the Fatty Acyl-CoA Reductase 1 gene/the quantitative value of the internal reference gene is more than or equal to 3;
the positive quality control substance is RNA of a clinical sample, and the quantitative value of the Fatty Acyl-CoA Reductase 1 gene/the quantitative value of the internal reference gene is less than 3.
The using method comprises the following steps: (1) preparing a reagent: 18 mul of PCR reaction solution and 2 mul of enzyme mixed solution are fully and evenly mixed for standby,
wherein the PCR reaction solution consists of primers (SEQ ID NO. 1-2 and SEQ ID NO. 4-5), probes (SEQ ID NO.3 and SEQ ID NO.6) and PCR buffer in Table 1, wherein the final concentration of an amplification system of the primers is 0.2 mu mol/L, the final concentration of an amplification system of the probes is 0.1 mu mol/L, and the final concentration of the PCR buffer is 1X;
the enzyme mixed solution consists of reverse transcriptase, an RNase inhibitor, hot start Taq enzyme and dNTPs, wherein the reaction final concentration of the reverse transcriptase is 2.0U, the reaction final concentration of the RNase inhibitor is 0.5U, the reaction final concentration of the hot start Taq enzyme is 2.0U, and the reaction final concentration of the dNTPs is 0.4 mM.
(2) Sample adding: adding 5 μ l of negative quality control product, positive quality control product, FAR1 quantitative reference product, internal reference gene quantitative reference product or sample to be tested (colorectal cancer tissue RNA) into PCR reaction tube, covering tube cover, placing into sample groove of instrument,
wherein, FAR1 quantitative reference: the standard was diluted with a defined amount of Fatty Acyl-CoA Reductase 1 plasmid at a concentration of 1X 106copies/mL、1×105copies/mL、1×104copies/mL、1×103copies/mL。
Quantitative reference products of internal reference genes: is a standard substance diluted with a predetermined amount of reference plasmid at a concentration of 1 × 106copies/mL、1×105copies/mL、1×104copies/mL、1×103copies/mL。
(3) And (3) PCR detection: the PCR reaction tube was placed in a fluorescent quantitative PCR instrument (ABI7500), and the reaction program was set:
opening a 'Setup' window, setting negative quality control and positive quality control according to the corresponding sequence of the samples, setting unknown samples as 'unknown', setting FAR1 and quantitative reference products of reference genes as 'Standard' and 'Quantity' columns to fill in corresponding concentrations, and setting Sample names in the column of 'Sample Name'; the probe detection mode is set as follows: reporter Dye 1: FAM, Quencher Dye 1: NONE, Reporter Dye 2: VIC, Quencher Dye 2: none, Passive Reference: NONE. Opening an instrument window, and setting the circulation conditions as follows:
Figure BDA0003465575270000041
note: indicates collected fluorescence signal
3. And (4) analyzing results:
and storing the detection data file after the reaction is finished. An Amplification Plot window is opened under Results. The location of the sample of interest to be analyzed is selected. Changing the Baseline value to start: 3, stop: 10, and opens the manual setting Threshold: 2-6e + 3. And (3) double clicking numerical values on Rn coordinates to open a Graph Settings window, changing Log in Post Run Settings into Linear, opening an Analysis preferences window after OK, selecting an Analysis automatic Analysis result under an Analysis menu, viewing the result on a Report interface, and recording an unknown sample quantitative value.
4. Interpretation of results
Negative result interpretation: the quantitative value of the Fatty Acyl-CoA Reductase 1 gene/quantitative value of the reference gene of the same sample is greater than or equal to the drug resistance of 3, 5-FU chemotherapy.
Positive result interpretation: the quantitative value of the Fatty Acyl-CoA Reductase 1 gene/quantitative value of the internal reference gene of the same sample is less than 3, 5-FU chemotherapy sensitivity.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a kit for evaluating 5-FU treatment sensitivity/drug resistance, which is feasible for detecting Fatty Acyl-CoA Reductase 1 gene in a colorectal cancer sample. The invention can help a clinician to determine whether a colorectal cancer patient is suitable for the treatment of fluorouracil medicaments according to the expression condition of the Fatty Acyl-CoA Reductase 1 gene in the tumor tissue of the colorectal cancer patient, thereby formulating a personalized treatment scheme. The kit is simple and convenient to operate, short in detection time, capable of realizing high-throughput detection, low in price, mainly applied to detection of the Fatty Acyl-CoA Reductase 1 gene, and capable of guiding a clinician to be used for 5-FU medication guidance.
Drawings
FIG. 1 is a graph showing the amplification curve of the negative control in example 1.
FIG. 2 is a graph showing the amplification curve of the positive control in example 1.
FIG. 3 is a graph showing the amplification of the fat Acyl-CoA Reductase 1 gene standard of example 1.
FIG. 4 is a graph showing the amplification of the reference gene standard of example 1.
FIG. 5 is a graph showing the calibration curve of the Fatty Acyl-CoA Reductase 1 gene of example 1.
FIG. 6 is a reference gene calibration graph of example 1.
FIG. 7 is a graph showing the quantitative values of the negative and positive quality control materials in example 2.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 method for detecting Fatty Acyl-CoA Reductase 1 Gene
1. Primer design
Specific primers and probes were designed using Oligodt7.0 software based on the Fatty Acyl-CoA Reductase 1 gene (FAR1, NCBI: NM-032228.6) and reference gene (GAPDH, NCBI: NM-001256799.3) sequences registered in GenBank, and their specificity was preliminarily identified by the BLAST function of NCBI, and the primer-probe sequences are shown in Table 1.
Table 1:
Figure BDA0003465575270000051
Figure BDA0003465575270000061
2. real-time fluorescent quantitative PCR amplification and detection of Fatty Acyl-CoA Reductase 1 gene
(1) Preparation of reagents: 18 mul of PCR reaction solution and 2 mul of enzyme mixed solution are fully and evenly mixed for standby,
wherein the PCR reaction solution consists of primers (SEQ ID NO. 1-2 and SEQ ID NO. 4-5), probes (SEQ ID NO.3 and SEQ ID NO.6) and PCR buffer in Table 1, wherein the final concentration of an amplification system of the primers is 0.2 mu mol/L, the final concentration of an amplification system of the probes is 0.1 mu mol/L, and the final concentration of the PCR buffer is 1X;
the enzyme mixed solution consists of reverse transcriptase, an RNase inhibitor, hot start Taq enzyme and dNTPs, wherein the reaction final concentration of the reverse transcriptase is 2.0U, the reaction final concentration of the RNase inhibitor is 0.5U, the reaction final concentration of the hot start Taq enzyme is 2.0U, and the reaction final concentration of the dNTPs is 0.4 mM.
(2) Sample adding: adding 5 μ l of negative quality control product, positive quality control product, FAR1 quantitative reference product or internal reference gene quantitative reference product into PCR reaction tube, covering tube cover, placing into sample groove of instrument,
wherein, FAR1 quantitative reference: the standard was diluted with a defined amount of Fatty Acyl-CoA Reductase 1 plasmid at a concentration of 1X 106copies/mL、1×105copies/mL、1×104copies/mL、1×103copies/mL；
Internal reference gene quantitative reference: is a standard substance diluted with a predetermined amount of reference plasmid at a concentration of 1 × 106copies/mL、1×105copies/mL、1×104copies/mL、1×103copies/mL；
The internal reference gene quantitative reference product is obtained by diluting artificially constructed plasmid containing an internal reference target gene sequence to a corresponding concentration by using 1 × TE, wherein the internal reference gene target gene sequence is shown as SEQ ID NO. 7;
the FAR1 quantitative reference is obtained by diluting artificially constructed plasmid containing a Fatty Acyl-CoA Reductase 1 target gene sequence to a corresponding concentration by 1 × TE, wherein the Fatty Acyl-CoA Reductase 1 target gene sequence is shown in SEQ ID NO. 8;
the negative quality control product is RNA of a clinical sample, and the quantitative value of the Fatty Acyl-CoA Reductase 1 gene/the quantitative value of the internal reference gene is more than or equal to 3;
the positive quality control product is RNA of a clinical sample, and the quantitative value of the Fatty Acyl-CoA Reductase 1 gene/quantitative value of the reference gene of the positive quality control product is less than 3.
(3) And (3) PCR detection: placing the PCR reaction tube in a fluorescent quantitative PCR instrument, and setting a reaction program:
Figure BDA0003465575270000062
Figure BDA0003465575270000071
note: indicates collected fluorescence signal
3. And (4) analyzing results:
and storing the detection data file after the reaction is finished. An Amplification Plot window is opened under Results. The location of the sample of interest to be analyzed is selected. Changing the Baseline value to start: 3, stop: 10, and opens the manual setting Threshold: 2-6e + 3. And (3) double clicking numerical values on Rn coordinates to open a Graph Settings window, changing Log in Post Run Settings into Linear, opening an Analysis preferences window after OK, selecting an Analysis automatic Analysis result under an Analysis menu, viewing the result on a Report interface, and recording an unknown sample quantitative value.
4. Results of the experiment
Amplification curves of FAM and VIC detection channels of the negative and positive quality control products are obvious S-shaped curves (see figure 1 and figure 2), the amplification curve of the FAM detection channel of the Fatty Acyl-CoA Reductase 1 gene quantitative reference product is S-shaped (see figure 3), the correlation coefficients of the standard curves are 0.997 respectively, and the slope is-3.74 (see figure 4); the amplification curve of the VIC detection channel of the reference gene quantitative reference substance is S-shaped (see figure 5), the correlation coefficient of the standard curve is 0.999, and the slope is-3.77 (see figure 6).
The kit is used for detecting the expression quantity of mRNA of the Fatty Acyl-CoA Reductase 1 gene based on a relative quantification method of a standard curve method. The quantitative value of the Fatty Acyl-CoA Reductase 1 gene/quantitative value of the reference gene of the negative quality control product is more than 3; the quantitative value of the Fatty Acyl-CoA Reductase 1 gene/quantitative value of the internal reference gene of the positive quality control product is less than 3.
The correlation coefficients of the Fatty Acyl-CoA Reductase 1 gene and the internal reference gene amplification curve in the kit are both larger than 0.98, and the negative and positive quality control products meet the requirements, the quality of the kit in the specification is reliable, and the quantitative result is accurate.
Example 2 kit for detecting Fatty Acyl-CoA Reductase 1 Gene
A, make up
The FAR1 PCR reaction solution 1 tube consists of primers, probes and PCR buffers, wherein the final concentration of an amplification system of the primers is 0.2 mu mol/L, the final concentration of an amplification system of the probes is 0.1 mu mol/L, the final concentration of the PCR buffers is 1 x, and the specific primers and probes are as follows:
the upstream primer of the Fatty Acyl-CoA Reductase 1 gene: 5'-TGTGCAGCTACAGTAAGGTTT-3' (SEQ ID NO.1),
downstream primer of Fatty Acyl-CoA Reductase 1 gene: 5'-GATTCTTCATTTGTTGTGCAAGG-3' (SEQ ID NO.2),
fatty Acyl-CoA Reductase 1 gene probe: 5 '-FAM-ATGTGATTGCAACGCGACAGCT T-BHQ 1-3' (SEQ ID NO.3),
internal reference gene upstream primer: 5'-AAAACCTGCCAAATATGATGACA-3' (SEQ ID NO.4),
the downstream primer of the internal reference gene: 5'-TGAAGTCAGAGGAGACCACC-3' (SEQ ID NO.5),
internal reference gene probe: 5 '-VIC-CAGTGTAGCCCAGGATGCCCTT-BHQ 1-3' (SEQ ID NO. 6);
the FAR1 enzyme mixed solution 1 tube consists of reverse transcriptase, RNase inhibitor, hot start Taq enzyme and dNTPs, wherein the reaction final concentration of the reverse transcriptase is 2.0U, the reaction final concentration of the RNase inhibitor is 0.5U, the reaction final concentration of the hot start Taq enzyme is 2.0U, and the reaction final concentration of the dNTPs is 0.4 mM.
FAR1 quantitative reference: the standard substance is diluted by a defined amount of Fatty Acyl-CoA Reductase 1 plasmid, and the concentration is 1X 106copies/mL、1×105copies/mL、1×104copies/mL、1×103The cotty Acyl-CoA Reductase 1 plasmid is a plasmid containing a artificially constructed target gene sequence of cotty Acyl-CoA Reductase 1 and is diluted to a corresponding concentration by 1 xTE, and the cotty Acyl-CoA Reductase 1 target gene sequence of the plasmid isAs shown in SEQ ID NO. 8;
quantitative reference products of internal reference genes: is a standard substance diluted with a predetermined amount of reference plasmid, and has a concentration of 1 × 106copies/mL、1×105copies/mL、1×104copies/mL、1×103The copies/mL, the reference gene quantitative reference product is that the artificially constructed plasmid containing the reference target gene sequence is diluted to the corresponding concentration by 1 × TE, and the reference gene target gene sequence is shown as SEQ ID NO. 7;
negative quality control 1 tube: RNA of a clinical sample, wherein the quantitative value of the Fatty Acyl-CoA Reductase 1 gene/quantitative value of an internal reference gene is greater than or equal to 3;
positive quality control 1 tube: is RNA of clinical sample, and the quantitative value of Fatty Acyl-CoA Reductase 1 gene/quantitative value of reference gene is less than 3.
Second, use method
(1) Preparation of reagents: 18 mul of PCR reaction solution and 2 mul of enzyme mixed solution are fully and evenly mixed for standby,
(2) sample adding: and respectively adding 5 mul of a negative quality control product, a positive quality control product, a FAR1 quantitative reference product, an internal reference gene quantitative reference product and a sample to be detected (colorectal cancer tissue RNA) into the PCR reaction tube, covering a tube cover tightly, and putting into an instrument sample groove.
(3) And (3) PCR detection: placing the PCR reaction tube in a fluorescent quantitative PCR instrument, and setting a reaction program:
Figure BDA0003465575270000081
Figure BDA0003465575270000091
note: denotes the collection of fluorescence signals
3. And (4) analyzing results:
and storing the detection data file after the reaction is finished. An Amplification Plot window is opened under Results. The location of the sample of interest to be analyzed is selected. Changing the Baseline value to start: 3, stop: 10, and opens the manual setting Threshold: 2-6e + 3. And (3) double clicking numerical values on Rn coordinates to open a Graph Settings window, changing Log in Post Run Settings into Linear, opening an Analysis preferences window after OK, selecting an Analysis automatic Analysis result under an Analysis menu, viewing the result on a Report interface, and recording an unknown sample quantitative value.
4. Interpretation of results
Negative result interpretation: the amount of the Fatty Acyl-CoA Reductase 1 gene/amount of the internal reference gene (see FIG. 7) in the same sample was 3.25, 5-FU chemotherapy-resistant.
Positive result interpretation: the amount of fat Acyl-CoA Reductase 1 gene/amount of internal reference gene (see FIG. 7) in the same sample was 1.75, and 5-FU was chemosensitive.
Example 35-detection of chemotherapy-resistant samples of FU
First, experiment method
15 clinical 5-FU chemotherapy-resistant tissue samples were selected, all samples were subjected to nucleic acid extraction, the kit of example 2 was used for detection, and negative and positive quality controls were used for quality control.
Second, experimental results
Amplification curves of FAM and VIC detection channels of the negative and positive quality control products are obvious S-shaped curves, wherein the quantitative value of the negative quality control product/the quantitative value of the internal reference gene is 3.25 and is more than 3; the quantitative value of the positive quality control product/the quantitative value of the internal reference gene is 1.75 and less than 3. The negative and positive quality control products meet the quality control requirements of the kit, so the detection result of the sample to be detected is effective.
As can be seen from the results of the 15 samples tested, the Fatty Acyl-CoA Reductase 1 gene quantitation value/reference gene quantitation value detected by all the 5-FU chemotherapy-resistant samples were all greater than 3, and the detailed results are shown in Table 2.
TABLE 2
Figure BDA0003465575270000092
Figure BDA0003465575270000101
The results show that the Fatty Acyl-CoA Reductase 1 gene quantitative value/internal reference gene quantitative value of 15 cases of 5-FU chemotherapy drug-resistant specimens are all larger than 3, and the detection results are completely consistent with clinical results, which indicates that the kit is feasible for detecting specificity and has good specificity.
Example 35-detection of chemotherapy-sensitive samples for FU
First, experiment method
20 clinical samples of colorectal cancer tissue sensitive to 5-FU chemotherapy were selected, all samples were subjected to nucleic acid extraction, the kit of example 2 was used for detection, and negative and positive quality controls were used for quality control.
Second, experimental results
The negative and positive quality control products meet the quality control requirements of the kit, so the detection result of the sample to be detected is effective. The detection results of 20 cases of 5-FU chemotherapy-sensitive colorectal cancer positive tissue samples are less than 3, and the detection results are shown in Table 3.
TABLE 3
Figure BDA0003465575270000102
Figure BDA0003465575270000111
The results show that the Fatty Acyl-CoA Reductase 1 gene quantitative value/internal reference gene quantitative value of 20 cases of 5-FU chemotherapy sensitive samples are less than 3, and the detection results are completely consistent with clinical results, which indicates that the kit is feasible for detecting specificity and has good specificity.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
SEQ ID NO.1 5’-TGTGCAGCTACAGTAAGGTTT-3’
SEQ ID NO.2 5’-GATTCTTCATTTGTTGTGCAAGG-3’
SEQ ID NO.3 5’-FAM-ATGTGATTGCAACGCGACAGCTT-BHQ1-3’
SEQ ID NO.4 5’-AAAACCTGCCAAATATGATGACA-3’
SEQ ID NO.5 5’-TGAAGTCAGAGGAGACCACC-3’
SEQ ID NO.6 5’-VIC-CAGTGTAGCCCAGGATGCCCTT-BHQ1-3’
Shown as SEQ ID NO. 7.
AAGCTCACTGGCATGGCCTTCCGTGTCCCCACTGCCAACGTGTCAGTGGTGGACCTGACCTGCCGTCTAGAAAAACCTGCCAAATATGATGACATCAAGAAGGTGGTGAAGCAGGCGTCGGAGGGCCCCCTCAAGGGCATCCTGGGCTACACTGAGCACCAGGTGGTCTCCTCTGACTTCAACAGCGACACCCACTCCTCCACCTTTGACGCTGGGGCTG。
Shown as SEQ ID NO. 8.
ACCAATATTATATTCCACTGTGCAGCTACAGTAAGGTTTAATGAAAATTTAAGAGATGCTGTTCAGTTAAATGTGATTGCAACGCGACAGCTTATTCTCCTTGCACAACAAATGAAGAATCTGGAAGTGTTCATGCATGTATCAACAGCATATGCCTACTGTAATCGCAAGCATATTGATGAAGTAGTCTATCCACCACCTGTGGATCCCAAGAAGCTGATTGATTCTTTAGAGTGGATGGATGATGGCCTAGTAAATGATATCACGCCAAAATTGATAGGAGACAGACCTAATACA。
Sequence listing
<110> southern hospital of southern medical university
<120> a kit for evaluating 5-FU treatment sensitivity/drug resistance
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tgtgcagcta cagtaaggtt t 21
<210> 2
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gattcttcat ttgttgtgca agg 23
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgtgattgc aacgcgacag ctt 23
<210> 4
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aaaacctgcc aaatatgatg aca 23
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tgaagtcaga ggagaccacc 20
<210> 6
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
cagtgtagcc caggatgccc tt 22
<210> 7
<211> 220
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
aagctcactg gcatggcctt ccgtgtcccc actgccaacg tgtcagtggt ggacctgacc 60
tgccgtctag aaaaacctgc caaatatgat gacatcaaga aggtggtgaa gcaggcgtcg 120
gagggccccc tcaagggcat cctgggctac actgagcacc aggtggtctc ctctgacttc 180
aacagcgaca cccactcctc cacctttgac gctggggctg 220
<210> 8
<211> 297
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
accaatatta tattccactg tgcagctaca gtaaggttta atgaaaattt aagagatgct 60
gttcagttaa atgtgattgc aacgcgacag cttattctcc ttgcacaaca aatgaagaat 120
ctggaagtgt tcatgcatgt atcaacagca tatgcctact gtaatcgcaa gcatattgat 180
gaagtagtct atccaccacc tgtggatccc aagaagctga ttgattcttt agagtggatg 240
gatgatggcc tagtaaatga tatcacgcca aaattgatag gagacagacc taataca 297

Claims (10)

1. The application of a product for detecting the expression quantity of the Fatty Acyl-CoA Reductase 1 gene in preparing a kit for evaluating the treatment sensitivity/drug resistance of 5-FU.
2. The use according to claim 1, wherein the expression level of the Fatty Acyl-CoA Reductase 1 gene is the expression level of mRNA of the Fatty Acyl-CoA Reductase 1 gene.
3. The use of claim 1, wherein assessing 5-FU treatment susceptibility/resistance is assessing colorectal cancer 5-FU treatment susceptibility/resistance.
4. The use according to claim 1, wherein the product for detecting the expression level of the Fatty Acyl-CoA Reductase 1 gene is a detection kit for the expression level of mRNA of the Fatty Acyl-CoA Reductase 1 gene.
5. The use of claim 4, wherein the kit for detecting the expression level of mRNA of the Fatty Acyl-CoA Reductase 1 gene comprises a primer having a nucleotide sequence of SEQ ID NO. 1-2 and a probe having a nucleotide sequence of SEQ ID NO. 3.
6. The use according to claim 5, wherein the kit for detecting the expression level of mRNA of the Fatty Acyl-CoA Reductase 1 gene further comprises a primer having a nucleotide sequence of SEQ ID No.4 to 5 and a probe having a nucleotide sequence of SEQ ID No. 6.
7. The use according to claim 5 or 6, wherein the 5' end of the probe is labeled with any one of FAM, VIC, HEX, or CY5 fluorophores.
8. The use according to claim 5 or 6, wherein the 3' of the probe is labeled with any one of a quencher group BHQ1 and BHQ 2.
9. The use according to claim 5 or 6, wherein the kit for detecting the expression level of mRNA of the Fatty Acyl-CoA Reductase 1 gene further comprises a negative quality control substance and/or a positive quality control substance.
10. The use according to claim 5 or 6, wherein the kit for detecting the expression level of mRNA of the Fatty Acyl-CoA Reductase 1 gene further comprises a FAR1 quantitative reference and/or an internal reference gene quantitative reference.
CN202210028785.8A 2022-01-11 2022-01-11 Kit for evaluating 5-FU treatment sensitivity/drug resistance Pending CN114592059A (en)

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Application publication date: 20220607