Extraction-free qPCR (quantitative polymerase chain reaction) detection kit for HSD3B1 genotype detection and detection method thereof

Abstract

The invention relates to the field of molecular biology, and discloses an extraction-free qPCR detection kit for HSD3B1 genotype detection and a detection method thereof.The extraction-free qPCR detection kit for HSD3B1 genotype detection comprises PCR water, a wild type primer probe, a mutant type primer probe, 2XqPCR Mix and a positive quality control product, the 2XqPCR Mix comprises PCR buffer, dNTPs, MgCl2 and efficient stress-resistant DNA polymerase, and the positive quality control product comprises a PCR buffer, dNTPs, MgCl2 and efficient stress-resistant DNA polymerase. The kit comprises a wild type primer probe and a mutant type primer probe, the wild type primer probe comprises an HSD3B1 (A1245C) site primer probe which is shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.4, and the mutant type primer probe comprises an HSD3B1 (A1245C) site primer probe. The HSD3B1 gene (1245C) site genotype fragment with the concentration of 500 copies / mL can be detected through the mutation specific primer and the probe, the sensitivity is high, the detection process is that fluorescent PCR detection is carried out on a human serum sample without extraction, the experiment steps are reduced, the pollution possibility is greatly reduced, the operation is simple and rapid, and the process from sample inspection to result obtaining can be completed within one hour.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to an extraction-free qPCR detection kit for HSD3B1 genotype detection and a detection method thereof. Background Art

[0002] The HSD3B1 gene encodes 3β-hydroxysteroid dehydrogenase 1 (3βHSD1), which catalyzes the conversion of extragonadal precursors into dihydrotestosterone (DHT); HSD3B1 (1245A>C) mutations cause the encoded amino acid to change from aspartic acid to threonine, which reduces the degradation of the 3βHSD1 enzyme, thereby increasing the synthesis of DHT from extragonadal (such as adrenal) androgen precursors. This ultimately promotes the occurrence and development of CRPC, as well as the response and outcome of ADT treatment. The treatment and prognosis of many diseases are related to genetics. Different groups and individuals have different susceptibility to diseases and responsiveness to drugs. The genetic basis is the variability of the human genome DNA sequence, of which the most common mutation is SNP. By detecting the genotype of the HSD3B1 (A1245C) site, people with good ADT treatment effects can be screened out, and the efficacy of patients can be predicted, thereby achieving personalized treatment guidance for prostate cancer.

[0003] Commonly used detection methods for genotype loci include direct sequencing of PCR products and arms-qPCR. Direct DNA sequencing is cumbersome, time-consuming and has low sensitivity, while arms-qPCR has the characteristics of high sensitivity and easy operation and is widely used in various medical testing fields. Currently, there are hundreds of fluorescent PCR detection reagents and methods used in clinical practice. Conventional methods for detecting nucleic acids through fluorescent PCR generally involve further adding nucleic acid templates to the reaction system for PCR detection based on nucleic acid purification. Generally, the nucleic acid purification process requires steps such as lysis, purification, washing and collection. Sample processing often takes tens of minutes. Due to the numerous operating steps, mutual contamination between samples is inevitable during the large sample processing process. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides an extraction-free qPCR detection kit for HSD3B1 genotype detection and a detection method thereof, which solves the problem that the conventional method for detecting nucleic acids by fluorescent PCR is generally to further add a nucleic acid template into a reaction system for PCR detection on the basis of nucleic acid purification, and the general nucleic acid purification process requires steps such as lysis, purification, washing and collection. The processing of samples often takes tens of minutes. Due to the numerous operating steps, mutual contamination between samples is also an inevitable problem in the process of large sample processing.

[0005] To achieve the above objectives, the present invention is implemented through the following technical scheme: an extraction-free qPCR detection kit for HSD3B1 genotype detection and a detection method thereof, comprising PCR water, wild-type primer probe, mutant primer probe, 2XqPCR Mix and positive quality control products, the 2XqPCR Mix comprising PCR buffer, dNTPs, MgCl2, and high-efficiency anti-reverse DNA polymerase, the wild-type primer probe comprising HSD3B1 (A1245C) site primer probe, SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.4, and the mutant primer probe comprising HSD3B1 (A1245C) site primer probe.

[0006] Preferably, the contents of PCR buffer, dNTPs, MgCl2, and high-efficiency anti-reverse DNA polymerase in the 2XqPCR Mix are 2.5 μL, 2 μL, 3 μL, and 1 μL, respectively, and are supplemented to 12.5 μL with water. The dNTPs include each at a concentration of 10 mmol / L, and the concentration of MgCl2 is 25 mM.

[0007] Preferably, the contents of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.4 in the wild-type primer probe are 0.2-0.8 μL, 0.2-0.8 μL and 0.2-0.4 μL, respectively, and the concentrations are all 10 μmol / L; the HSD3B1 (A1245C) site primer probe includes SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.4, and the contents are 0.2-0.8 μL, 0.2-0.8 μL and 0.2-0.4 μL, respectively, and the concentrations are all 10 μmol / L.

[0008] A primer for detecting human HSD3B1 (A1245C) site variation, wherein the common forward primer sequence for HSD3B1 (A1245C) site detection is SEQ ID NO.1, the reverse wild-type primer sequence is SEQ ID NO.2, and the reverse mutant-specific primer sequence is SEQ ID NO.3.

[0009] A probe for detecting human HSD3B1 (A1245C) site variation, wherein the probe sequence for detecting the HSD3B1 (A1245C) site is SEQ ID NO.4.

[0010] The extraction-free qPCR detection method for HSD3B1 genotype detection comprises the following steps: using human serum or plasma samples, without going through a nucleic acid extraction process, directly adding them into a reaction system as a PCR template and adding them into a fluorescent PCR detection reaction system for detection.

[0011] Preferably, the nucleic acid extraction process refers to the process of extracting and purifying nucleic acid from a sample outside the qPCR detection reaction system, the sample processing process and the qPCR detection reaction are integrated into the same reaction system, and the sample used as an amplification template for qPCR includes a human serum or plasma sample.

[0012] Preferably, the method specifically comprises the following steps: S1. Add 2 µL of serum, plasma sample or positive control into wild-type and mutant PCR reaction tubes respectively; S2. Add PCR water, 2XqPCR Mix, and wild-type primer probe to the wild-type PCR reaction tube, and add PCR water, 2XqPCR Mix, and mutant primer probe to the mutant PCR reaction tube, with a total system of 25 μL; S3. Cover the tube cap and perform fluorescent PCR detection.

[0013] Preferably, the added amounts of PCR water and 2XqPCR Mix in S2 are 8 μL and 12.5 μL, respectively, the added amount of the wild-type primer probe is 2.5 μL, and the added amount of the mutant primer probe is 2.5 μL. The amplification reaction conditions for fluorescent PCR detection in S3 are: pre-denaturation at 95-97°C for 5 min, denaturation at 95-97°C for 5 s, annealing at 57-62°C for 20-30 s, and 40 cycles.

[0014] The determination method of an extraction-free qPCR detection kit for HSD3B1 genotype detection includes validity determination and result interpretation, wherein the validity determination is that the Ct value of a wild-type PCR reaction tube and a mutant-type PCR reaction tube detected by the sample is Undet or 40, the detection fails, no sample is added, and the detection is repeated, and the result interpretation is that the mutant-type and wild-type ΔCt (2, +∞) indicates that the sample is a wild pure type AA, the mutant-type and wild-type ΔCt [-2, 2] indicates that the sample is a heterozygous type AC, and the mutant-type and wild-type ΔCt (-∞, -2) indicates that the sample is a mutant pure type CC.

[0015] The present invention provides an extraction-free qPCR detection kit and a detection method thereof for HSD3B1 genotype detection, which has the following beneficial effects: 1. The present invention can detect the genotype fragment of the HSD3B1 gene (1245C) site at a concentration of 500 copies / mL through mutation-specific primers and probes. It has high sensitivity and the detection process is a fluorescent PCR detection without extraction of human serum samples, which reduces the experimental steps and greatly reduces the possibility of contamination. The operation is simple and fast, and the process from sample submission to result acquisition can be completed within 1 hour, thereby solving the problem of mutual contamination between samples during large sample processing due to the numerous operating steps in the conventional method of detecting nucleic acids through fluorescent PCR.

[0016] 2. The kit of the present invention can detect the genotype of the human HSD3B1 gene (1245C) site rapidly, accurately and with high sensitivity, and in particular, it does not require DNA extraction and can be directly detected using samples.

[0017] 3. The present invention uses human serum or plasma directly as a template and adds it to an optimized PCR reaction system for detection. The process is completed in one step without opening the lid, which can not only reduce the experimental operation steps and save time, but also avoid the sample contamination that may be caused by the opening operation.

[0018] 4. The primer probe of the present invention is designed for the genotype region sequence of the HSD3B1 gene (1245C) site, and one or more base mutations are introduced at the 3' end of the mutation-specific downstream primer to specifically amplify the mutant allele. A mutation-nonspecific downstream primer is designed a few bases outside the mutation site to simultaneously amplify the wild-type and mutant alleles, thereby detecting the wild type and mutant types of the HSD3B1 gene (1245C) site with strong specificity.

[0019] 5. The results of the present invention are clearly interpreted and objective, and the results can also be quantitatively analyzed. The entire system does not contain toxic and harmful substances, does not require post-processing of PCR products, is harmless to operators and the environment, and has strong practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is an amplification curve diagram of the wild-type HSD3B1 (A1245C) in normal human serum samples detected by the extraction-free qPCR detection kit of the present invention; Figure 2 This is an amplification curve diagram of the HSD3B1 (A1245C) site mutation type detected by the extraction-free qPCR detection kit of the present invention in normal human serum samples. DETAILED DESCRIPTION

[0021] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Embodiment:

[0022] The embodiment of the present invention provides an extraction-free qPCR detection kit for HSD3B1 genotype detection and a detection method thereof, comprising PCR water, a wild-type primer probe, a mutant primer probe, 2XqPCR Mix and a positive quality control product, characterized in that: the 2XqPCR Mix comprises PCR buffer, dNTPs, MgCl2, and a high-efficiency anti-reverse DNA polymerase, the wild-type primer probe comprises an HSD3B1 (A1245C) site primer probe, SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.4, and the mutant primer probe comprises an HSD3B1 (A1245C) site primer probe.

[0023] The contents of PCR buffer, dNTPs, MgCl2, and high-efficiency anti-reverse DNA polymerase in 2XqPCR Mix are 2.5 μL, 2 μL, 3 μL, and 1 μL, respectively, and are supplemented to 12.5 μL with water. The dNTPs include each at a concentration of 10 mmol / L, and the concentration of MgCl2 is 25 mM.

[0024] The contents of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.4 in the wild-type primer probe are 0.2-0.8μL, 0.2-0.8μL, and 0.2-0.4μL, respectively, and the concentrations are all 10μmol / L. The HSD3B1 (A1245C) site primer probe includes SEQID NO.1, SEQ ID NO.3, and SEQ ID NO.4, and the contents are 0.2-0.8μL, 0.2-0.8μL, and 0.2-0.4μL, respectively, and the concentrations are all 10μmol / L.

[0025] A primer for detecting a human HSD3B1 (A1245C) site variation, wherein the common forward primer sequence for HSD3B1 (A1245C) site detection is SEQ ID NO.1, the reverse wild-type primer sequence is SEQ ID NO.2, and the reverse mutant-specific primer sequence is SEQ ID NO.3.

[0026] A probe for detecting human HSD3B1 (A1245C) site variation. The probe sequence for detecting the HSD3B1 (A1245C) site is SEQ ID NO.4.

[0027] The extraction-free qPCR detection method for HSD3B1 genotype detection comprises the following steps: using human serum or plasma samples, without going through a nucleic acid extraction process, directly adding them into a reaction system as a PCR template and adding them into a fluorescent PCR detection reaction system for detection.

[0028] The nucleic acid extraction process refers to the process of extracting and purifying nucleic acids from samples outside the qPCR detection reaction system. The sample processing process and the qPCR detection reaction are integrated into the same reaction system. Samples used as qPCR amplification templates include human serum or plasma samples.

[0029] The specific steps include: S1. Add 2 µL of serum, plasma sample or positive control into wild-type and mutant PCR reaction tubes respectively; S2. Add PCR water, 2XqPCR Mix, and wild-type primer probe to the wild-type PCR reaction tube, and add PCR water, 2XqPCR Mix, and mutant primer probe to the mutant PCR reaction tube, with a total system of 25 μL; S3. Cover the tube cap and perform fluorescent PCR detection.

[0030] The amount of PCR water and 2XqPCR Mix added in S2 was 8 μL and 12.5 μL, the amount of wild-type primer probe added was 2.5 μL, and the amount of mutant primer probe added was 2.5 μL. The amplification reaction conditions for fluorescent PCR detection in S3 were: pre-denaturation at 95-97°C for 5 min, denaturation at 95-97°C for 5 s, annealing at 57-62°C for 20-30 s, and 40 cycles.

[0031] A determination method for an extraction-free qPCR detection kit for HSD3B1 genotype detection includes validity determination and result interpretation, wherein the validity determination is that the Ct values ​​of a wild-type PCR reaction tube and a mutant-type PCR reaction tube in a sample detection are Undet or 40, the detection fails, no sample is added, and the detection is repeated, and the result is interpreted as mutant-type and wild-type ΔCt (2, +∞), and this sample is a wild pure type AA, mutant-type and wild-type ΔCt [-2, 2], and this sample is a heterozygous type AC, mutant-type and wild-type ΔCt (-∞, -2), and this sample is a mutant pure type CC.

[0032] The following is further introduced in conjunction with specific embodiments: Example 1

[0033] The probe used in the present invention is a TaqMan probe labeled with FAM at the 5' end, and oligonucleotides labeled with a fluorescent reporter group (R) and a fluorescent quencher group (Q) at both ends of the probe. When the probe is intact, that is, in a random state and a state without PCR product hybridization, the fluorescence emitted by the reporter group is absorbed by the quencher group. During the fluorescent PCR amplification process, when the specific PCR product and the TaqMan probe undergo hybridization reaction, the 5' end exonuclease activity of the Taq enzyme also cleaves the probe, and the fluorescence released by the reporter group can be detected by a fluorometer built into a quantitative detector. After each cycle of PCR, the fluorescent signal has a synchronous exponential growth process like the target fragment, and the strength of the fluorescent signal represents the number of copies of the template RNA. Therefore, the present invention can not only be used for simple qualitative detection, but also for quantitative detection of the specific content of the sample.

[0034] The present invention provides an extraction-free qPCR detection kit for detecting the genotype of the HSD3B1 gene (1245C) site, which can clarify the genotype of the HSD3B1 gene (1245C) site of a nucleic acid fragment in a sample.

[0035] The present invention provides an extraction-free qPCR detection kit for detecting the genotype of the HSD3B1 gene (1245C) locus, adopts the extraction-free qPCR technology as the detection method, directly uses the serum sample as the template for detection, simplifies the operation steps and shortens the PCR detection time.

[0036] The present invention provides an extraction-free qPCR detection kit for detecting the genotype of the HSD3B1 gene (1245C) site, wherein the template used for the fluorescent PCR amplification is a human serum sample collected clinically.

[0037] In order to achieve the purpose of the present invention, the following technical solutions are adopted: Provided is an extraction-free qPCR detection kit for HSD3B1 gene (1245C) site genotype detection, the kit includes PCR water, wild-type primer probe, mutant primer probe, 2XqPCR Mix and positive quality control; wherein 2XqPCR Mix includes: PCR buffer, dNTPs, MgCl2, high-efficiency anti-reversal DNA polymerase; wild-type primer probe includes HSD3B1 (A1245C) site primer probe, mutant primer probe includes HSD3B1 (A1245C) site primer probe. The reaction system is 25μL, including: 2XqPCR Mix 12.5μL, wild-type primer probe 2.5μL, PCR water 9μL, sample / positive quality control 2μL; The universal upstream primer sequence for genotype detection of the HSD3B1 gene (1245C) site is SEQ ID NO.1; the downstream wild-type primer sequence is SEQ ID NO.2; the downstream mutant-specific primer sequence is SEQ ID NO.3; the sequence of the TaqMan probe for genotype detection of the HSD3B1 gene (1245C) site is SEQ ID NO.4; The primer probes completely matched the sequence template of the specific conserved region of the HSD3B1 gene (1245C) locus (GeneBank accession numbers AC011445.6 and AY336714.1); The positive quality control products were plasmids corresponding to the wild-type and mutant forms of the aforementioned HSD3B1 gene (1245C) loci; The negative control product is the wild-type plasmid corresponding to the aforementioned HSD3B1 gene (1245C) site.

[0038] The primer sequences are listed below: SEQ ID NO.1: 5'-TCACCTTCTTCTTATAAGAAGGC-3' SEQ ID NO.2: 5'-TGAGTCTTGGACTTCAGAG-3' SEQ ID NO.3: 5'-TGAGTCTTGGACTTCAGCT-3' SEQ ID NO.4: 5'-FAM-TCCACAAGGGAACCAACCCA-BHQl-3' Example 2

[0039] The present invention also provides an extraction-free qPCR detection method for the HSD3B1 gene (1245C) locus genotype based on the aforementioned kit, the method specifically comprising the following steps: (1) Add 2 µL of sample (serum / plasma) and positive control to wild-type and mutant-type extraction-free fluorescent PCR reaction tubes, respectively. Add 12.5 µL of 2XqPCR Mix, 2.5 µL of wild-type primer probe, 9 µL of PCR water, and 2 µL of sample / positive control to the wild-type tube, and add 12.5 µL of 2XqPCR Mix, 2.5 µL of mutant primer probe, 9 µL of PCR water, and 2 µL of sample / positive control to the mutant tube. Prepare a 25 µL system, cover the tubes, and perform fluorescent PCR detection.

[0040] (2) The conditions of PCR amplification reaction are as follows: pre-denaturation at 95-97℃ for 5 min; pre-denaturation at 95-97℃ for 5 min; denaturation at 95-97℃ for 5 s; annealing at 57-62℃ (fluorescence collection point) for 20-30 s; 40 cycles.

[0041] (3) Validity determination: If the Ct value of the wild-type tube and mutant tube detected by the sample is Undet or 40, the test fails, no sample is added, and the test is repeated; and the Ct value of the wild-type tube and mutant tube detected by the positive quality control is ≤35, otherwise the experiment is considered invalid; (4) Interpretation of results: The mutant and wild type ΔCt (2, +∞), this sample is the wild pure type AA, the mutant and wild type ΔCt [-2, 2], this sample is the heterozygous type AC, the mutant and wild type ΔCt (-∞, -2), this sample is the mutant pure type CC. Example 3

[0042] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods.

[0043] Unless otherwise specified, the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources.

[0044] 1. Primer probe design and screening for specific detection: According to the genotype of the HSD3B1 gene (1245C) site, introduce one or two base mutations at the 3' end of the mutation-specific downstream primer to specifically amplify the mutant allele. Design and screen multiple pairs of primers and probes, and the primer length is generally 18-22bp.

[0045] The universal upstream primer sequence for genotype detection of the HSD3B1 gene (1245C) locus is shown in SEQ ID NO.1: 5'-TCACCTTCTCTTATAAGAAGGC-3' The downstream wild-type specific primer sequence for genotype detection of the HSD3B1 gene (1245C) site is shown in SEQ ID NO.2: 5'- TGAGTCTTGGACTTCAGAG-3' The downstream mutant-specific primer sequence for genotype detection of the HSD3B1 gene (1245C) site is shown in SEQ ID NO.3: 5'- TGAGTCTTGGACTTCAGCT-3' The sequence of the TaqMan probe for genotype detection of the HSD3B1 gene (1245C) site is shown in SEQ ID NO.4: 5'-FAM-TCCACAAGGGAACCAACCCA-BHQl-3' 2. Optimization of the reaction system: Serum from 30 normal subjects was collected and the genotype of the HSD3B1 gene (1245C) locus was detected by extraction-free qPCR.

[0046] (1) Optimization of primer concentration: When other conditions in the reaction system are the same, the primer concentration is serially diluted from 0.1uM to 1.6uM. By analyzing and comparing the test results, it is determined that the optimal primer concentration is 0.5uM.

[0047] (2) Optimization of probe concentration: When other conditions in the reaction system were the same, the probe concentration was serially diluted from 0.05 uM to 0.5 uM. By analyzing and comparing the test results, it was determined that the optimal probe concentration was 0.4 uM.

[0048] (3) Optimization of annealing temperature: When other conditions in the reaction system are the same, gradient PCR (54°C-62°C annealing temperature) is performed. By analyzing and comparing the experimental results, it is determined that the optimal annealing temperature is 60°C.

[0049] (4) Enzyme optimization: When other conditions in the reaction system are the same, various enzymes are added to the reaction system. By analyzing and comparing the experimental results, it is determined that the best enzyme is a highly efficient and stress-resistant DNA polymerase.

[0050] The reaction system was established using the above primers and probes. The extraction-free fluorescent PCR reaction system for genotype detection of the HSD3B1 gene (1245C) site was determined to be 25 μL. The reaction solution was configured according to the fluorescent quantitative PCR reagent, including: 2XqPCR Mix 12.5 μL, primer probe 2.5 μL, PCR water 9 μL, and sample / positive quality control product 2 μL; qPCR Mix components and their final concentrations are: The final concentration of PCR buffer is 1×; The final concentration of dNTPs was 0.2 mM; The final concentration of MgCl2 was 5 mM; The final concentration of high-efficiency DNA polymerase is 0.2U / µL 3. Analysis of test results: If the sample to be tested contains a mutation in the HSD3B1 gene (1245C) site, a positive amplification curve will be displayed when amplified with specific primers, and the detection sensitivity can reach 500 copies / mL; if there is no mutation in the HSD3B1 gene (1245C) site in the sample to be tested, a negative amplification curve will be displayed when amplified with mutation-specific primers, that is, the Ct value is ≥38, indicating that the above primers have good specificity and sensitivity for the probe. The total template amount of the sample is detected by the non-specific primer amplification curve. Samples with a total template amount ≤104 copies / mL are considered unqualified samples (discarded or concentrated and then analyzed), and samples with a total template amount ≥106 copies / mL are appropriately diluted and re-analyzed.

[0051] The detection sensitivity of the mutation-specific primers and probes provided by the present invention can reach 500 copies / mL, indicating that they have good sensitivity.

[0052] When the mutation-specific primers and probes provided by the present invention detect samples without HSD3B1 gene (1245C) site mutation, the Ct value of the amplification curve is ≥38, indicating that the specificity is strong.

[0053] Since the present invention simultaneously designs mutation non-specific primers for detecting the total template amount of the sample, false negative results are avoided and quality control is convenient.

[0054] The detection reagent provided by the present invention can be directly used for detection with samples, and the method is fast, the operation is simple, the pollution is small, and the result interpretation is objective.

[0055] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An extraction-free qPCR detection kit for HSD3B1 genotype detection, comprising PCR water, wild-type primer probe, mutant primer probe, 2XqPCR Mix and positive quality control product, characterized in that: The 2XqPCR Mix includes PCR buffer, dNTPs, MgCl2, and a high-efficiency anti-reverse DNA polymerase; the wild-type primer probe includes a HSD3B1 (A1245C) site primer probe, SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.4; and the mutant primer probe includes a HSD3B1 (A1245C) site primer probe.

2. The extraction-free qPCR detection kit for HSD3B1 genotype detection according to claim 1, characterized in that: The contents of PCR buffer, dNTPs, MgCl2, and high-efficiency anti-reverse DNA polymerase in the 2XqPCR Mix are 2.5 μL, 2 μL, 3 μL, and 1 μL, respectively, and are supplemented to 12.5 μL with water. The dNTPs include each with a concentration of 10 mmol / L, and the concentration of MgCl2 is 25 mM.

3. The extraction-free qPCR detection kit for HSD3B1 genotype detection according to claim 1, characterized in that: The contents of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.4 in the wild-type primer probe are 0.2-0.8 μL, 0.2-0.8 μL and 0.2-0.4 μL, respectively, and the concentrations are all 10 μmol / L. The HSD3B1 (A1245C) site primer probe includes SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.4, and the contents are 0.2-0.8 μL, 0.2-0.8 μL and 0.2-0.4 μL, respectively, and the concentrations are all 10 μmol / L.

4. A primer for detecting human HSD3B1 (A1245C) site variation, characterized in that: The common forward primer sequence for detecting the HSD3B1 (A1245C) site is SEQ ID NO.1, the reverse wild-type primer sequence is SEQ ID NO.2, and the reverse mutant-specific primer sequence is SEQ ID NO.

3.

5. A probe for detecting human HSD3B1 (A1245C) site variation, characterized in that: The probe sequence for detecting the HSD3B1 (A1245C) site is SEQ ID NO.

4.

6. An extraction-free qPCR detection method for HSD3B1 genotype detection, characterized in that: The extraction-free qPCR detection kit for HSD3B1 genotype detection as described in any one of claims 1 to 3 comprises the following steps: using human serum or plasma samples, without going through a nucleic acid extraction process, directly adding them to a reaction system as a PCR template and adding them to a fluorescent PCR detection reaction system for detection.

7. The extraction-free qPCR detection method for HSD3B1 genotype detection according to claim 6, characterized in that: The nucleic acid extraction process refers to the process of extracting and purifying nucleic acid from a sample outside the qPCR detection reaction system. The sample processing process and the qPCR detection reaction are integrated into the same reaction system. The sample used as the amplification template for qPCR includes a human serum or plasma sample.

8. The extraction-free qPCR detection method for HSD3B1 genotype detection according to claim 6, characterized in that: The specific steps include: S1. Add 2 µL of serum, plasma sample or positive control into wild-type and mutant PCR reaction tubes respectively; S2. Add PCR water, 2XqPCR Mix, and wild-type primer probe to the wild-type PCR reaction tube, and add PCR water, 2XqPCR Mix, and mutant primer probe to the mutant PCR reaction tube, with a total system of 25 μL; S3. Cover the tube cap and perform fluorescent PCR detection.

9. The extraction-free qPCR detection method for HSD3B1 genotype detection according to claim 8, characterized in that: The amount of PCR water and 2XqPCR Mix added in S2 is 8 μL and 12.5 μL, the amount of the wild-type primer probe added is 2.5 μL, and the amount of the mutant primer probe added is 2.5 μL. The amplification reaction conditions of the fluorescent PCR detection in S3 are: pre-denaturation at 95-97°C for 5 min, denaturation at 95-97°C for 5 s, annealing at 57-62°C for 20-30 s, and 40 cycles.

10. A determination method for an extraction-free qPCR detection kit for HSD3B1 genotype detection, characterized in that: The extraction-free qPCR detection method for HSD3B1 genotype detection as described in claim 6 includes validity judgment and result interpretation, the validity judgment is that the Ct value of the wild-type PCR reaction tube and the mutant PCR reaction tube of the sample detection is Undet or 40, the detection fails, no sample is added, and the test is re-tested, and the result interpretation is mutant and wild-type ΔCt (2, +∞), this sample is wild pure type AA, mutant and wild type ΔCt [-2, 2], this sample is heterozygous type AC, mutant and wild type ΔCt (-∞, -2), this sample is mutant pure type CC.

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