阅读说明：本技术 用于y染色体微缺失检测的引物组、试剂盒及基于数字pcr的检测方法 (Primer group and kit for detecting microdeletion of Y chromosome and detection method based on digital PCR ) 是由 殷建 尹焕才 袁通阔 刘荻 于 2019-09-27 设计创作，主要内容包括：本发明公开了一种用于Y染色体微缺失检测的引物组、试剂盒及基于数字PCR的检测方法。本发明针对AZFa亚区sY84和sY86位点,AZFb亚区sY127和sY134位点,AZFc亚区sY254和sY255位点设计了引物组,且其具有高灵敏度、准确客观的优点。本发明提供的检测方法可快速、高灵敏且高准确度地检测无精子症病人的Y染色体微缺失情况,并可有效降低对样本量的要求,从而可将外周血采集进一步降低至指尖血,减小病人痛苦,且可避免病人DNA样本较少时造成的假阴性结果；本发明采用数字PCR方法进行检查,检测结果不依赖于Ct值即可获得,并可直观反映DNA样本的原始拷贝数,从而减少了检测者的工作量。(The invention discloses a primer group and a kit for detecting microdeletion of a Y chromosome and a detection method based on digital PCR. The invention designs a primer group aiming at the sites sY84 and sY86 of the AZFa subregion, the sites sY127 and sY134 of the AZFb subregion and the sites sY254 and sY255 of the AZFc subregion, and has the advantages of high sensitivity, accuracy and objectivity. The detection method provided by the invention can quickly, highly sensitively and accurately detect the Y chromosome microdeletion condition of the azoospermia patient, and can effectively reduce the requirement on the sample volume, thereby further reducing the peripheral blood collection to the fingertip blood, reducing the pain of the patient, and avoiding the false negative result caused by less DNA samples of the patient; the invention adopts a digital PCR method for inspection, the detection result can be obtained without depending on the Ct value, and the original copy number of the DNA sample can be visually reflected, thereby reducing the workload of a detector.)

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.

Technical Field

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

Background

Infertility is a globally important reproductive health problem, and about 15% of couples suffer from difficulty in or inability to breed, while 30% to 50% of infertility are caused by male aspermia or oligospermia. Currently, one important cause of male infertility is chromosomal structural abnormalities and microdeletions of chromosomal genes. Clinical results indicate that about 10% to 15% of patients with primary azoospermia and oligospermia have Y chromosome microdeletion, and the symptom can be transmitted to the next generation along with assisted reproductive technology. Therefore, the Y chromosome microdeletion detection has important significance for clinical treatment of male reproductive genetics, improvement of the curative effect and safety of an assisted reproductive technology and development of genetic diagnosis before embryo implantation.

The traditional detection method of Y chromosome microdeletion is mainly developed on the basis of PCR technology. In the national clinical laboratory, peripheral blood samples are collected and detected by methods such as multiple PCR gel electrophoresis, fluorescent quantitative (q) PCR, capillary electrophoresis and the like according to the scheme provided by European society of academic and academic (EAA) and European Molecular genetic Quality collaboration Network (EMQN). However, these techniques are long in time consumption, have high subjectivity in result judgment and low sensitivity, and do not meet the development direction that the current clinical examination techniques are faster and more sensitive and have less sample requirements.

The digital PCR is a novel nucleic acid quantitative technology developed in the beginning of the 20 th century, and compared with the traditional PCR, the method can be used for carrying out absolute quantitative analysis on a micro template and has higher accuracy, specificity, sensitivity and detection speed. Meanwhile, the automation degree of the digital PCR detection and analysis process is higher, so that the error of artificial result judgment is reduced, and the misjudgment of the result caused by the fact that the low copy number cannot be detected in the fluorescent quantitative PCR detection is reduced. Therefore, the use of digital PCR for the detection of Y chromosome microdeletions is a research direction, but deletion-related protocols are known in the art.

Disclosure of Invention

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

In order to solve the technical problems, the invention adopts the technical scheme that: a primer group for detecting microdeletion of Y chromosome is provided, 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.

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

Preferably, the kit further comprises: PCR premix, positive control, negative control, and microdroplet generation oil.

Preferably, the PCR premix solution comprises dye, Taq enzyme, dNTP, MgCl₂。

Preferably, the reaction concentration of Taq enzyme is 6U, the reaction concentration of dNTP is 0.8mM, and MgCl is used₂The final concentration was 5.6 mM.

Preferably, 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.

The invention also provides a detection method based on digital PCR for detecting the microdeletion of the Y chromosome, which utilizes the kit to detect the microdeletion of the Y chromosome by a digital PCR method.

Preferably, the method of the invention comprises 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.

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

Preferably, 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.

The invention has the beneficial effects that:

the invention can detect the Y chromosome microdeletion condition of the patient without spermatozoon disease rapidly, sensitively and accurately, and can effectively reduce the requirement on the sample size, thereby further reducing the peripheral blood collection to the fingertip blood, reducing the pain of the patient, and avoiding the false negative result caused by less DNA samples of the patient;

the invention adopts a digital PCR method for inspection, the detection result can be obtained without depending on the Ct value, and the original copy number of the DNA sample can be intuitively reflected, thereby reducing the workload of a detector;

the kit obtained by the invention can be stably stored for more than half a year at the temperature of 20 ℃ below zero, and has good product development potential.

Drawings

FIG. 1 is a diagram showing the results of peripheral blood samples of AZFb-deficient patients in example 2 of the present invention;

FIG. 2 is a diagram showing the result of fluorescent quantitative PCR detection of AZFb-deficient samples in example 2 of the present invention;

FIGS. 3a, 3b and 3c are graphs showing the results of detecting samples with concentrations of 10ng/ul, 1ng/ul and 0.1ng/ul in sequence by the fluorescent quantitative PCR method in example 3 of the present invention, respectively;

FIGS. 4a, 4b, and 4c are graphs showing the results of detecting samples with concentrations of 0.001ng/ul, 0.0001ng/ul, and 0.00001ng/ul in this order by the digital PCR-based detection method in example 3 of the present invention.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.

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