A kit and method for detecting Y chromosome microdeletion

Abstract

The invention discloses a kit for detecting Y chromosome microdeletion and a detection method. The kit for detecting Y chromosome microdeletion comprises: a PCR reaction solution, a primer-probe mixture, a positive control substance and a negative control substance; the primer-probe mixture comprises primers and probes for detecting SY84 and SY86 sites in an AZFa region, primers and probes for detecting SY127 and SY134 sites in an AZFb region, primers and probes for detecting SY254 and SY255 sites in an AZFc region, primers and probes for detecting SRY genes, and primers and probes for detecting ZFX / Y sex chromosome genes; the kit for detecting Y chromosome microdeletion only requires one PCR reaction well to detect six STS basic sites and two quality control sites, and the result is judged by using the Tm value in a melting curve instead of the Ct value in an amplification curve, so the qualitative analysis is accurate, and the result is intuitive and easy to interpret.

Description

Technical Field

[0001] The present invention relates to the technical field of kits, and in particular to a kit and a detection method for detecting Y chromosome microdeletions. Background Art

[0002] Statistics from the World Health Organization show that about 15% of couples worldwide suffer from infertility, of which about half are male infertility. The factors that cause male infertility are complex and varied, of which genetic abnormalities account for more than 30%. Such genetic abnormalities include Y chromosome microdeletions, abnormal chromosome structure and number, gene mutations, etc. Among patients with azoospermia or oligospermia, Y chromosome microdeletions account for about 10-15%, which is the second largest genetic factor after other factors, second only to Klinefelter's syndrome. In the general population, 1 in every 4,000 men has a Y chromosome microdeletion, but in infertile men, the frequency of deletions is significantly increased. Currently, Y chromosome microdeletion detection has become a routine examination item for male infertility patients.

[0003] Y chromosome microdeletion refers to the loss of a tiny DNA fragment in the azoospermia factor (AZF) on the long arm of the Y chromosome, which is closely related to sperm production. This loss can cause sperm production disorders, leading to oligospermia, asthenospermia, teratozoospermia and even azoospermia. The AZF region includes the AZFa, AZFb, and AZFc regions. Microdeletions in any of these regions may cause oligospermia or azoospermia. The probability of AZFa region deletion is low, but the loss of the entire region often leads to Sertoli cell only syndrome (SCOS), which is clinically manifested as azoospermia. In this case, it is almost impossible to obtain sperm from the testicles for assisted reproductive technology. Patients with AZFb region deletions will show spermatogenesis blockage, and sperm production stagnates at the spermatocyte stage, making it impossible to produce sperm. AZFc region deletion is relatively common and is one of the important causes of sperm formation disorders. Patients with deletions in this region still retain the ability to produce sperm, so they can be treated with assisted reproductive methods. However, this will pass the AZFc region deletion to male offspring, causing male infertility in the offspring. For patients with complete deletions in the AZFa, AZFb, and AZFc regions, they will inevitably show azoospermia and it is impossible to obtain sperm from the testicles by any means. The chance of assisted reproduction for such patients to obtain sperm is almost zero. It can be seen that Y chromosome microdeletion detection is of vital importance for exploring the causes of male infertility, avoiding unnecessary drug and surgical treatments, providing guidance for clinical assisted reproduction, and conducting preimplantation genetic diagnosis.

[0004] The development of assisted reproductive technology, such as testicular sperm extraction (TESE), intracytoplasmic sperm injection (ICSI), and in-vitro fertilization (IVF), has brought the possibility of giving birth to the next generation to many patients with oligospermia or even azoospermia, which is a revolutionary breakthrough in the field of male infertility treatment. However, for patients with complete AZFa deletion, complete AZFb deletion, AZFc region deletion, and AZFb+c deletion, although ICSI can be performed, this operation will pass the relevant deletions to the next generation, resulting in male infertility in the offspring. In addition, for patients with AZFa and AZFa, AZFb, and AZFc deletions, they will inevitably show azoospermia, and the chance of obtaining sperm through TESE for such patients is almost zero. Therefore, screening for Y chromosome microdeletions before surgery is of great value for the selection of male infertility treatment methods. At present, many countries in Europe and the United States have widely carried out the detection of Y chromosome microdeletions. The detection of Y chromosome microdeletions can, on the one hand, identify the causes of male infertility in some clinical patients with unknown causes, and on the other hand, it can also provide a basis and guidance for the clinical diagnosis of male infertility patients.

[0005] The three versions of the "Guidelines for Molecular Diagnosis of Y Chromosome Microdeletions" jointly issued by the European Association of Andrology and the European Molecular Genetics Quality Control Network (EAA / EMQN) all recommend testing a total of six sequence tag sites (STS) including sY84 and sY86 in the AZFa region, sY127 and sY134 in the AZFb region, and sY254 and sY255 in the AZFc region to diagnose Y chromosome microdeletions. If two STSs in each region are missing at the same time, it indicates that the region is missing, and the above six STS sites can detect more than 95% of AZF microdeletions, which is sufficient to meet routine clinical diagnosis needs.

[0006] (1) AZFa region deletion is relatively rare, accounting for about 0.5%-4% of patients with Y chromosome microdeletion, but the consequences of deletion are the most serious. The clinical manifestations of patients are: Sertoli cell-only syndrome (SCOS), accompanied by testicular volume reduction and azoospermia. There is currently no good treatment for this type of patients, and sperm donation or adoption is recommended. (2) AZFb region deletion is relatively common, accounting for about 3%-5% of patients with Y chromosome microdeletion. The phenotype is mainly manifested as: rare spermatogenic epithelial cells after meiosis, spermatogenesis is blocked at the spermatocyte stage, and mature sperm production is rare. Even if this disease is assisted by testicular sperm injection (ICSI) technology, the effect is still poor. The treatment methods for this type of patients are similar to those for AZF a region deletion. Sperm donation or adoption is recommended. (3) AZFc region deletion is the most common, accounting for more than 60%. The clinical phenotypes and testicular histological types of this type of patients are diverse, with azoospermia, severe oligospermia, and progressive decline in sperm count being the most common. Among azoospermia patients, those with AZFc deletion have a much greater chance of obtaining sperm through TESE, and can also undergo ICSI conception. However, the male offspring of these patients will be carriers of AZFc deletion, and there is even a possibility that the deletion area will increase, causing reproductive dysfunction in male offspring, but healthy female offspring can be obtained through preimplantation diagnosis. In addition, studies have found that the number of sperm in oligospermia patients with AZFc region deletion tends to decrease progressively, and finally develops into azoospermia. Therefore, oligospermia patients with AZFc region deletion should be treated as soon as possible or their semen should be frozen and preserved.

[0007] At present, there are many methods for detecting Y chromosome microdeletions, mainly including multiplex PCR-gel electrophoresis, multiplex PCR-capillary electrophoresis, multiplex fluorescence PCR, gene chip method, etc. Multiplex PCR-gel electrophoresis has problems such as being easily contaminated and producing false positive results, poor specificity and sensitivity, high requirements for DNA templates, low degree of automation, and not suitable for large-scale screening; although multiplex PCR-capillary electrophoresis and gene chip methods have the characteristics of high throughput and high precision, they have strict technical requirements and expensive instruments and equipment, which are not conducive to promotion; multiplex fluorescence PCR has a high degree of automation and does not require electrophoresis detection and hybridization steps, which can effectively reduce the risk of misjudgment of results due to contamination, but due to the limitation of fluorescence channels, at least two PCR reactions are required to detect 6 STS basic sites, which increases the detection cost, makes the operation steps more complicated, and reduces the detection throughput.

[0008] In view of this, the present invention is proposed. Summary of the invention

[0009] The purpose of the present invention is to provide a kit and a detection method for detecting Y chromosome microdeletions. The kit only requires one PCR reaction well to detect six STS basic sites (sY84 and sY86 in the AZFa region, sY127 and sY134 in the AZFb region, and sY254 and sY255 in the AZFc region) and two quality control sites (SRY and ZFX / Y). The reagent composition is simple and the reagent cost is low. In addition, the kit has strong compatibility with sample types and nucleic acid extraction methods, simple detection operation steps, and intuitive and clear result interpretation.

[0010] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0011] The present invention provides a kit for detecting Y chromosome microdeletions, and the kit for detecting Y chromosome microdeletions comprises:

[0012] PCR reaction solution, primer-probe mixture, positive control and negative control;

[0013] The primer-probe mixture includes primers and probes for detecting SY84 and SY86 sites in the AZFa region, primers and probes for detecting SY127 and SY134 sites in the AZFb region, primers and probes for detecting SY254 and SY255 sites in the AZFc region, primers and probes for detecting the SRY gene, and primers and probes for detecting the ZFX / Y sex chromosome gene.

[0014] The nucleotide sequence of the upstream primer for detecting the SY84 site is shown in SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the probe is shown in SEQ ID NO: 3;

[0015] The nucleotide sequence of the upstream primer for detecting the SY86 site is shown in SEQ ID NO: 4, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 5, and the nucleotide sequence of the probe is shown in SEQ ID NO: 6;

[0016] The nucleotide sequence of the upstream primer for detecting the SY127 site is shown in SEQ ID NO: 7, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 8, and the nucleotide sequence of the probe is shown in SEQ ID NO: 9;

[0017] The nucleotide sequence of the upstream primer for detecting the SY134 site is shown in SEQ ID NO: 10, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 11, and the nucleotide sequence of the probe is shown in SEQ ID NO: 12;

[0018] The nucleotide sequence of the upstream primer for detecting the SY254 site is shown in SEQ ID NO: 13, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 14, and the nucleotide sequence of the probe is shown in SEQ ID NO: 15;

[0019] The nucleotide sequence of the upstream primer for detecting the SY255 site is shown in SEQ ID NO: 16, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 17, and the nucleotide sequence of the probe is shown in SEQ ID NO: 18;

[0020] The nucleotide sequence of the upstream primer for detecting the SRY gene is shown in SEQ ID NO: 19, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 20, and the nucleotide sequence of the probe is shown in SEQ ID NO: 21;

[0021] The nucleotide sequence of the upstream primer for detecting the ZFX / Y sex chromosome gene is shown in SEQ ID NO: 22, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 23, and the nucleotide sequence of the probe is shown in SEQ ID NO: 24.

[0022] Compared with the prior art, the beneficial effects of the present invention include at least:

[0023] The kit for detecting Y chromosome microdeletions of the present invention only needs one PCR reaction well to detect six STS basic sites (sY84 and sY86 in the AZFa region, sY127 and sY134 in the AZFb region, and sY254 and sY255 in the AZFc region) and two quality control sites (SRY and ZFX / Y). The result is judged by using the Tm value in the melting curve instead of the Ct value in the amplification curve, and the qualitative analysis is accurate, and the result is intuitive and easy to interpret. In addition, the reagent composition is simple, the reagent cost is low, and the compatibility with sample types and nucleic acid extraction methods is strong. The detection operation steps are simple, and the result interpretation is intuitive and clear. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the specific embodiments or the description of the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale.

[0025] Figure 1 This is a melting curve diagram of four samples in Example 2 of the present invention. DETAILED DESCRIPTION

[0026] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0027] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the invention belongs.

[0028] The embodiment of the present invention provides a kit for detecting Y chromosome microdeletions, and the kit for detecting Y chromosome microdeletions comprises:

[0029] PCR reaction solution, primer-probe mixture, positive control and negative control;

[0030] The primer-probe mixture includes primers and probes for detecting SY84 and SY86 sites in the AZFa region, primers and probes for detecting SY127 and SY134 sites in the AZFb region, primers and probes for detecting SY254 and SY255 sites in the AZFc region, primers and probes for detecting the SRY gene, and primers and probes for detecting the ZFX / Y sex chromosome gene.

[0031] The nucleotide sequence of the upstream primer for detecting the SY84 site is shown in SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the probe is shown in SEQ ID NO: 3;

[0032] The nucleotide sequence of the upstream primer for detecting the SY86 site is shown in SEQ ID NO: 4, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 5, and the nucleotide sequence of the probe is shown in SEQ ID NO: 6;

[0033] The nucleotide sequence of the upstream primer for detecting the SY127 site is shown in SEQ ID NO: 7, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 8, and the nucleotide sequence of the probe is shown in SEQ ID NO: 9;

[0034] The nucleotide sequence of the upstream primer for detecting the SY134 site is shown in SEQ ID NO: 10, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 11, and the nucleotide sequence of the probe is shown in SEQ ID NO: 12;

[0035] The nucleotide sequence of the upstream primer for detecting the SY254 site is shown in SEQ ID NO: 13, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 14, and the nucleotide sequence of the probe is shown in SEQ ID NO: 15;

[0036] The nucleotide sequence of the upstream primer for detecting the SY255 site is shown in SEQ ID NO: 16, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 17, and the nucleotide sequence of the probe is shown in SEQ ID NO: 18;

[0037] The nucleotide sequence of the upstream primer for detecting the SRY gene is shown in SEQ ID NO: 19, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 20, and the nucleotide sequence of the probe is shown in SEQ ID NO: 21;

[0038] The nucleotide sequence of the upstream primer for detecting the ZFX / Y sex chromosome gene is shown in SEQ ID NO: 22, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 23, and the nucleotide sequence of the probe is shown in SEQ ID NO: 24.

[0039] The kit for detecting Y chromosome microdeletions of the present invention only needs one PCR reaction well to detect six STS basic sites (sY84 and sY86 in the AZFa region, sY127 and sY134 in the AZFb region, and sY254 and sY255 in the AZFc region) and two quality control sites (SRY and ZFX / Y). The result is judged by using the Tm value in the melting curve instead of the Ct value in the amplification curve, and the qualitative analysis is accurate, and the result is intuitive and easy to interpret. In addition, the reagent composition is simple, the reagent cost is low, and the compatibility with sample types and nucleic acid extraction methods is strong. The detection operation steps are simple, and the result interpretation is intuitive and clear.

[0040] In some embodiments, the nucleotide sequence of the upstream primer for detecting the SY84 site is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 1 or the complementary sequence, the nucleotide sequence of the downstream primer is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 2 or the complementary sequence, and the nucleotide sequence of the probe is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 3 or the complementary sequence;

[0041] The nucleotide sequence of the upstream primer for detecting the SY86 site is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 4 or the complementary sequence, the nucleotide sequence of the downstream primer is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 5 or the complementary sequence, and the nucleotide sequence of the probe is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 6 or the complementary sequence;

[0042] The nucleotide sequence of the upstream primer for detecting the SY127 site is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 7 or the complementary sequence, the nucleotide sequence of the downstream primer is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 8 or the complementary sequence, and the nucleotide sequence of the probe is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 9 or the complementary sequence;

[0043] The nucleotide sequence of the upstream primer for detecting the SY134 site is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 10 or the complementary sequence, the nucleotide sequence of the downstream primer is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 11 or the complementary sequence, and the nucleotide sequence of the probe is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 12 or the complementary sequence;

[0044] The nucleotide sequence of the upstream primer for detecting the SY254 site is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 13 or the complementary sequence, the nucleotide sequence of the downstream primer is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 14 or the complementary sequence, and the nucleotide sequence of the probe is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 15 or the complementary sequence;

[0045] The nucleotide sequence of the upstream primer for detecting the SY255 site is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 16 or the complementary sequence, the nucleotide sequence of the downstream primer is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 17 or the complementary sequence, and the nucleotide sequence of the probe is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 18 or the complementary sequence;

[0046] The nucleotide sequence of the upstream primer for detecting the SRY gene is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 19 or the complementary sequence, the nucleotide sequence of the downstream primer is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 20 or the complementary sequence, and the nucleotide sequence of the probe is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 21 or the complementary sequence;

[0047] The nucleotide sequence of the upstream primer for detecting the ZFX / Y sex chromosome gene is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 22 or its complementary sequence, the nucleotide sequence of the downstream primer is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 23 or its complementary sequence, and the nucleotide sequence of the probe is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 24 or its complementary sequence.

[0048] In some embodiments, the primer probe mixture is prepared by mixing SY84 primer probe mixture, SY86 primer probe mixture, SY127 primer probe mixture, SY134 primer probe mixture, SY254 primer probe mixture, SY255 primer probe mixture, SRY gene primer probe mixture and ZFX / Y sex chromosome gene primer probe mixture in a volume ratio of 2:2:2:2:1:1:1:1;

[0049] The SY84 primer-probe mixture, SY86 primer-probe mixture, SY127 primer-probe mixture, SY134 primer-probe mixture, SY254 primer-probe mixture, SY255 primer-probe mixture, SRY gene primer-probe mixture and ZFX / Y sex chromosome gene primer-probe mixture are respectively prepared by mixing the corresponding upstream primer solution, downstream primer solution and probe solution in a volume ratio of 1: (8-12): (4-6);

[0050] The concentrations of the upstream primer solution, the downstream primer solution and the probe solution are 5-15 μM respectively.

[0051] In some embodiments, the positive control is a genomic DNA solution of a normal male cell line, and the concentration is 4-6 ng / μl.

[0052] In some embodiments, the negative control is a genomic DNA solution of a normal female cell line, and the concentration is 4-6 ng / μl.

[0053] In some embodiments, the PCR reaction solution is 2xTaq enzyme mix.

[0054] In some embodiments, the 5' ends of the probes corresponding to different detection sites are respectively modified with fluorescent groups, and the 3' ends are respectively modified with quenching groups.

[0055] In some embodiments, the fluorescent group includes but is not limited to FAM, VIC, Pacific Blue, Atto425, Bodipy FL, Alexa Fluor488, TET, JOE, HEX, NED, Cy3, Quasar570, TAMRA, Texas Red, ROX, AquaPhluor593, Cy5, Quasar 570 and Quasar670.

[0056] In some embodiments, the quenching group includes but is not limited to TAMRA, BHQ1, BHQ2, BHQ3, ECLIPS, MGB and Dabcyl.

[0057] Another embodiment of the present invention provides a method for detecting Y chromosome microdeletions for non-diagnostic purposes, the detection method comprising using the kit for detecting Y chromosome microdeletions to detect whether SY84 and SY86 in the AZFa region, SY127 and SY134 in the AZFb region, and SY254 and SY255 in the AZFc region are deleted.

[0058] The technical solution of the present invention is further described in detail below through specific embodiments.

[0059] Example 1

[0060] This embodiment is a kit for detecting Y chromosome microdeletions, and the kit for detecting Y chromosome microdeletions includes:

[0061] PCR reaction solution, primer-probe mixture, positive control and negative control;

[0062] The primer-probe mixture includes primers and probes for detecting SY84 and SY86 sites in the AZFa region, primers and probes for detecting SY127 and SY134 sites in the AZFb region, primers and probes for detecting SY254 and SY255 sites in the AZFc region, primers and probes for detecting the SRY gene, and primers and probes for detecting the ZFX / Y sex chromosome gene. The primers and probes are specifically shown in Table 1;

[0063] Table 1

[0064]

[0065]

[0066] The primer probe mixture is prepared by mixing SY84 primer probe mixture, SY86 primer probe mixture, SY127 primer probe mixture, SY134 primer probe mixture, SY254 primer probe mixture, SY255 primer probe mixture, SRY gene primer probe mixture and ZFX / Y sex chromosome gene primer probe mixture in a volume ratio of 2:2:2:2:1:1:1:1;

[0067] The SY84 primer-probe mixture, SY86 primer-probe mixture, SY127 primer-probe mixture, SY134 primer-probe mixture, SY254 primer-probe mixture, SY255 primer-probe mixture, SRY gene primer-probe mixture and ZFX / Y sex chromosome gene primer-probe mixture are prepared by mixing the corresponding upstream primer solution, downstream primer solution and probe solution in a volume ratio of 1:10:5 respectively;

[0068] The concentrations of the upstream primer solution, downstream primer solution and probe solution are 10 μM respectively;

[0069] The positive control substance is a normal male cell line genomic DNA solution, and the concentration is 5ng / μl;

[0070] The negative control substance is a genomic DNA solution of a normal female cell line, and the concentration is 5 ng / μl;

[0071] The PCR reaction solution is 2xTaq enzyme mix, Hieff Universal gPCR master mix, catalog number is 11211E;

[0072] The preparation method of the kit for detecting Y chromosome microdeletion comprises the following steps:

[0073] (a) Preparation of PCR reaction solution:

[0074] Will Hieff Universal gPCR master mix (from Yisheng Biotechnology, catalog number 11211E) was packaged and labeled at 270 μL / tube to obtain PCR reaction solution;

[0075] (b) Preparation of primer-probe mixture:

[0076] The synthesized primer probe dry powder was diluted to 10 μM with 10 mM Tris buffer. The primer probe mixture for each locus was first prepared by mixing the F, R, and P volume ratio of 1:10:5. Then, the primer probe mixtures of the eight loci were mixed at a volume ratio of 2:2:2:2:1:1:1:1 to prepare the primer probe mixture of this kit. 110 μL was dispensed into each tube.

[0077] (c) Preparation of positive control:

[0078] A normal male cell line was cultured for nucleic acid extraction. The extracted DNA was quantified using qubit and diluted with double distilled water to a final concentration of 5 ng / μL. 20 μL was dispensed into each tube to obtain a positive control.

[0079] (d) Preparation of negative control:

[0080] A normal female cell line was cultured for nucleic acid extraction. The extracted DNA was quantified using qubit and diluted with double distilled water to a final concentration of 5 ng / μL. 20 μL was dispensed into each tube to obtain a negative control.

[0081] Example 2

[0082] This embodiment is a method for detecting Y chromosome microdeletion using the kit of Example 1, and the method comprises the following steps:

[0083] Using the kit of Example 1, using the kit of the present invention, one normal sample and one deletion sample with known Y chromosome microdeletion status, and positive and negative control substances, a PCR reaction system was prepared according to Table 1;

[0084] Table 1

[0085] Reagents Single reaction (μL) 2xPCR reaction solution 10 Primer probe mixture 4 Template DNA 2 NF Water 4 total 20

[0086] After mixing by oscillation and instant centrifugation, amplification and detection were performed using the Shanghai Hongshi Fluorescence Quantitative PCR Instrument SLAN-96. The PCR reaction procedure was as follows:

[0087] 95℃10min; (95℃15s, 59℃15s to collect FAM, HEX, ROX and CY5 channel fluorescence, 72℃20s)x50; 95℃1min, 40℃1min, 40-95℃ at a heating rate of 0.04℃ / s to collect FAM, HEX, ROX and CY5 channel fluorescence.

[0088] Test results such as Figure 1 As shown, Figure 1 In the figure, A, B, C, and D are the melting curves of FAM, HEX, ROX, and CY5 channels of normal samples, deletion samples, positive controls, and negative controls, respectively;

[0089] Depend on Figure 1 It can be seen that each fluorescence channel of Figure A and Figure C has a melting curve peak in the two preset temperature regions, and there is no deletion at all sites; Figure D has a melting curve peak only in the temperature region of the ZFX / Y site, which is consistent with the expected results; the ROX fluorescence channel of Figure B has no melting curve peak, and the HEX fluorescence channel has no melting curve peak at 56-60°C, indicating that the sample has a deletion of Y chromosome AZFb (sY134) and AZFc (sY254 and sY255), both samples and positive controls have amplification curves and melting curves, and the negative control has an amplification curve and melting curve only in the CY5 channel. The corresponding relationship between the temperature of each melting peak and the detection site is shown in Table 2:

[0090] Table 2

[0091] area Location Fluorescence channels Tm value range AZFa SY84 FAM 62-66℃ AZFa SY86 FAM 69-73℃ FWf SY127 HEX 66-70℃ FWf SY134 HEX 56-60℃ FqV SY254 ROX 69-73℃ FqV SY255 ROX 59-63℃ SRY SRY CY5 61-65℃ ZFX / Y ZFX / Y CY5 69-73℃

[0092] The CY5 channel (red) is the internal reference gene detection channel. When the melting curve of the CY5 channel has a peak between 69-73°C, it indicates that the sample extraction and amplification reactions are normal. When the melting curve of the CY5 channel has a peak between 61-65°C, it indicates that the Y chromosome exists in the sample. When both of the above peaks exist, it means that the Y chromosome microdeletion results can be interpreted. Otherwise, it indicates that the reaction is abnormal and the experiment needs to be repeated or the cause needs to be investigated and then repeated.

[0093] When the reference gene result of the CY5 channel is normal, according to the correspondence between the detection site and the fluorescence channel and the melting curve temperature Tm value in the above table, the presence of a peak indicates that the corresponding site is not missing, and the absence of a peak indicates that the corresponding site is missing.

[0094] Example 3

[0095] This example is a study on the accuracy of the detection method of Example 2;

[0096] 50 different blood samples were used. After nucleic acid extraction, the samples were respectively tested by the detection method of Example 2 of the present invention and sent to Guangzhou Yunkang Daan Medical Testing Laboratory for capillary electrophoresis. The test results of the 50 samples were statistically analyzed, as shown in Table 3 below:

[0097] Table 3

[0098]

[0099]

[0100]

[0101] From Table 3, we can see that:

[0102] The kit of the present invention detected 11 samples with Y chromosome microdeletions, of which 6 were sY254 and sY255 deletions in the AZFc region, 2 were sY127 and sY134 deletions in the AZFb region, 2 were co-deletions in the AZFb and AZFc regions, and 1 was a partial deletion of AZFb and a deletion of AZFc. The detection results of 50 samples using the kit method of the present invention were completely consistent with the capillary electrophoresis method, proving that the accuracy of the detection method is 100%.

[0103] Example 4

[0104] This example is a compatibility test of the detection method of the present invention with different sample types and nucleic acid extraction methods;

[0105] Saliva samples and whole blood samples were collected from 5 volunteers. After verification by capillary electrophoresis, 1 of the 5 volunteers had a Y chromosome microdeletion, and the other 4 samples were normal. A portion of DNA was extracted from each sample using the magnetic bead extraction method (Guangzhou Meiji Biological Extraction Kit) and the boiling crude extraction method (self-prepared reagents). The above 20 DNAs were tested using the kit of the present invention, and the results are shown in Table 4;

[0106] Table 4

[0107]

[0108] From Table 4, we can see that:

[0109] The detection results of the DNA samples obtained by the two sample types and two extraction methods using the present kit were consistent with the results of the capillary electrophoresis method, indicating that the kit of the present invention has high compatibility with sample types and nucleic acid extraction methods.

[0110] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. A kit for detecting Y chromosome microdeletions, characterized in that: The kit for detecting Y chromosome microdeletion comprises: PCR reaction solution, primer-probe mixture, positive control and negative control; The primer-probe mixture includes primers and probes for detecting SY84 and SY86 sites in the AZFa region, primers and probes for detecting SY127 and SY134 sites in the AZFb region, primers and probes for detecting SY254 and SY255 sites in the AZFc region, primers and probes for detecting the SRY gene, and primers and probes for detecting the ZFX / Y sex chromosome gene. The nucleotide sequence of the upstream primer for detecting the SY84 site is shown in SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the probe is shown in SEQ ID NO: 3; The nucleotide sequence of the upstream primer for detecting the SY86 site is shown in SEQ ID NO: 4, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 5, and the nucleotide sequence of the probe is shown in SEQ ID NO: 6; The nucleotide sequence of the upstream primer for detecting the SY127 site is shown in SEQ ID NO: 7, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 8, and the nucleotide sequence of the probe is shown in SEQ ID NO: 9; The nucleotide sequence of the upstream primer for detecting the SY134 site is shown in SEQ ID NO: 10, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 11, and the nucleotide sequence of the probe is shown in SEQ ID NO: 12; The nucleotide sequence of the upstream primer for detecting the SY254 site is shown in SEQ ID NO: 13, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 14, and the nucleotide sequence of the probe is shown in SEQ ID NO: 15; The nucleotide sequence of the upstream primer for detecting the SY255 site is shown in SEQ ID NO: 16, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 17, and the nucleotide sequence of the probe is shown in SEQ ID NO: 18; The nucleotide sequence of the upstream primer for detecting the SRY gene is shown in SEQ ID NO: 19, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 20, and the nucleotide sequence of the probe is shown in SEQ ID NO: 21; The nucleotide sequence of the upstream primer for detecting the ZFX / Y sex chromosome gene is shown in SEQ ID NO: 22, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 23, and the nucleotide sequence of the probe is shown in SEQ ID NO:

24.

2. The kit for detecting Y chromosome microdeletions according to claim 1, characterized in that The nucleotide sequence of the upstream primer for detecting the SY84 site is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 1 or the complementary sequence, the nucleotide sequence of the downstream primer is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 2 or the complementary sequence, and the nucleotide sequence of the probe is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 3 or the complementary sequence; The nucleotide sequence of the upstream primer for detecting the SY86 site is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 4 or the complementary sequence, the nucleotide sequence of the downstream primer is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 5 or the complementary sequence, and the nucleotide sequence of the probe is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 6 or the complementary sequence; The nucleotide sequence of the upstream primer for detecting the SY127 site is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 7 or the complementary sequence, the nucleotide sequence of the downstream primer is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 8 or the complementary sequence, and the nucleotide sequence of the probe is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 9 or the complementary sequence; The nucleotide sequence of the upstream primer for detecting the SY134 site is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 10 or the complementary sequence, the nucleotide sequence of the downstream primer is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 11 or the complementary sequence, and the nucleotide sequence of the probe is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 12 or the complementary sequence; The nucleotide sequence of the upstream primer for detecting the SY254 site is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 13 or the complementary sequence, the nucleotide sequence of the downstream primer is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 14 or the complementary sequence, and the nucleotide sequence of the probe is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 15 or the complementary sequence; The nucleotide sequence of the upstream primer for detecting the SY255 site is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 16 or the complementary sequence, the nucleotide sequence of the downstream primer is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 17 or the complementary sequence, and the nucleotide sequence of the probe is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 18 or the complementary sequence; The nucleotide sequence of the upstream primer for detecting the SRY gene is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 19 or the complementary sequence, the nucleotide sequence of the downstream primer is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 20 or the complementary sequence, and the nucleotide sequence of the probe is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 21 or the complementary sequence; The nucleotide sequence of the upstream primer for detecting the ZFX / Y sex chromosome gene is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 22 or its complementary sequence, the nucleotide sequence of the downstream primer is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 23 or its complementary sequence, and the nucleotide sequence of the probe is to add, delete or replace 1 to 5 bases on the sequence shown in SEQ ID NO: 24 or its complementary sequence.

3. The kit for detecting Y chromosome microdeletion according to claim 1, characterized in that The primer probe mixture is prepared by mixing SY84 primer probe mixture, SY86 primer probe mixture, SY127 primer probe mixture, SY134 primer probe mixture, SY254 primer probe mixture, SY255 primer probe mixture, SRY gene primer probe mixture and ZFX / Y sex chromosome gene primer probe mixture in a volume ratio of 2:2:2:2:1:1:1:1; The SY84 primer-probe mixture, SY86 primer-probe mixture, SY127 primer-probe mixture, SY134 primer-probe mixture, SY254 primer-probe mixture, SY255 primer-probe mixture, SRY gene primer-probe mixture and ZFX / Y sex chromosome gene primer-probe mixture are respectively prepared by mixing the corresponding upstream primer solution, downstream primer solution and probe solution in a volume ratio of 1: (8-12): (4-6); The concentrations of the upstream primer solution, the downstream primer solution and the probe solution are 5-15 μM respectively.

4. The kit for detecting Y chromosome microdeletion according to claim 1, characterized in that The positive control substance is a normal male cell line genomic DNA solution, and the concentration is 4-6 ng / μl.

5. The kit for detecting Y chromosome microdeletion according to claim 1, characterized in that The negative control substance is a genomic DNA solution of a normal female cell line, and the concentration is 4-6 ng / μl.

6. The kit for detecting Y chromosome microdeletions according to claim 1, characterized in that The PCR reaction solution is 2xTaq enzyme mix.

7. The kit for detecting Y chromosome microdeletion according to claim 1, characterized in that The 5' ends of the probes corresponding to different detection sites are modified with fluorescent groups, and the 3' ends are modified with quenching groups.

8. The kit for detecting Y chromosome microdeletions according to claim 7, characterized in that: The fluorescent groups include but are not limited to FAM, VIC, Pacific Blue, Atto 425, BodipyFL, Alexa Fluor488, TET, JOE, HEX, NED, Cy3, Quasar 570, TAMRA, Texas Red, ROX, AquaPhluor593, Cy5, Quasar 570 and Quasar 670.

9. The kit for detecting Y chromosome microdeletions according to claim 7, characterized in that: The quenching groups include, but are not limited to, TAMRA, BHQ1, BHQ2, BHQ3, ECLIPS, MGB and Dabcyl.

10. A method for detecting Y chromosome microdeletions for non-diagnostic purposes, characterized in that: The detection method comprises using any one of the kits for detecting Y chromosome microdeletions described in claims 1 to 9 to detect whether SY84 and SY86 in the AZFa region, SY127 and SY134 in the AZFb region, and SY254 and SY255 in the AZFc region are deleted.

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