CN110656167A - Primer group and kit for detecting microdeletion of Y chromosome and detection method based on digital PCR - Google Patents

Abstract

The invention discloses a primer set, a kit and a detection method based on digital PCR for Y chromosome microdeletion detection. The present invention designs primer sets for the sY84 and sY86 sites of the AZFa subregion, the sY127 and sY134 sites of the AZFb subregion, and the sY254 and sY255 sites of the AZFc subregion, and has the advantages of high sensitivity, accuracy and objectivity. The detection method provided by the present invention can detect the Y chromosome microdeletion of azoospermia patients with high speed, high sensitivity and high accuracy, and can effectively reduce the requirement on the sample volume, so that the peripheral blood collection can be further reduced to fingertip blood , reduce the pain of the patient, and can avoid false negative results when the patient's DNA samples are few; the invention adopts the digital PCR method for inspection, and the detection results can be obtained independently of the Ct value, and can directly reflect the original copy of the DNA sample number, thereby reducing the workload of the inspector.

Description

Primer sets, kits and digital PCR-based assays for Y chromosome microdeletion detection Detection method

technical field

The invention relates to the technical field of biological detection, in particular to a primer set, a kit and a detection method based on digital PCR for Y chromosome microdeletion detection.

Background technique

Infertility is an important reproductive health problem in the world. About 15% of couples will encounter difficulties in fertility or infertility, while 30% to 50% of infertility is caused by male infertility or oligospermia. At present, an important cause of male infertility is abnormal chromosomal structure and microdeletion of chromosomal genes. Clinical results show that about 10% to 15% of patients with primary azoospermia and oligospermia have Y chromosome microdeletions, and this symptom can be passed on to the next generation with assisted reproductive technology. Therefore, the detection of Y chromosome microdeletion is of great significance for the clinical treatment of male reproductive genetics, improving the efficacy and safety of assisted reproductive technology, and carrying out preimplantation genetic diagnosis.

The traditional detection method of Y chromosome microdeletion is mainly carried out on the basis of PCR technology. Domestic clinical laboratories mostly collect peripheral blood samples for multiplex PCR gel electrophoresis and fluorescence quantification according to the protocols proposed by the European Academy of Andrology (EAA) and the European Molecular Genetics Quality Network (EMQN). (q) PCR and capillary electrophoresis were used for detection. However, these techniques take a long time, the result determination is highly subjective, and the sensitivity is low, which does not conform to the current development direction of faster, more sensitive, and less sample requirements for clinical testing techniques.

Digital PCR is a new nucleic acid quantification technology developed in the early 20th century. Compared with traditional PCR, it can perform absolute quantitative analysis of trace templates, and has higher accuracy, specificity, sensitivity and detection speed. At the same time, the digital PCR detection and analysis process has a relatively high degree of automation, which reduces the error of manual result judgment, and also reduces the misjudgment of the results due to the low copy number detection caused by the fluorescence quantitative PCR detection. Therefore, the use of digital PCR for Y chromosome microdeletion detection is a research direction, but there are related solutions in the existing technology.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a primer set, a kit and a detection method based on digital PCR for Y chromosome microdeletion detection in view of the above-mentioned deficiencies in the prior art.

In order to solve the above-mentioned technical problems, the technical scheme adopted in the present invention is: a primer set for Y chromosome microdeletion detection is provided, including the following primer sets:

The primer pair used to detect the SY84 site, the sequence is:

Upstream primer AGAAGCGTCTGATAGCAGGT, downstream primer GCCTACTACCTGGAGGCTTC;

The primer pair used to detect the SY86 site, the sequence is:

upstream primer GTGACACACAGACTATGCTTC, downstream primer ACACACAGAGTGACAACACT;

The primer pair used to detect the SY127 site, the sequence is:

Upstream primer GGCTCACAAACGACGAGAAA, downstream primer CTGCAGGCAGTAATAAGTGA;

The primer pair used to detect the SY134 site, the sequence is:

Upstream primer GTTCTGCCTCACCATATCACG, downstream primer ACCACTGCCAAAACTGTCAA;

The primer pair used to detect the SY254 site, the sequence is:

Upstream primer TGGTGTTACCAGAAGGCAAA, downstream primer GAACCGTATCTACCATAGCAGC;

The primer pair used to detect the SY255 locus, the sequence is:

Upstream primer GTTACAGGATTCGGCGTGAT, downstream primer CTCGTCATGTGCAGCGAC.

Another object of the present invention is to provide a kit for Y chromosome microdeletion detection, which includes the primer set as described above.

Preferably, the kit further includes: PCR master mix, positive control, negative control and droplet generation oil.

Preferably, the PCR master mix includes dye, Taq enzyme, dNTP, MgCl ₂ .

Preferably, the reaction concentration of the Taq enzyme is 6 U, the reaction concentration of dNTP is 0.8 mM, and the final concentration of MgCl ₂ is 5.6 mM.

Preferably, the positive control is: blood cell DNA obtained from the peripheral blood of a healthy adult male, the concentration is: 10-80 ng/ul, and the negative control is sterile deionized water.

The present invention also provides a digital PCR-based detection method for Y chromosome microdeletion detection, which utilizes the above-mentioned kit to perform Y chromosome microdeletion detection by means of digital PCR.

Preferably, the method of the present invention comprises the following steps:

1) Collect the blood of the patient to be tested, perform DNA extraction, and obtain the DNA sample to be tested;

2) Evenly mix the obtained DNA sample with PCR master mix, primer set and sterile water;

3) the mixed solution obtained in step 2) generates droplets;

4) placing the generated droplets in a digital PCR amplifier for amplification to obtain PCR products;

5) Use a droplet reader to detect and analyze the obtained PCR products.

Preferably, in the step 1), the concentration of the DNA sample to be tested is greater than or equal to 0.0001ng/ul.

Preferably, in the step 4), the PCR amplification procedure is: the enzyme is activated at 95°C for 5 minutes; then at 95°C for 30 seconds, at 60°C for 1 minute, and performing 40 cycles; at 4°C for 5 minutes ; 90°C for 5 minutes.

The beneficial effects of the present invention are:

The invention can quickly, highly sensitively and accurately detect the Y chromosome microdeletion of azoospermic patients, and can effectively reduce the requirement for sample volume, so that the collection of peripheral blood can be further reduced to fingertip blood. Pain for the patient and avoid false negative results when the patient's DNA sample is small;

The invention adopts the digital PCR method for inspection, and the detection result can be obtained independently of the Ct value, and can directly reflect the original copy number of the DNA sample, thereby reducing the workload of the inspector;

The kit obtained by the invention can be stably stored at -20° C. for more than half a year, and has good product development potential.

Description of drawings

1 is a schematic diagram of the detection results of peripheral blood samples of AZFb-deficient patients in Example 2 of the present invention;

FIG. 2 is a schematic diagram of the results of fluorescence quantitative PCR detection of AZFb deletion samples in Example 2 of the present invention;

Figures 3a, 3b, and 3c are respectively the result diagrams of samples with a concentration of 10ng/ul, 1ng/ul, and 0.1ng/ul detected by the fluorescence quantitative PCR method in Example 3 of the present invention;

Figures 4a, 4b, and 4c are the results of detecting samples with concentrations of 0.001 ng/ul, 0.0001 ng/ul, and 0.00001 ng/ul in sequence by using the digital PCR-based detection method in Example 3 of the present invention.

Detailed ways

The present invention will be further described in detail below with reference to the embodiments, so that those skilled in the art can implement according to the description.

It should be understood that terms such as "having", "comprising" and "including" as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

Example 1

A primer set for Y chromosome microdeletion detection was designed to detect gene deletions at sites sY84 and sY86 in the AZFa subregion, sY127 and sY134 in the AZFb subregion, and sY254 and sY255 in the AZFc subregion. The following primer combinations:

The primer pair used to detect the SY84 site, the sequence is:

Upstream primer AGAAGCGTCTGATAGCAGGT, downstream primer GCCTACTACCTGGAGGCTTC;

The primer pair used to detect the SY86 site, the sequence is:

upstream primer GTGACACACAGACTATGCTTC, downstream primer ACACACAGAGTGACAACACT;

The primer pair used to detect the SY127 site, the sequence is:

Upstream primer GGCTCACAAACGACGAGAAA, downstream primer CTGCAGGCAGTAATAAGTGA;

The primer pair used to detect the SY134 site, the sequence is:

Upstream primer GTTCTGCCTCACCATATCACG, downstream primer ACCACTGCCAAAACTGTCAA;

The primer pair used to detect the SY254 site, the sequence is:

Upstream primer TGGTGTTACCAGAAGGCAAA, downstream primer GAACCGTATCTACCATAGCAGC;

The primer pair used to detect the SY255 locus, the sequence is:

Upstream primer GTTACAGGATTCGGCGTGAT, downstream primer CTCGTCATGTGCAGCGAC.

The above primer combination has strong complementary pairing properties with the 6 sites of the Y chromosome microdeletion, ensuring the accuracy of the target site.

Example 2

A kit for Y chromosome microdeletion detection was constructed, the kit including the primer set described in Example 1. In a further preferred embodiment, the kit further includes: PCR master mix, positive control, negative control and droplet generation oil.

Wherein, the PCR premix solution includes dye, Taq enzyme, dNTP, MgCl ₂ . More specifically, the dyes are: EvaGreen dye 2x (20x EvaGreen dye diluted 10 times) 0, the reaction concentration of Taq enzyme is 6U, the reaction concentration of dNTP is 0.8mM, and the final concentration of _MgCl2 is 5.6mM.

Wherein, the positive control is blood cell DNA obtained from the peripheral blood of healthy adult males, and the concentration is 10-80 ng/ul, and the negative control is sterile deionized water (without DNA and RNA).

The droplet generation oil, QX200 Droplet Generation Oil for EvaGreen, was purchased from Bole Corporation, USA.

Example 3

A digital PCR-based detection method for Y chromosome microdeletion detection is provided. The method utilizes the kit obtained in Example 2 to perform Y chromosome microdeletion detection by means of digital PCR. It specifically includes the following steps:

1) Collect peripheral blood or fingertip blood of the patient to be tested, the volume range: 50μl-500μl, DNA extraction can be carried out using a universal nucleic acid column extraction kit (Shanghai Sangong, B518253-0050), and the obtained DNA is subjected to ultraviolet ( 260nm) absorption detection to obtain concentration and purity, to obtain DNA samples to be tested, the concentration of which is ≥ 0.0001ng/ul;

2) Evenly mix the obtained DNA sample with PCR premix, primer set and sterile water, and the volumes are respectively 10ul PCR premix, 0.4ul primer set (10uM), 2ul DNA extract, 7.2ul sterile water, Vortex to mix and keep at 4°C until use;

3) The mixed solution obtained in step 2) generates droplets, specifically:

Put the droplet generation card into the holder, and add 20ul reaction system to each hole in the middle row of pores; add 70ul of microdroplet generation oil to each hole in the bottom row of the generation card; after installing the gasket on the card holder, put it in Droplet generator; after the droplet generation is completed, slowly suck out the droplets from the top row of holes and transfer them to the eight-connected tube; as shown in the figure, it is the droplet generation card, and the droplet generation oil is added to the bottom row and mixed. The uniform liquid to be tested is added to the middle row. After the droplet generator is generated, the generated droplets are in the top row, and the generated droplets are gently transferred to the eight-connected tube for subsequent operations;

4) Place the generated droplets in a digital PCR amplifier for amplification. The PCR amplification procedure is as follows: the enzyme is activated at 95°C and kept for 5 minutes; then kept at 95°C for 30 seconds, 60°C for 1 minute, and performed for 40 minutes. cycle; the signal is stable at 4°C for 5 minutes; at 90°C for 5 minutes; PCR products are obtained;

5) Use the droplet reader to detect and analyze the obtained PCR products: remove the cap of the eight-tube tube, put it into the droplet reader, fasten the fixed lock, and close the hatch; set the experimental parameters, sample name and target Name, Supermix chooses EvaGreen; run the program to detect. The test results are shown in Figures 1 and 2. Figure 1 shows the detection results of peripheral blood samples from patients with AZFb deletion, where the concentration of the DNA samples to be tested is 0.0001 ng/ul. Figure 2 shows the results of fluorescence quantitative PCR detection of AZFb-deficient samples, and AZFb in the figure is two straight lines.

Compared with the conventional fluorescent quantitative PCR, the detection method of the present invention can directly reflect the original copy number of the DNA sample, thereby reducing the workload of the detector, and has lower detection limit and higher sensitivity. In this example, a comparison between the conventional fluorescent quantitative PCR and the digital PCR-based detection method of the present invention is carried out. Wherein, both methods use the kit obtained in Example 2. The DNA samples to be tested are all the same, and are blood cell DNA obtained from the peripheral blood of healthy adult males, that is, there is no deletion of the Y chromosome.

Referring to Figures 3a, 3b, and 3c, the fluorescence quantitative PCR method is used, and the concentration of the DNA sample to be tested is 10ng/ul, 1ng/ul, 0.1ng/ul in turn, and then according to the current level of qPCR (fluorescence quantitative PCR) testing , basically if the target gene does not successfully take off before Ct32, it can be seen from the figure that when the concentration is 10ng/ul and 1ng/ul, Ct32 successfully takes off before, and the detection can be realized. When the concentration is 0.1ng/ul, the Without success, the detection could not be achieved, so in this example, the detection limit of qPCR was 0.1 ng/ul.

Referring to Figures 4a, 4b, and 4c, the invented detection method based on digital PCR is adopted, and the concentration of the DNA sample to be tested is 0.001ng/ul, 0.0001ng/ul, 0.00001ng/ul in sequence. It can be seen from the figure that 0.0001 The results can still be detected at 0.00001ng/ul, and the AZFb site can not be detected at 0.00001ng/ul, so the detection limit of this method can reach 0.0001ng/ul, which is much lower than 0.1ng/ul of qPCR, which can highlight the Advantages of the high sensitivity of the inventive detection method.

Although the embodiment of the present invention has been disclosed as above, it is not limited to the application listed in the description and the embodiment, and it can be applied to various fields suitable for the present invention. For those skilled in the art, it can be easily Therefore, the invention is not limited to the specific details without departing from the general concept defined by the appended claims and the scope of equivalents.

Invention Title: Primer set, kit and digital PCR-based detection method for y chromosome microdeletion detection

The primer pair used to detect the SY84 site, the sequence is:

Upstream primer AGAAGCGTCTGATAGCAGGT, downstream primer GCCTACTACCTGGAGGCTTC.

The primer pair used to detect the SY86 site, the sequence is:

Upstream primer GTGACACACAGACTATGCTTC, downstream primer AACACACAGAGTGACAACACT.

The primer pair used to detect the SY127 site, the sequence is:

Upstream primer GGCTCACAAACGACGAGAAA, downstream primer CTGCAGGCAGTAATAAGTGA.

The primer pair used to detect the SY134 site, the sequence is:

Upstream primer GTCTGCCTCACCATATCACG, downstream primer ACCACTGCCAAAACTGTCAA.

The primer pair used to detect the SY254 site, the sequence is:

Upstream primer TGGTGTTACCAGAAGGCAAA, downstream primer GAACCGTATCTACCATAGCAGC.

The primer pair used to detect the SY255 locus, the sequence is:

Upstream primer GTTACAGGATTCGGCGTGAT, downstream primer CTCGTCATGTGCAGCGAC.

Claims (10)

1. A primer group for detecting microdeletion of Y chromosome, which comprises the following primer groups:

the primer pair for detecting the SY84 site has the sequence as follows:

an upstream primer AGAAGCGTCTGATAGCAGGT, a downstream primer GCCTACTACCTGGAGGCTTC;

the primer pair for detecting the SY86 site has the sequence as follows:

an upstream primer GTGACACACAGACTATGCTTC, a downstream primer ACACACAGAGTGACAACACT;

the primer pair for detecting the SY127 site has the sequence as follows:

an upstream primer GGCTCACAAACGACGAGAAA, a downstream primer CTGCAGGCAGTAATAAGTGA;

the primer pair for detecting the SY134 site has the sequence as follows:

an upstream primer GTCTGCCTCACCATATCACG, a downstream primer ACCACTGCCAAAACTGTCAA;

the primer pair for detecting the SY254 site has the sequence as follows:

an upstream primer TGGTGTTACCAGAAGGCAAA, a downstream primer GAACCGTATCTACCATAGCAGC;

the primer pair for detecting the SY255 site has the sequence:

an upstream primer GTTACAGGATTCGGCGTGAT and a downstream primer CTCGTCATGTGCAGCGAC.

2. A kit for detecting microdeletion of Y chromosome, comprising the primer set according to claim 1.

3. The kit for detecting microdeletion of the Y chromosome of claim 2, wherein the kit further comprises: PCR premix, positive control, negative control, and microdroplet generation oil.

4. The kit for detecting microdeletion of Y chromosome of claim 3, wherein the PCR pre-mix solution comprises dye, Taq enzyme, dNTP, MgCl₂。

5. The kit for detecting microdeletion of Y chromosome of claim 4, wherein the reaction concentration of Taq enzyme is 6U, the reaction concentration of dNTP is 0.8mM, and MgCl is used as the reagent₂The final concentration was 5.6 mM.

6. The kit for detecting microdeletion of the Y chromosome of claim 3, wherein the positive control is: blood cell DNA obtained from peripheral blood of healthy adult males at concentrations of: 10-80ng/ul, and the negative control is sterile deionized water.

7. A digital PCR-based detection method for the detection of microdeletion of the Y chromosome, which is characterized by performing microdeletion of the Y chromosome by a digital PCR method using the kit according to any one of claims 2 to 6.

8. The digital PCR-based detection method for Y chromosome microdeletion detection according to claim 7, comprising the steps of:

1) collecting blood of a patient to be detected, and performing DNA extraction to obtain a DNA sample to be detected;

2) uniformly mixing the obtained DNA sample with the PCR premix solution, the primer group and sterile water;

3) generating microdroplets from the mixed solution obtained in the step 2);

4) putting the generated microdroplets into a digital PCR amplification instrument for amplification to obtain a PCR product;

5) the obtained PCR product was subjected to detection analysis using a droplet reader.

9. The digital PCR-based detection method for Y chromosome microdeletion detection according to claim 8, wherein in the step 1), the concentration of the DNA sample to be detected is not less than 0.0001 ng/ul.

10. The digital PCR-based detection method for Y chromosome microdeletion detection according to claim 8, wherein in the step 4), the PCR amplification procedure is: activating enzyme at 95 deg.C for 5 min; then keeping the temperature at 95 ℃ for 30 seconds and 60 ℃ for 1 minute, and performing 40 cycles; holding at 4 deg.C for 5 min; the temperature was maintained at 90 ℃ for 5 minutes.

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