Human DEFB126 gene mutation detection kit and application thereof

Abstract

The invention discloses a human DEFB126 gene mutation detection kit and application thereof. The human DEFB126 gene mutation detection kit comprises PCR reaction liquid, and the PCR reaction liquid contains two pairs of specific primers and fluorescent probes designed for DEFB126 genes. According to the human DEFB126 gene mutation detection kit and the application thereof, a PCR-molecular beacon method is adopted, and the human DEFB126 gene mutation detection kit has the advantages of being high in sensitivity and specificity, easy and convenient to operate and the like; the kit provides an effective auxiliary means for clinical diagnosis of male infertility, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to a kit for detecting human DEFB126 gene mutations and its application. Background Art

[0002] Male infertility is an important problem affecting fertility worldwide, and disorders in spermatogenesis, maturation, and transportation are one of the main causes. β-defensin 126 (DEFB126) protein is encoded by the DEFB126 gene, and this protein forms a protective layer on the surface of sperm, helping sperm penetrate the cervical mucus of females. Research shows that frameshift mutations in the DEFB126 gene can lead to abnormal expression of this protein, thereby affecting sperm function and causing infertility.

[0003] Currently, gene detection technology is increasingly widely used in reproductive medicine, but specific detection kits for DEFB126 gene mutations are still limited. Existing detection methods have problems such as insufficient sensitivity and complex operation, and there is an urgent need for an efficient and simple detection method. Summary of the Invention

[0004] The purpose of the present invention is to provide a kit for detecting human DEFB126 gene mutations and its application, which uses the PCR-molecular beacon method for in vitro qualitative detection of frameshift mutations in the DEFB126 gene.

[0005] To achieve the above purpose, the present invention provides a kit for detecting human DEFB126 gene mutations, which includes a PCR reaction solution, Taq DNA polymerase, a positive control, and a negative control. The PCR reaction solution includes Tris-HCl, MgCl2, dNTPs, primers, and a fluorescent probe. The primers are two primer pairs designed for the DEFB126 gene. Among them,

[0006] Primer pair 1 is: DEFF1, the nucleotide sequence is as shown in SEQ ID NO.1; DEFR1, the nucleotide sequence is as shown in SEQ ID NO.2;

[0007] The fluorescent probe sequence of primer pair 1 is: DEFP1, the nucleotide sequence is as shown in SEQ ID NO.3;

[0008] Primer pair 2 is: DEFF2, the nucleotide sequence is as shown in SEQ ID NO.4; DEFR2, the nucleotide sequence is as shown in SEQ ID NO.5;

[0009] The fluorescent probe sequence of primer pair 2 is: DEFP2, the nucleotide sequence is as shown in SEQ ID NO.6.

[0010] Preferably, the PCR-molecular beacon method is used for detection.

[0011] Preferably, the detection result is determined by melting curve analysis.

[0012] On the other hand, the present invention provides an application of a kit for detecting human DEFB126 gene mutations. The kit for detecting human DEFB126 gene mutations is used for in vitro qualitative detection of frameshift mutations of the human DEFB126 gene.

[0013] Therefore, a kit for detecting human DEFB126 gene mutations and its application according to the present invention have the following beneficial effects:

[0014] (1) The kit of the present invention uses real-time fluorescence quantitative PCR technology based on molecular beacons, combined with asymmetric PCR and melting curve analysis technology to qualitatively detect frameshift mutations of the DEFB126 gene in DNA samples;

[0015] (2) The kit of the present invention uses specific primer pairs to perform highly accurate PCR amplification on the target sequence, and discriminates SNP typing by analyzing and comparing the difference in complementary pairing force between the beacon and the target sequence bases to determine the result;

[0016] (3) The kit for detecting human DEFB126 gene mutations provided by the present invention adopts the PCR-molecular beacon method, and has the characteristics of high sensitivity, strong specificity and simple operation;

[0017] (4) The kit of the present invention provides an effective auxiliary means for clinical diagnosis of male infertility and has broad application prospects.

[0018] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0020] Figure 1 It is the detection map of the real-time fluorescence quantitative PCR instrument in Test Example 1 of the present invention;

[0021] Figure 2 It is the detection map of the real-time fluorescence quantitative PCR instrument in Test Example 2 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and examples.

[0023] In order to make the objectives, technical solutions, and advantages of the present application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all descriptions of embodiments, aiming to provide further detailed explanations for the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs.

[0024] The instrument and equipment, reagent materials used in the embodiments are all obtained through commercial channels.

[0025] Example 1

[0026] A kit for detecting human DEFB126 gene mutations, and its components are shown in Table 1 below:

[0027] Table 1 Composition of the kit

[0028]

[0029] The sequences of the primers and fluorescent probes are shown in Table 2 below:

[0030] Table 2 Sequences of primers and fluorescent probes

[0031]

[0032] The kit for detecting human DEFB126 gene mutations adopts real-time fluorescence quantitative PCR technology based on molecular beacons, combined with asymmetric PCR and melting curve analysis technology. The specific principle is as follows:

[0033] (1) Molecular beacon design: Design molecular beacons for the genomic sequence containing SNP sites, and its structure includes a "stem region" and a "loop region", as well as fluorescent groups and quenching groups at both ends.

[0034] (2) Fluorescence quenching: When the molecular beacon is in a free state, the two ends of the hairpin structure bring the fluorescent molecule and the quenching molecule close, and the fluorescence is quenched.

[0035] (3) Fluorescence emission: When the molecular beacon is completely complementary to the target sequence and binds, the complementary region of the beacon stem is pulled apart, the distance between the fluorescent molecule and the quenching molecule increases, and the fluorescence is released and detected.

[0036] Example 2

[0037] An application of a kit for detecting human DEFB126 gene mutations, including the following steps:

[0038] S1. Sample processing: Extract DNA from human peripheral blood using a commercial nucleic acid extraction reagent, and the extraction product can be stored at -20°C for more than 6 months.

[0039] S2. Reagent Preparation:

[0040] Take out the PCR reaction solution and Taq DNA polymerase from the human DEFB126 gene mutation detection kit. After the reaction solution is melted at room temperature and gently shaken and mixed evenly, centrifuge at 2000 rpm for 10 seconds. Calculate the required number of test reactions n for the reaction reagents. The reaction system for each test is configured as shown in Table 3 below:

[0041] Table 3 Reaction System Configuration

[0042] Component Reaction tube PCR reaction solution 17 μL Taq DNA polymerase 1 μL Total volume 18 μL

[0043] The calculation formula for the required number of test reactions n for the reaction reagents is:

[0044] n = number of samples + number of negative controls + number of positive controls.

[0045] Calculate the dosage of each reagent in the reaction system according to n tests, add it to a centrifuge tube with an appropriate volume and mix evenly. Aliquot 18 μL into a PCR thin-walled octuplet tube, and then transfer it to the sample processing area for subsequent sample addition processing.

[0046] S3. Sample Addition:

[0047] The total volume of a single reaction is 20 μL. Therefore, add 2 μL of negative control, 2 μL of positive control, and 2 μL of each sample in sequence to the PCR thin-walled tube containing 18 μL of reaction solution in step S2. Tighten the cap of the octuplet tube, then gently mix the octuplet tube and centrifuge instantaneously, and finally transfer it to the PCR detection area.

[0048] S4. PCR Amplification and Fluorescence Detection:

[0049] Put each reaction tube into a fluorescence quantitative PCR instrument in turn for PCR amplification. The amplification program is as shown in Table 4 below:

[0050] Table 4 PCR Reaction Program

[0051]

[0052] The reaction volume is 20 μL. For fluorescence detection, select the FAM channel and HEX channel, select none for the quenching group, and select none for the reference fluorescence (no quenching group and reference fluorescence).

[0053] Quality Control Requirements:

[0054] (1) Threshold Setting: Set the threshold line just above the highest point of the normal negative control. The automatic analysis mode or manual analysis mode can be adjusted according to the situation.

[0055] (2) Negative control: There should be no obvious melting peak corresponding to Tm. If a melting peak appears, it may be due to reagent contamination or operation contamination. After excluding the pollution source, re-detection is required.

[0056] (3) Result determination: If the above requirements are met simultaneously, it indicates that the test is successful and the test samples can be interpreted; otherwise, it is regarded as invalid and the errors in aspects such as the instrument, reagents, and amplification conditions need to be checked.

[0057] S5. Result analysis

[0058] Melting curve analysis: By analyzing the melting curve, the Tm values of different genotypes can be distinguished, thereby determining the genotype of the sample.

[0059] The target genes corresponding to each reaction tube and the Tm values of the melting peaks are shown in Table 5 below. According to the Tm value ranges shown in the following table, the corresponding genotype interpretation is carried out:

[0060] Table 5 Target gene and Tm value ranges of the melting peaks of the positive control

[0061]

[0062] Note: " / " indicates no obvious melting peak. If the detected peak is at the critical value between the high peak and the low peak, re-detection is required.

[0063] Test Example 1

[0064] The application of a kit for detecting human DEFB126 gene mutations is the same as that in Example 2. The detection map using the Hongshi SLAN-96P real-time fluorescence quantitative PCR instrument is as Figure 1 shown, where the blue curve is the FAM channel and the green curve is the HEX channel.

[0065] FAM channel: The temperatures at which melting peaks appear for DEFB126 (delCC) are 64.44 °C and 72.37 °C. According to the interpretation rules, the result should be the CC / -- type (heterozygous mutation).

[0066] HEX channel: The temperature at which a melting peak appears for DEFB126 (delCAAA) is 64.88 °C. According to the interpretation rules, the result should be the CAAA type (wild type).

[0067] Test Example 2

[0068] The application of a kit for detecting human DEFB126 gene mutations is the same as that in Example 2. The detection map using the Hongshi SLAN-96P real-time fluorescence quantitative PCR instrument is as Figure 2 shown, where the blue curve is the FAM channel and the green curve is the HEX channel.

[0069] FAM channel: The melting peak temperatures of DEFB126 (delCC) are 64.38 °C and 72.33 °C. According to the interpretation rules, the result should be of the CC / -- type (heterozygous mutation).

[0070] HEX channel: The melting peak temperatures of DEFB126 (delCAAA) are 47.71 °C and 64.85 °C. According to the interpretation rules, the result should be of the CAAA / ---- type (heterozygous mutation).

[0071] Therefore, the human DEFB126 gene mutation detection kit of the present invention adopts the PCR-molecular beacon method, and has the characteristics of high sensitivity, strong specificity, and simple operation; this kit provides an effective auxiliary means for the clinical diagnosis of male infertility and has broad application prospects.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A kit for detecting human DEFB126 gene mutations, comprising a PCR reaction solution, Taq DNA polymerase, a positive control, and a negative control, characterized in that, The PCR reaction solution contains 2 pairs of specific primers and a fluorescent probe designed for the DEFB126 gene.

2. The human DEFB126 gene mutation detection kit according to claim 1, wherein, Primer pair 1 is: DEFF1, the nucleotide sequence is shown in SEQ ID NO.1; DEFR1, the nucleotide sequence is shown in SEQ ID NO.2; The fluorescent probe sequence of primer pair 1 is: DEFP1, the nucleotide sequence is shown in SEQ ID NO.3; Primer pair 2 is: DEFF2, the nucleotide sequence is shown in SEQ ID NO.4; DEFR2, the nucleotide sequence is shown in SEQ ID NO.5; The fluorescent probe sequence of primer pair 2 is: DEFP2, the nucleotide sequence is shown in SEQ ID NO.

6.

3. The human DEFB126 gene mutation detection kit according to claim 1, characterized in that: The PCR-molecular beacon method is used for detection.

4. A kit for detecting human DEFB126 gene mutations according to claim 1, characterized in that: The test results are determined by melting curve analysis.

5. Use of a kit for detecting human DEFB126 gene mutations according to any one of claims 1 to 4, characterized in that: The human DEFB126 gene mutation detection kit is used for in vitro qualitative detection of frameshift mutations of the human DEFB126 gene.

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