Primer-probe combination and kit for rapid detection of human y chromosome microdeletion at constant temperature

Abstract

The present application relates to a kind of primer-probe composition and kit for rapid detection of human Y chromosome microdeletion at constant temperature.The base sequence of specific primer and probe for different STS sites (SY84, SY86, SY127, SY134, SY25, SY255) and SRY, ZFX / Y of chromosome microdeletion is provided.The detection kit is also provided, including the specific primer-probe combination obtained by design screening, enzyme freeze-dried powder, dissolution solution and activator for micro-volume reaction.Only a small amount of sample DNA can be detected within 15 minutes in the constant temperature amplification fluorescence detection instrument to realize the type of Y chromosome microdeletion, with good specificity and high accuracy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of in vitro nucleic acid molecule detection, and relates to a primer-probe composition and a kit for rapid detection of human Y chromosome microdeletion at constant temperature. BACKGROUND

[0002] Y chromosome microdeletion is one of the most important genetic factors leading to male infertility. Y chromosome microdeletion refers to the deletion of the azoospermia factor (AZF) region on the Y chromosome, which can be divided into three regions, AZFa, AZFb and AZFc, and each region has a specific sequence tagged site (STS) for identification. According to the 1999 version, 2004 version and 2013 version of the Y chromosome microdeletion molecular diagnosis practice guidelines jointly issued by the European Academy of Andrology (EAA) and the European Molecular Genetics Quality Network (EMQN), the method of detecting STS by multiplex PCR-agarose gel is recommended for detecting Y chromosome microdeletion. At the same time, the guidelines recommend detecting 2 STS sites in each region, sY84 and sY86 sites in the AZFa region, sY127 and sY134 sites in the AZFb region, and sY254 and sY255 sites in the AZFc region. If the 2 STS sites in each region are missing at the same time, it means that the region has a deletion. It is pointed out that through the above 6 STS, almost all clinically relevant microdeletions or 95% of the AZF microdeletions reported in the literature can be detected, which is sufficient for routine diagnosis.

[0003] At present, the existing human Y chromosome microdeletion detection methods in the prior art mainly include PCR derived detection technology and sequencing detection. 1) Multiple PCR-agarose gel electrophoresis method, although it is recommended by the Y chromosome microdeletion molecular diagnosis practice guide, but the operation is complex, the detection time is long and slow, and automation cannot be realized. 2) Real-time fluorescent quantitative PCR technology, although the sensitivity is higher than that of multiple PCR-agarose gel electrophoresis method, the degree of automation is high, and there is no need for electrophoresis detection; but the turnaround time is longer, at least one hour is needed, professional detection instrument equipment and professional operation of personnel, which limits the application of detection scene. There is a multiple fluorescent quantitative PCR for detecting Y chromosome microdeletion, such as in the application number 2018109907722, a multiple PCR primer group for rapidly detecting human Y chromosome microdeletion, a kit and application, a multiple PCR primer group for rapidly detecting human Y chromosome microdeletion is provided, which can simultaneously amplify 6 STS gene sites on the Y chromosome and the internal reference gene SRY; each STS site contains specific amplification PCR primers and specific amplification RCR nested primers, which can reduce the difference in amplification efficiency of each gene, promote balanced, efficient and stable amplification, but the concentration and purity of the extracted genomic DNA sample required are high, the DNA concentration is more than 5 ng / μL, and the purity A260 / A280 is between 1.8-2.0. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a Y chromosome microdeletion detection primer-probe combination with short detection time, accurate results, high sensitivity and simple operation, and to provide a related detection kit.

[0005] In order to achieve the above purpose, the present application provides the following technical solutions:

[0006] 1) The specific amplification of the upstream primer of the SY84 region of the AZFa region is shown as SEQ ID NO: 1, the downstream primer is shown as SEQ ID NO: 2, and the fluorescent probe is shown as SEQ ID NO: 25;

[0007] 1) The specific amplification of the upstream primer of the SY84 region of the AZFa region is shown as SEQ ID NO: 1, the downstream primer is shown as SEQ ID NO: 2, and the fluorescent probe is shown as SEQ ID NO: 25;

[0008] 2) The specific amplification of the upstream primer of the SY86 region of the AZFa region is shown as SEQ ID NO: 4, the downstream primer is shown as SEQ ID NO: 5, and the fluorescent probe is shown as SEQ ID NO: 26;

[0009] 2) The specific amplification of the upstream primer of the SY86 region of the AZFa region is shown as SEQ ID NO: 4, the downstream primer is shown as SEQ ID NO: 5, and the fluorescent probe is shown as SEQ ID NO: 26;

[0010] 2) The specific amplification of the upstream primer of the SY86 region of the AZFa region is shown as SEQ ID NO: 4, the downstream primer is shown as SEQ ID NO: 5, and the fluorescent probe is shown as SEQ ID NO: 26;

[0011] 3) the upstream primer for specifically amplifying SY127 in AZFb region is shown as SEQ ID NO: 7, the downstream primer is shown as SEQ ID NO: 8, and the fluorescent probe is shown as SEQ ID NO: 27;

[0012] 4) the upstream primer for specifically amplifying SY134 in AZFc region is shown as SEQ ID NO: 10, the downstream primer is shown as SEQ ID NO: 11, and the fluorescent probe is shown as SEQ ID NO: 28;

[0013] 5) the upstream primer for specifically amplifying SY254 in AZFc region is shown as SEQ ID NO: 13, the downstream primer is shown as SEQ ID NO: 14, and the fluorescent probe is shown as SEQ ID NO: 29;

[0014] 6) the upstream primer for specifically amplifying SY255 is shown as SEQ ID NO: 16, the downstream primer is shown as SEQ ID NO: 17, and the fluorescent probe is shown as SEQ ID NO: 30;

[0015] 7) the upstream primer for specifically amplifying SRY is shown as SEQ ID NO: 19, the downstream primer is shown as SEQ ID NO: 20, and the fluorescent probe is shown as SEQ ID NO: 31;

[0016] 8) the upstream primer for specifically amplifying ZFX / Y is shown as SEQ ID NO: 22, the downstream primer is shown as SEQ ID NO: 23, and the fluorescent probe is shown as SEQ ID NO: 32.

[0017] Further, the fluorescent probe is modified with a thymine at a position about 30 nt from the 5' end for a fluorescent group, with a thymine at a position about 15 nt from the 3' end for a quenching group, with a base replaced by THF as a cleavage site for exonuclease exo at a position within 2-4 nt between the fluorescent group and the quenching group, and with a blocking group at the 3' end of the probe.

[0018] Further, in the primer-probe combination for rapid detection of human Y chromosome microdeletion at constant temperature, the fluorescent group is selected from FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy5; the quenching group is selected from BHQ1, BHQ2, BHQ3 or DABCYL; and the blocking group is selected from C3-spacer, amine group, biotin-triethylene glycol or phosphate group.

[0019] Preferably, the fluorescent reporter group is FAM, the fluorescent quenching group is BHQ1, and the 3' end blocking group is C3-spacer.

[0020] Preferably, the fluorescent probe sequence is shown as SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24.

[0021] 2. The kit for rapid detection of microdeletion of human Y chromosome at constant temperature is also provided, comprising the primer-probe combination of any of the above.

[0022] Further, the kit for rapid detection of microdeletion of human Y chromosome at constant temperature further comprises dNTPs, buffer, and enzyme composition.

[0023] Further, in the kit for rapid detection of microdeletion of human Y chromosome at constant temperature, the buffer is polyethylene glycol 1.5-5%; Tris 10-50mM; dithiothreitol 1-10mM; ATP 1.5-3.5mM; phosphocreatine disodium salt 20-75mM; phosphokinase 10-200ng / μL; trehalose 2.5-7.5%; mannitol 40-50ng / μL.

[0024] Further, in the kit for rapid detection of microdeletion of human Y chromosome at constant temperature, the enzyme composition comprises recombinase 100-500ng / μL; single-stranded DNA binding protein 100-200ng / μL; DNA polymerase 50-250ng / μL; exonuclease 200-300ng / μL.

[0025] Further, the kit for rapid detection of microdeletion of human Y chromosome at constant temperature further comprises an activator.

[0026] Preferably, the activator is magnesium acetate. The activator is 80-160mM of magnesium acetate.

[0027] Preferably, the magnesium acetate is 80-160mM.

[0028] More preferably, the magnesium acetate is 140mM.

[0029] Further, in the kit for rapid detection of microdeletion of human Y chromosome at constant temperature, the reaction system of the kit is 10μL, comprising: 5-6μL of mixture of dNTPs, buffer, and enzyme composition; 0.5-1μL of activator; 2-3μL of sample DNA, or 2-3μL of negative / positive control; 0.2-0.4μL of upstream primer; 0.2-0.4μL of upstream primer; 0.08-0.12μL of fluorescent probe; and ddH2O to make up to 10μL.

[0030] The present application has the beneficial effects that: in view of the problems existing in the prior art Y chromosome microdeletion detection technology, the primer-probe composition for constant temperature amplification Y chromosome microdeletion detection is invented, the primer pair and the probe composition have strong specificity, the detection sensitivity is higher than that of the conventional PCR, the sample DNA only needs 0.2 ng / μL, and the purity requirement of the sample DNA is not strict. The kit provided by the present application has the advantages of simple detection operation method and short reaction time. The primer-probe composition of the present application is in the independent reaction well, and the detection of multiple sites can be completed in 15 minutes under the constant temperature in the 10 μl micro-volume system. Whether the deletion exists on the Y chromosome is judged by detecting the fluorescence signal, and the rapid result reading is realized in 15 minutes. BRIEF DESCRIPTION OF DRAWINGS

[0031] In order to make the purpose, technical scheme and beneficial effects of the present application clearer, the present application provides the following drawings for illustration:

[0032] Figure 1 The minimum concentration detection limit of the kit for normal male blood samples.

[0033] Figure 2 The amplification result of the normal male sample.

[0034] Figure 3 The amplification result of the AZF a region deletion sample.

[0035] Figure 4 The amplification result of the AZF b region deletion sample.

[0036] Figure 5 The amplification result of the AZF c region deletion sample.

[0037] Figure 6 The amplification result of the AZF b+c region deletion sample.

[0038] Figure 7 The amplification result of the AZF a+b+c region deletion sample.

[0039] Figure 8 The amplification result of the normal female sample.

[0040] Figure 9 The result of the blank group.

[0041] Figure 10 The amplification result of different amplification reaction volumes. DETAILED DESCRIPTION

[0042] The preferred embodiments of the present application will be described in detail below with reference to the attached drawings. The experimental methods in the examples not specified in the specific conditions are generally in accordance with the conventional conditions or in accordance with the conditions recommended by the manufacturers. Unless otherwise specified, all raw materials and reagents of the present application are conventional market raw materials and reagents.

[0043] Example 1 primer probe design and detection scheme

[0044] According to the 2013 version of the Y chromosome microdeletion detection guidelines published by the European Association of Andrology (EAA) and EMQN guidelines, the classic 6-site (sY84, sY86, sY127, sY124, sY254, sY255) detection is recommended. The above-mentioned 6 sites can diagnose all clinically relevant ZAF deletion conditions reported in more than 95% of the literature, which is sufficient to meet the needs of routine diagnosis, and set the male sex-determining gene SRY as a gender abnormality control. Some XY male transsexual patients may not carry the SRY site. Human zinc finger protein gene ZFX / Y is used as a control for sample collection, nucleic acid extraction and amplification. The sequences of the primer-probe combinations are shown in Table 1. The fluorescent probe sequences are shown in SEQ ID NO: 25-SEQ ID NO: 32. The fluorescent group is modified by thymine at about 30 nt (nucleotides) from the 5' end, and the fluorescent group includes FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy5; the quenching group is modified by thymine at about 15 nt from the 3' end, and the quenching group includes BHQ1, BHQ2, BHQ3 or DABCYL; any base in the range of 2-4 nt between the fluorescent group and the quenching group is replaced by THF (tetrahydrofuran) as the cutting site of the exonuclease exo, and the 3' end of the probe is labeled with a blocking group to prevent DNA polymerase catalytic extension. The 3' end blocking group includes C3-spacer, amine, biotin-triglycol or phosphate group. Preferably, the fluorescent reporter group is FAM, the fluorescent quenching group is BHQ1, and the 3' end blocking group is Spacer C3.

[0045] Table 1 primer-probe sequence

[0046]

[0047]

[0048]

[0049] The detection scheme is designed according to the primer-probe combination listed in Table 1; Table 2 is the reaction tube design for the corresponding STS site of the present application, the parameters are optimized through experiments, and the optimal pre-freeze enzyme mix is finally determined, Table 3 is the components and component volume of the pre-freeze enzyme mix, and the dNTPs, energy buffer and various engineering enzymes are mixed into the pre-freeze enzyme mix according to the volume ratio in Table 3, wherein the dNTPs is 1-1.8mM, the concentration ranges of the components in the energy buffer are as follows: polyethylene glycol 1.5-5%; Tris 10-50mM; dithiothreitol 1-10mM; ATP 1.5-3.5mM; phosphocreatine disodium salt 20-75mM; phosphokinase 10-200ng / μL; trehalose 2.5-7.5%; mannitol 40-50ng / μL. In the composition of the engineering enzyme, the concentration ranges of the components are as follows: recombinant enzyme 100-500ng / μL; single-stranded DNA binding protein 100-200ng / μL; DNA polymerase 50-250ng / μL; exonuclease 200-300ng / μL. Table 4 is the component and component volume of each reaction tube in the enzyme freeze-dried mixture, and the primers and probes corresponding to the STS site of each reaction tube in Table 2 are added to the pre-freeze enzyme mix according to the component amount in Table 4, and then freeze-dried into an enzyme freeze-dried mixture.

[0050] Table 2 is the detection design of each reaction tube corresponding to the STS site:

[0051] Reaction tube name Point Fluorophore A sY84 FAM B sY86 FAM C sY127 FAM D sY134 FAM E sY255 FAM F sY254 FAM G SRY FAM H ZFX / Y FAM

[0052] Table 3 is the component of the pre-freeze enzyme mix:

[0053]

[0054]

[0055] Table 4 is the component of each reaction tube in the enzyme freeze-dried mixture:

[0056]

[0057] *Note: The pre-freeze enzyme mix contains the above-mentioned pre-freeze enzyme mix containing dNTPs 1-1.8mM, energy buffer and various engineering enzymes.

[0058] Principle of the test: the present application adopts technology to amplify and detect human Y chromosome STS sequence, according to the 2013 version of Y chromosome microdeletion detection guidelines issued by the European Association of Assisted Reproduction (EAA) and EMQN, the recommended classic 6 sites (sY86, sY84, sY134, sY127, sY255, sY254), and the male sex determination gene SRY, human zinc finger protein gene ZFX / Y double target monitoring sample collection and extraction process, the probe is labeled with FAM, under the condition of 39℃ optimal reaction temperature, the recombination enzyme and the primer form a protein-DNA complex, after searching for the homologous sequence on the DNA template, the DNA double strand is opened, the upstream and downstream primers start to specifically recognize the nucleic acid target base sequence, under the synergistic action of single-strand binding protein, the polymerase synthesizes DNA according to the Watson-Crick base pairing principle from 5' to 3', so as to realize the exponential amplification of the target region. The fluorescent probe carries out real-time fluorescence detection on the amplification process. The human Y chromosome microdeletion detection kit can be prepared according to table 5.

[0059] Table 5 Composition of human Y chromosome microdeletion detection kit

[0060]

[0061]

[0062] The dissolving agent contains Tris 10-60mM; potassium acetate 50-150mM; 2.5%-7.5% polyethylene glycol, preferably the molecular weight of polyethylene glycol is 20000-35000; the activator is 80-160mM magnesium acetate, preferably the magnesium acetate in the activator is 140mM. The reaction final concentration of the upstream primer and the downstream primer in each primer-probe combination in the kit is 300nM-500nM, and the reaction final concentration of the probe is 60nM-240nM.

[0063] Example 2 Sample processing, detection method and result analysis

[0064] 1) Preparation of human peripheral blood sample genomic DNA

[0065] Commercial nucleic acid rapid release agent is used for sample processing and DNA extraction, and the steps of releasing nucleic acid from EDTA anticoagulated blood sample are as follows:

[0066] a) The EDTA anticoagulated blood sample is first diluted 10 times with DEPC water.

[0067] b) Take 50μL of the diluted and treated blood sample, add 150μL of sample release agent, and mix evenly.

[0068] c) After mixing, stand for 3 minutes and take the supernatant.

[0069] Sample requirement: peripheral whole blood, sample collection requirement: EDTA anticoagulant tube, after collection, the collection tube needs to be inverted and mixed for 3-4 times to make the blood fully mixed with the anticoagulant. The whole blood sample is recommended to be stored at -20°C or below for 6 months, and repeated freeze-thawing is not more than 5 times.

[0070] Sample processing: commercial whole blood genomic DNA extraction kit is selected, and the extracted DNA OD 260 / OD 280 is between 1.6-2.0. The extracted DNA is used for detection within 48 hours, and is stored at 4°C. If the detection time is delayed to more than 48 hours, it is stored at -20°C or below for standby, and the storage validity period is not more than half a year.

[0071] 2) Reagent use method

[0072] a) Add 6 μL of dissolving agent to each of the reaction tubes (A-H) preloaded with enzyme freeze-dried mixture in turn.

[0073] b) Take 3 μL of the sample treated with sample release agent, and add it to each of the reaction tubes (A-H) in turn; or 3 μL of negative / positive control (G, H), the positive control is normal male whole blood extracted genomic DNA, and the negative control is normal female whole blood extracted genomic DNA.

[0074] c) Add 1 μL of activator to the inside of the cover of each reaction tube and mix (preferably invert the reaction tube up and down and shake for 8-10 times for mixing).

[0075] d) After mixing, shake the reaction liquid to the bottom of the tube (or centrifuge quickly), and then immediately place the reaction tube into the fluorescence detection equipment for amplification fluorescence signal collection.

[0076] 3) Amplification procedure

[0077] The fluorescence detection procedure is set as: constant temperature 39°C; collect fluorescence signal every 30 seconds, for 30 times in succession; the fluorescence detection channel is FAM channel. The amplification temperature range can be 37°C-42°C, preferably 39°C.

[0078] Suitable instrument: fluorescence constant temperature amplifier, ABI 7500, ABI Q3.

[0079] 4) Result analysis, which can be compared with Table 6 for detection result.

[0080] Table 6 Test result explanation

[0081]

[0082] From the above Table 6, using constant temperature amplification method for amplification, various types of AZFa, AZFb, AZFc, AZFb+c, AZFa+b+c deletion and female samples can be judged.

[0083] Deletion determination: the reaction unit in which no fluorescence signal curve is produced indicates that the corresponding AZF region is deleted.

[0084] Performance index of product of Example 3

[0085] 1. Sensitivity analysis

[0086] The normal male sample was selected for sensitivity analysis, and the detection reagent of the application can stably detect the minimum concentration of human genomic DNA of 0.2 ng / μL. Figure 1 The amplification test results of the minimum concentration detection limit of the normal male blood sample of the application kit (0.2 ng / μL).

[0087] 2. Specificity

[0088] Different samples were detected by the detection reagent of the application:

[0089] 1. Amplification of normal male samples

[0090] Figure 2 The amplification results of the normal male sample are as follows.

[0091] A B C D E F G H Detected Detected Detected Detected Detected Detected Detected Detected

[0092] 2. Amplification of AZFa region deletion samples

[0093] Figure 3 The amplification results of the AZFa region deletion sample are as follows.

[0094] A B C D E F G H Not detected Not detected Detected Detected Detected Detected Detected Detected

[0095] 3. Amplification of AZFb region deletion samples

[0096] Figure 4 The amplification results of the AZFb region deletion sample are as follows.

[0097] A B C D E F G H Detected Detected Not detected Not detected Detected Detected Detected Detected

[0098] 4. Amplification of AZFc region deletion samples

[0099] Figure 5 The amplification results of the AZFc region deletion sample are as follows.

[0100] A B C D E F G H Detected Detected Detected Detected Not detected Not detected Detected Detected

[0101] 5) Amplification of samples with AZFb+c deletion

[0102] Figure 6 The amplification results of the samples with AZFb+c region deletion are as follows.

[0103] A B C D E F G H Detected Detected Not detected Not detected Not detected Not detected Detected Detected

[0104] 6) Amplification of samples with AZFa+b+c deletion

[0105] Figure 7 The amplification results of the samples with AZFa+b+c region deletion are as follows.

[0106] A B C D E F G H Not detected Not detected Not detected Not detected Not detected Not detected Detected Detected

[0107] 7) Amplification of normal female samples

[0108] Figure 8 The amplification results of the normal female samples are as follows.

[0109] A B C D E F G H Not detected Not detected Not detected Not detected Not detected Not detected Not detected Detected

[0110] 8) Amplification with sterile water as template

[0111] Figure 9 The amplification results of the blank experiment with sterile water as template are as follows.

[0112] A B C D E F G H Not detected Not detected Not detected Not detected Not detected Not detected Not detected Not detected

[0113] 3) Reproducibility

[0114] Two concentrations (2 ng / μL and 0.2 ng / μL) of normal male DNA samples were selected for amplification detection. In different detection institutions, 3 groups of samples were each tested twice, and each test was repeated 4 times for different concentrations. The results were consistent under different experimental conditions. All STS sites could be effectively detected.

[0115] 4) Stability

[0116] The reagent should be stored at -20°C away from light, and the effective period is 6 months.

[0117] The un-used product after opening can be stored at 2-8°C for 3 days.

[0118] After reconstitution, the sample addition should be completed within 10 minutes, and the detection should be performed as soon as possible.

[0119] The freeze-dried enzyme reaction tube should not be repeated freeze-thawed more than 3 times.

[0120] Example 4

[0121] Determine the best amplification reaction volume, under the condition of the same amount of nucleic acid template, amplify the effect of different volume reaction comparison. Test results as shown in Figure 10 Not detected Not detected Not detected Not detected Not detected Not detected Not detected Detected As shown, the 10 μl system under the amplification fluorescence signal intensity and peak time are better than 50 μl system and 25 μl system.

[0122]

[0123]

[0124] Among them, the primer probe mix is configured in the proportion of 2 μl upstream primer, 2 μl downstream primer and 0.6 μl probe.

[0125] Finally, it should be pointed out that the above preferred embodiments are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present application.

Claims

1. A primer-probe composition for rapid isothermal detection of human Y chromosome microdeletions, characterized in that, The primer and probe sequences in the primer-probe composition are as follows: 1) The upstream primer for specifically amplifying the SY84 region of AZFa is shown in SEQ ID NO:1, the downstream primer is shown in SEQ ID NO:2, and the fluorescent probe is shown in SEQ ID NO:25; 2) The upstream primer for specifically amplifying the SY86 region of AZFa is shown in SEQ ID NO:4, the downstream primer is shown in SEQ ID NO:5, and the fluorescent probe is shown in SEQ ID NO:26; 3) The upstream primer for specifically amplifying the SY127 region of AZFb is shown in SEQ ID NO:7, the downstream primer is shown in SEQ ID NO:8, and the fluorescent probe is shown in SEQ ID NO:27; 4) The upstream primer for specifically amplifying the SY134 region of AZFc is shown in SEQ ID NO:10, the downstream primer is shown in SEQ ID NO:11, and the fluorescent probe is shown in SEQ ID NO:28; 5) The upstream primer for specifically amplifying the SY254 region of AZFc is shown in SEQ ID NO:13, the downstream primer is shown in SEQ ID NO:14, and the fluorescent probe is shown in SEQ ID NO:29; 6) The upstream primer for specific amplification of SY255 is shown in SEQ ID NO:16, the downstream primer is shown in SEQ ID NO:17, and the fluorescent probe is shown in SEQ ID NO:30; 7) The upstream primer for specific amplification of SRY is shown in SEQ ID NO:19, the downstream primer is shown in SEQ ID NO:20, and the fluorescent probe is shown in SEQ ID NO:31; 8) The upstream primer for specific amplification of ZFX / Y is shown in SEQ ID NO:22, the downstream primer is shown in SEQ ID NO:23, and the fluorescent probe is shown in SEQ ID NO:

32.

2. A kit for rapid detection of human Y chromosome microdeletions at an isothermal temperature, characterized in that, Contains the primer-probe composition of claim 1.

3. The isothermal rapid detection kit for human Y chromosome microdeletions according to claim 2, characterized in that, The kit also includes dNTPs, buffer solutions, and engineered enzyme compositions.

4. The isothermal rapid detection kit for human Y chromosome microdeletions according to claim 3, characterized in that, The buffer solution consists of: polyethylene glycol 1.5-5%; Tris 10-50 mM; dithiothreitol 1-10 mM; ATP 1.5-3.5 mM; creatine phosphate disodium salt 20-75 mM; phosphokinase 10-200 ng / µL; trehalose 2.5-7.5%; and mannitol 40-50 ng / µL.

5. The isothermal rapid detection kit for human Y chromosome microdeletions according to claim 3, characterized in that, The engineered enzyme composition contains 100-500 ng / µL of recombinant enzyme; 100-200 ng / µL of single-stranded DNA binding protein; 50-250 ng / µL of DNA polymerase; and 200-300 ng / µL of exonuclease.

6. The isothermal rapid detection kit for human Y chromosome microdeletions according to claim 3, characterized in that, The kit also includes an activator.

7. The isothermal rapid detection kit for human Y chromosome microdeletions according to claim 6, characterized in that, The activator is magnesium acetate.

8. The isothermal rapid detection kit for human Y chromosome microdeletions according to claim 6, characterized in that, The reaction system of the kit is 10 µL, comprising: 5-6 µL of a mixture of dNTPs, buffer and engineered enzyme composition, 0.5-1 µL of activator, 2-3 µL of sample DNA, or 2-3 µL of negative / positive control, 0.2-0.4 µL of upstream primer, 0.08-0.12 µL of fluorescent probe, and ddH2O to bring the total to 10 µL.

Images