CN111500728A - Primer probe composition, kit and detection method for detecting human AR-V7 and AR gene expression - Google Patents
Primer probe composition, kit and detection method for detecting human AR-V7 and AR gene expression Download PDFInfo
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Abstract
The invention provides a primer probe composition, a kit and a detection method for detecting human AR-V7 and AR gene expression; the primer probe composition comprises an AR-V7 forward primer with a nucleotide sequence shown as SEQ ID NO.1, an AR-V7 reverse primer with a nucleotide sequence shown as SEQ ID NO.2, an AR-V7 probe with a nucleotide sequence shown as SEQ ID NO.3, an AR forward primer with a nucleotide sequence shown as SEQ ID NO.4, an AR reverse primer with a nucleotide sequence shown as SEQ ID NO.5, an AR probe with a nucleotide sequence shown as SEQ ID NO.6 and an internal reference primer probe. In the invention, the specific primers and probes are used for respectively amplifying the full length of AR and the molecules of AR-V7 spliceosome in the RNA of blood cells, and the method has the advantages of rapidness, high sensitivity, simple and convenient operation, reduction of sample pollution in the middle process, low risk of misoperation and the like.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a primer probe composition, a kit and a detection method for detecting human AR-V7 and AR gene expression.
Background
Prostate cancer is one of the common malignant tumors in European and American men, the incidence of prostate cancer in the United states is the first place, and the mortality rate is second to lung cancer. Although the incidence of the prostate cancer is lower than that of Europe and America in China, the incidence of the prostate cancer is increased in recent years, according to statistics, the incidence of the prostate cancer in 2013 is 8.58/10 ten thousand in China, the prostate cancer is the sixth highest incidence of the male cancer, the incidence of the prostate cancer in 2018 is 13.6/10 ten thousand, and the incidence is obviously higher than that in 2013. Prostate cancer belongs to Androgen-dependent tumors, and thus the current primary means of treating prostate cancer is Androgen Deprivation Therapy (ADT). However, after a median 18-24 months of treatment, almost all patients progress irreversibly to the hormone resistant stage, Castration-resistant prostate cancer (CRPC).
At present, abiraterone (an androgen biosynthesis inhibitor) and enzalutamide (an inhibitor of AR translocation and signal transduction) have a good treatment effect on CRPC patients, but after 20% -40% of patients are treated by abiraterone and enzalutamide, PSA is still maintained at a high level, no obvious curative effect is achieved, and most of patients treated by abiraterone and enzalutamide almost have acquired drug resistance in the later period. Research shows that positive androgen receptor shearing variant 7(AR-V7) is obviously and negatively correlated with response rate and survival rate of Abiraterone/enzalutamide PSA. In patients positive for AR-V7, the taxane chemotherapy was more effective than abiraterone and enzalutamide, while in patients negative for AR-V7, abiraterone and enzalutamide were more effective. Therefore, the detection of AR-V7 is of great significance to doctors for making reasonable and effective treatment schemes for CRPC patients and monitoring drug resistance of endocrine therapy (Abiraterone and Enzalutamide).
At present, the commonly used AR-V7 detection techniques are Circulating Tumor Cell (CTCs) method and exosome method. There are a number of problems with AR-V7 assays using CTC samples: (1) the sample requirement is high, and the sample must be subjected to CTC separation within hours of collection; (2) the operation is complicated, the technical requirement is high, a CTC separation platform needs to be equipped in a laboratory, and CTC separation is completed before RNA extraction, which is difficult to be carried out in a clinical laboratory; (3) the detection sensitivity is poor, and due to tumor heterogeneity, CTC isolation can only acquire part of tumor cells, thus failing to comprehensively reflect the disease state of patients. Exosome methods also require very specialized centrifugation equipment and very specialized centrifugation procedures, which are difficult to develop in clinical laboratories. Meanwhile, the representative problems of the exosome samples, the quality control problems of exosome separation and the like are not unified internationally. Therefore, an AR-V7 detection method with low sample requirement, simple operation, economy and high sensitivity is urgently needed.
Disclosure of Invention
The invention aims to disclose a primer probe composition, a kit and a detection method for detecting human AR-V7 and AR gene expression, which are used for realizing specific detection of AR-V7 and AR spliceosome molecules in human blood cell RNA, reducing the detection difficulty and improving the detection sensitivity and the reliability of a detection result.
In order to achieve the first object, the present invention provides a primer probe combination for detecting human AR-V7 and AR gene expression, comprising:
the forward primer AR-V7 has the nucleotide sequence shown in SEQ ID NO. 1;
AR-V7 reverse primer, having the nucleotide sequence shown in SEQ ID NO. 2;
an AR-V7 probe having a nucleotide sequence shown in SEQ ID NO. 3;
the AR forward primer has a nucleotide sequence shown as SEQ ID NO. 4;
the AR reverse primer has a nucleotide sequence shown as SEQ ID NO. 5;
an AR probe having a nucleotide sequence shown as SEQ ID NO. 6;
a GAPDH forward primer, which has a nucleotide sequence shown as SEQ ID NO. 7;
a GAPDH reverse primer, which has a nucleotide sequence shown as SEQ ID NO. 8;
the GAPDH probe has a nucleotide sequence shown as SEQ ID NO. 9.
Based on the same invention idea, the invention also discloses a kit for detecting human AR-V7 and AR gene expression, which comprises: the primer probe composition disclosed by the invention, DNA polymerase, dNTPs, reaction buffer, a positive control group containing a positive quality control substance, and a negative control group containing a negative quality control substance.
As a further improvement of the invention, the kit detects human AR-V7 and AR to a detection sample by using qRT-PCR or dPCR.
As a further improvement of the invention, the test sample is human peripheral whole blood or blood cells obtained by centrifugation of human peripheral whole blood.
As a further improvement of the present invention,
the positive quality control product is pseudoviral RNA of an AR-V7 target amplification region or RNA extracted from a known AR-V7 positive human cell strain;
the negative quality control product is pseudoviral RNA not containing AR-V7 target amplification region, RNA extracted from known AR-V7 negative human cell strain, buffer solution or RNase-Free ddH2O。
As a further improvement of the invention, the 3 'ends of the AR-V7 probe, the AR probe and the GAPDH probe are all marked with fluorescence quenching groups, and the 5' ends of the AR-V7 probe, the AR probe and the GAPDH probe are all marked with fluorescence reporters.
As a further improvement of the invention, the fluorescence quenching group is selected from any one of DABCY L, MGB, BHQ-1, BHQ-2, BHQ-3 or thiophonate, and the fluorescence reporter group is selected from any one of FAM, HEX, TET, JOE, NED, VIC, CY3, CY5, ROX or TAMRA.
Finally, the invention also discloses a method for detecting human AR-V7 and AR gene expression, which uses the kit for detecting human AR-V7 and AR gene expression disclosed by any one of the creations of the invention, and uses qRT-PCR or dPCR to detect a detection sample.
As a further improvement of the invention, the qRT-PCR or dPCR adds a pre-reaction step of the UNG enzyme in performing the amplification procedure.
As a further improvement of the invention, the detection method also comprises a sample pre-amplification step, which is carried out simultaneously with or separately from the pre-reaction step in which the UNG enzyme is added.
As a further improvement of the invention, the detection target regions of the AR-V7 forward primer, the AR-V7 reverse primer and the AR-V7 probe are the same or basically the same; the detection target regions of the AR forward primer, the AR reverse primer, the AR probe, the GAPDH forward primer, the GAPDH reverse primer and the GAPDH probe are the same or partially the same or completely different.
As a further improvement of the invention, the detection result obtained by the detection method is used for the auxiliary diagnosis or medication guidance of clinical tumors.
Compared with the prior art, the invention has the beneficial effects that:
in the invention, specific primers and probes are used for respectively amplifying the full length of AR and the molecules of AR-V7 spliceosome in blood cell RNA, and the proportion of AR-V7 is calculated.
Meanwhile, because part of prostate cancer AR is low expressed or not expressed, the problem that the AR signal is low or is lost as a sample amount or a tumor cell AR expression amount cannot be distinguished without an internal reference gene, the former needs to be re-sample-sent for detection, and the latter is because the re-sample-sent AR signal is still low or is lost due to the biological problem.
Meanwhile, the detection kit and the detection method are based on CTC existing in peripheral blood cells, and when no CTC is collected or the number of the CTC is extremely low in a sample, the amplification signals of AR-V7 and/or AR can be extremely low or lost. The normal amplification of the internal reference gene can eliminate the problems of insufficient sample size and few or missing CTCs in the sample, thereby ensuring that objective detection results can still be obtained under the objective conditions. The invention overcomes the non-popularity and the technical difficulty of the technical platform for separating CTC and exosome, directly adopts the RNA of peripheral blood cells as a reaction template, directly adopts qRT-PCR or dPCR for amplification, and is simple and easy to implement in a clinical laboratory.
Finally, an internal reference primer probe is simultaneously introduced in the application, so that objective detection results can still be obtained under the conditions that the AR of the detection sample is low in expression or not expressed and the CTC in the detection sample is few or deficient.
Drawings
FIG. 1 is an amplification curve of a positive quality control in a qRT-PCR assay using the detection method disclosed herein;
FIG. 2 is an amplification curve of negative quality control in qRT-PCR assay using the detection method disclosed herein;
FIG. 3 is a graph showing the amplification curves of AR-V7 and AR in clinical samples using qRT-PCR using the detection method disclosed herein.
Detailed Description
The present invention is described in detail with reference to the embodiments shown in the drawings, but it should be understood that these embodiments are not intended to limit the present invention, and those skilled in the art should understand that functional, methodological, or structural equivalents or substitutions made by these embodiments are within the scope of the present invention.
The applicant first explains the main technical terms referred to in the present application. qRT-PCR (reverse transcription-quantitative PCR), is a widely used method to study gene expression patterns, especially when the sample size is small or very precious. The qRT-PCR has the main advantages of wide range, high sensitivity, high accuracy, no PCR post-treatment step, avoidance of cross contamination, high yield, capability of performing multiple detection and the like. And the detection result of the dPCR (digital PCR) is judged independent of the cycle threshold (Ct) of an amplification curve, is not influenced by the amplification efficiency, has good accuracy and reproducibility, and can realize absolute quantitative analysis. Digital PCR allows direct readout of the number of DNA molecules, an absolute quantification of the nucleic acid molecules of the starting sample.
The first embodiment is as follows:
first, the present application discloses a primer probe composition for detecting human AR-V7 and AR gene expression, comprising:
the forward primer AR-V7 has the nucleotide sequence shown in SEQ ID NO. 1;
AR-V7 reverse primer, having the nucleotide sequence shown in SEQ ID NO. 2;
an AR-V7 probe having a nucleotide sequence shown in SEQ ID NO. 3;
the AR forward primer has a nucleotide sequence shown as SEQ ID NO. 4;
the AR reverse primer has a nucleotide sequence shown as SEQ ID NO. 5;
an AR probe having a nucleotide sequence shown as SEQ ID NO. 6;
a GAPDH forward primer, which has a nucleotide sequence shown as SEQ ID NO. 7;
a GAPDH reverse primer, which has a nucleotide sequence shown as SEQ ID NO. 8;
the GAPDH probe has a nucleotide sequence shown as SEQ ID NO. 9.
Specifically, the fluorescence quenching group is selected from any one of DABCY L, MGB, BHQ-1, BHQ-2, BHQ-3 or thiophosphate, and the fluorescence quenching group is selected from any one of FAM, HEX, TET, JOE, NED, VIC, CY3, CY5, ROX or TAMRA.
In the embodiment, based on the AR-V7 forward primer, the AR-V7 reverse primer, the AR-F L forward primer, the AR-F L reverse primer, the AR-V7 probe and the AR-F L probe, the method has the advantages of high primer amplification efficiency, short amplification sequence length, capability of avoiding the problem that a long fragment is not easy to amplify due to RNA fragmentation, capability of avoiding using a CTC separation platform, and capability of accurately and specifically detecting the full length of AR and the AR-V7 spliceosome molecules in human blood cell RNA, and the detection result has accurate clinical reference value for auxiliary diagnosis or medication guidance of clinical tumors.
Example two:
based on the primer probe composition for detecting human AR-V7 and AR gene expression disclosed in the first embodiment, the present embodiment also discloses a kit for detecting human AR-V7 and AR gene expression, which comprises: the primer probe composition, DNA polymerase, dNTPs, reaction buffer, positive control group containing positive quality control substances, and negative control group containing negative quality control substances as disclosed in the first embodiment of the present invention.
The kit detects human AR-V7 and AR to a detection sample by using qRT-PCR or dPCR.
The test sample is human peripheral whole blood or blood cells obtained by centrifugation of human peripheral whole blood.
The positive quality control product is pseudoviral RNA of an AR-V7 target amplification region or RNA extracted from a known AR-V7 positive human cell strain; the negative quality control product is pseudoviral RNA not containing AR-V7 target amplification region, RNA extracted from known AR-V7 negative human cell strain, buffer solution or RNase-Free ddH2O。
Specifically, in this example, the fluorescence quenching group is selected from any one of DABCY L, MGB, BHQ-1, BHQ-2, BHQ-3 or thiophosphate, the fluorescence quenching group is selected from any one of FAM, HEX, TET, JOE, NED, VIC, CY3, CY5, ROX or TAMRA, in this example, the AR-V7 forward primer and the AR-V7 reverse primer are mixed in proportion, the AR-F L forward primer and the AR-F L reverse primer are mixed in proportion, and the GAPDH forward primer and the GAPDH reverse primer are mixed in proportion.
Example three:
based on the technical solutions disclosed in the first and second embodiments, the present embodiment further discloses a method for detecting human AR-V7 and AR gene expression, using the kit for detecting human AR-V7 and AR gene expression described in the second embodiment, and using qRT-PCR or dPCR to detect a detection sample; and the qRT-PCR or dPCR adds a pre-reaction step of UNG enzyme in performing the amplification procedure. In this example, the pre-reaction step in which UNG enzyme was added serves to prevent contamination of the reaction system with PCR products. Meanwhile, in this embodiment, the detection method further comprises a sample pre-amplification step, which is performed separately from the pre-reaction step in which the UNG enzyme is added (i.e., in the same reaction tube) or separately (i.e., in two different reaction tubes), and which are similar to each other in effect.
In the detection method disclosed in this example, the detection target regions of the primers and probes in the kit are identical or substantially identical (the sequences may overlap by 70% or more), and the nucleotide sequences of the primers and/or probes may be partially or completely changed. The detection method is described in detail below.
The Kit is stored in 8 samples (total samples) to be detected of PAXgene Blood RNA Tube (BD, 762165), PAXgene Blood RNA Kit (BD, 762174), a positive quality control product and a negative quality control product, wherein the positive quality control product is prepared by mixing AR-V7 and AR-F L in equal proportion, the concentration of the positive quality control product is 1 ng/mu L pseudovirus RNA, and the negative quality control product is 50 ng/mu L RNA of AR-V7 negative cell line PC 3.
Firstly, collecting a whole Blood sample, namely collecting a human Blood sample according to the Blood sample collection requirement of PAXgene Blood RNA Tube (BD, 762165), extracting sample RNA according to the RNA extraction Protocol of the whole Blood sample of PAXgene Blood RNA Kit (BD, 762174), and finally eluting by using 30 mu L eluent.
Extracting RNA of the sample according to the instruction of the kit, and finally eluting by 30 mu L eluent, wherein the main component of the reaction system is
II U + One Step qRT-PCR Probe Kit (Novozan, Q222-CN), sampleThe reverse transcription and pre-amplification system involved in the pre-amplification step is shown in the following table:
| reagent | Amount used (ul) |
| 2×One Step U+Mix | 15.0 |
| One Step U+Enzyme Mix | 1.5 |
| AR-V7 forward primer | 0.5 |
| AR-V7 reverse primer | 0.5 |
| AR-F L forward primer | 0.5 |
| AR-F L reverse primer | 0.5 |
| GAPDH forward primer | 0.5 |
| GAPDH reverse primer | 0.5 |
| Total | 19.5 |
Watch 1
Subpackaging the premix solution and performing on-machine pre-amplification: taking a PCR tube, respectively adding 19.5ul of the premix and 10.5ul of the RNA template into each hole (the position of a sample hole is recorded), and shaking, mixing and instantly separating. The sample tubes were placed in a conventional PCR instrument and the sample pre-amplification procedure shown in table two below was run.
Watch two
According to the number (n) of samples to be tested, the following Table prepares the qPCR premix MIX (n +5) (wherein negative quality control, positive quality control 2, RNase-Free ddH2O), shaking and mixing uniformly, and instantly separating for later use. The qPCR reaction premix systems are shown in table three and table four below.
The reaction premix liquid in the AR/AR-V7 reaction tube performing the qPCR amplification procedure is shown in table three below:
| reagent | Amount used (ul) |
| 10 × PCR Buffer | 2.0 |
| dNTPs(10mM) | 1.0 |
| Mgcl2(50mM) | 1.6 |
| Taq polymerase(10U/ul) | 0.2 |
| AR-V7 forward primer | 0.4 |
| AR-V7 reverse primer | 0.4 |
| AR-V7 Probe | 0.4 |
| AR-F L forward primer | 0.4 |
| AR-F L reverse primer | 0.4 |
| AR-F L Probe | 0.3 |
| ddH2O | 8.9 |
| Pre-amplification product | 4.0 |
| Total | 20.0 |
Watch III
The reaction premix liquid in the inner control tube (containing GAPDH forward primer, GAPDH reverse primer and GAPDH probe) performing the qPCR amplification procedure is shown in table four below:
| reagent | Amount used (ul) |
| 10 × Buffer (reaction Buffer) | 2.0 |
| dNTPs(10mM) | 1.0 |
| Mgcl2(50mM) | 1.6 |
| Taq polymerase(10U/ul) | 0.2 |
| GAPDH forward primer | 0.4 |
| GAPDH reverse primer | 0.4 |
| GAPDH probe | 0.4 |
| ddH2O | 10 |
| Pre-amplification product | 4.0 |
| Total | 20.0 |
Watch four
The pre-amplification products in tables three and four are obtained from the pre-mixture and the RNA template in the pre-amplification procedure.
Subpackaging the premix liquid: a96-well plate is taken, 16ul of premix and 4ul of template are added into each well (the position of a sample well is recorded), a pipe cover is sealed, and the mixture is shaken, mixed and separated instantly.
Setting parameters of a real-time fluorescent quantitative PCR instrument of a qPCR amplification instrument (model ABI 7500), namely 95 ℃ for 5min, (95 ℃ for 10s, 60 ℃ for 45s)45cycles with the system of 20ul, an AR-V7+ AR-F L locus FAM channel, a VIC channel and a GAPDH locus VIC channel, putting a sample into the PCR amplification instrument, selecting the position where the sample is placed, storing the position to a specified folder, and starting to operate a program to perform PCR amplification reaction.
Watch five
The reaction system is used for detecting 8 samples, 2 positive samples and 6 negative samples can be accurately detected), and the specific interpretation is as follows:
the AR-F L in the negative quality control product has an obvious amplification curve and accords with the parameter range given in the following table, and the positive quality control products AR-V7 and AR-F L both have an obvious amplification curve and accords with the parameter range given in the following table.
The detection results accurately and efficiently detect the specificity of two spliceosome molecules of AR-V7 and AR-F L in the amplified blood RNA, and the detection judgment standards of whether the amplified curve of the qRT-PCR reaction program is judged to be AR-V7 and AR-F L are shown in the following table six.
| The result of the detection | Ct of AR-V7 | Ct of AR-F L |
| Effective positive control group | Ct≤38 | Ct≤34 |
| Effective negative control group | Ct no interpretation (no amplification curve) | Ct≤34 |
Table six: detection criterion
FIG. 1 shows that the Ct of the amplification curve of AR-V7 is about 27.5 (i.e. Ct is less than or equal to 38) and the Ct of AR-F L is about 26.5 (i.e. Ct is less than or equal to 34), which meets the corresponding interpretation standard, and thus the whole detection process is correct and the result is reliable.
FIG. 2 shows that the Ct of the amplification curve of AR-F L is about 14.8 (i.e. Ct is less than or equal to 34) and AR-V7 has no typical amplification curve or no Ct value, which indicates that the detection method can effectively and specifically detect AR-F L by using the kit disclosed in the second embodiment.
FIG. 3 shows that Ct of the amplification curve of AR-F L is about 14.8 (i.e. Ct is less than or equal to 34) and Ct of the amplification curve of AR-V7 is about 22.2 (i.e. Ct is less than or equal to 38), indicating that AR-F L and AR-V7 both have obvious amplification curves, indicating that AR-V7 is positive.
The same technical solutions as those in the first embodiment and/or the second embodiment are described above, and are not described herein again.
In conclusion, the primer probe composition, the kit and the detection method based on the kit disclosed by the invention realize the AR-V7 and AR full-length detection on human peripheral whole blood or blood cells obtained by using human peripheral whole blood centrifugation. Test results show that the primer probe composition has good specificity, completely has no non-specific amplification, and can be well adapted to rapid and efficient detection of clinical samples. In the invention, the specific primers and probes are used for respectively amplifying the full length AR and the molecules of the AR-V7 spliceosome in the RNA of the blood cells, and the method has the advantages of rapidness, high sensitivity, simple and convenient operation, reduction of sample pollution in the middle process, low risk of misoperation and the like.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
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Claims (12)
1. A primer probe combination for detecting human AR-V7 and AR gene expression is characterized by comprising:
the forward primer AR-V7 has the nucleotide sequence shown in SEQ ID NO. 1;
AR-V7 reverse primer, having the nucleotide sequence shown in SEQ ID NO. 2;
an AR-V7 probe having a nucleotide sequence shown in SEQ ID NO. 3;
the AR forward primer has a nucleotide sequence shown as SEQ ID NO. 4;
the AR reverse primer has a nucleotide sequence shown as SEQ ID NO. 5;
an AR probe having a nucleotide sequence shown as SEQ ID NO. 6;
a GAPDH forward primer, which has a nucleotide sequence shown as SEQ ID NO. 7;
a GAPDH reverse primer, which has a nucleotide sequence shown as SEQ ID NO. 8;
the GAPDH probe has a nucleotide sequence shown as SEQ ID NO. 9.
2. A kit for detecting human AR-V7 and AR gene expression, comprising: the primer probe composition of claim 1, DNA polymerase, dNTPs, reaction buffer, positive control group containing positive quality control, and negative control group containing negative quality control.
3. The kit of claim 2, wherein the kit detects human AR-V7 and AR in a test sample using qRT-PCR or dPCR.
4. The kit according to claim 3, wherein the test sample is human peripheral whole blood or blood cells obtained by centrifugation of human peripheral whole blood.
5. The kit according to claim 2,
the positive quality control product is pseudoviral RNA of an AR-V7 target amplification region or RNA extracted from a known AR-V7 positive human cell strain;
the negative quality control product is pseudoviral RNA not containing AR-V7 target amplification region, RNA extracted from known AR-V7 negative human cell strain, buffer solution or RNase-Free ddH2O。
6. The kit of claim 2, wherein the AR-V7 probe, the AR probe, and the GAPDH probe are each labeled with a fluorescence quencher at their 3 'ends, and the AR-V7 probe, the AR probe, and the GAPDH probe are each labeled with a fluorescence reporter at their 5' ends.
7. The kit according to claim 6, wherein the fluorescence quenching group is selected from DABCY L, MGB, BHQ-1, BHQ-2, BHQ-3 or thiophosphate, and the fluorescence reporter group is selected from FAM, HEX, TET, JOE, NED, VIC, CY3, CY5, ROX or TAMRA.
8. A method for detecting human AR-V7 and AR gene expression, characterized in that the kit for detecting human AR-V7 and AR gene expression of any one of claims 2 to 7 is used, and qRT-PCR or dPCR is used to detect a detection sample.
9. The detection method according to claim 8, wherein the qRT-PCR or dPCR adds a pre-reaction step of UNG enzyme in performing the amplification procedure.
10. The detection method according to claim 9, further comprising a sample pre-amplification step, which is performed simultaneously with or separately from the pre-reaction step in which the UNG enzyme is added.
11. The detection method according to claim 10, wherein the detection target regions of the AR-V7 forward primer, AR-V7 reverse primer, AR-V7 probe are the same or substantially the same; the detection target regions of the AR forward primer, the AR reverse primer, the AR probe, the GAPDH forward primer, the GAPDH reverse primer and the GAPDH probe are the same or partially the same or completely different.
12. The detection method according to any one of claims 8 to 11, wherein the detection result obtained by the detection method is used for auxiliary diagnosis or medication guidance of clinical tumors.
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