CN109136391B - Kit for evaluating fertility of patient by using ATP6 expression level and application thereof - Google Patents
Kit for evaluating fertility of patient by using ATP6 expression level and application thereof Download PDFInfo
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Abstract
The invention relates to application of ATP6 expression level as patient fertility evaluation and a kit thereof, which firstly proves that the ATP6 expression level can be used as a reference index for evaluating the fertility of a patient with normal semen parameters, and completes the detection of ATP6 expression level by purifying semen, extracting sperm RNA for sequencing, synthesizing cDNA, and carrying out fluorescence real-time quantitative PCR detection by using a SYBRGreen method, thereby diagnosing the patient with normal semen parameters and abnormal fertility. Its advantages are: (1) the invention provides a new application of ATP6 expression level. (2) The sample is convenient, easily detects. (3) The invention can provide theoretical basis for treating abnormal fertility and has good application prospect.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a kit for evaluating the fertility of a patient by taking an ATP6 expression level and application thereof.
Background
The data from the past 20 years shows that approximately 30% of these clinical cases occur in men alone and 20% in people with abnormalities in both couples. Thus, men are responsible for at least half of the total 50% of infertile couples. Male infertility is an important medical problem affecting most people. The causes of approximately half of male infertility often do not show any specific cause determined even after a comprehensive examination. Most of the current studies on these factors have been at the monogenic level and at the genomic level. Studies have shown that male infertility is not an independent disease, but a result of multiple diseases or a combination of multiple factors. Semen routine is a method commonly used in andrology laboratories for assessing male fertility, and the ability of corresponding semen to enable a female to conceive can be assessed through assessment of parameters such as sperm concentration, motility and morphology. There are still situations in the clinic where the semen parameters are normal, but the female is not allowed to conceive. A batch of genes with different expressions in two groups of sperms are found by extracting sperm RNA for sequencing through purifying sperms with normal semen parameters but with two different birth fates.
The Chinese patent application: CN1790022A discloses a sperm binding antibody detection reagent, which is characterized in that: the reagent is a liquid double reagent consisting of sensitized carboxylation latex and goat anti-human gamma globulin serum, and the sensitized carboxylation latex contains carboxylation latex for marking human gamma globulin. The invention has the following advantages: can meet the research and clinical requirements of various domestic hospitals and family planning departments, provides important basis for diagnosing the male immune infertility and whether the male can recover the fertility after receiving the vas deferens reproduction, has larger economic value, and has simple, convenient, rapid, sensitive and specific operation.
The Chinese patent application: CN101563134A discloses a pharmaceutical composition for the treatment of male sub-fertility comprising an agent causing a reduction in the effect of extracellular DNA on sperm cells. The agent may be, for example, an enzyme that degrades DNA (e.g., dnase), a substance that blocks the interaction between cell-free DNA and sperm cell surface receptors, a substance that binds DNA, a substance that inhibits endogenous sperm cell dnase, a substance that inhibits a member of a signaling pathway mediated by DNA bound to sperm cell surface receptors, or an agent that stimulates the production of endogenous substances that cause a reduction in the antifertility effect of cell-free DNA on sperm cells. The invention also provides a method of treating male sub-fertility comprising administering the pharmaceutical composition of the invention. The invention also provides methods of determining the fertility status of a male subject, methods of assisted reproduction, methods of selecting an Assisted Reproduction Technology (ART), and methods of selecting sperm cells in a population of sperm cells for use in an assisted reproduction technology. However, the ATP6 expression level as a kit for evaluating the fertility of patients and the application thereof are not reported at present.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides application of a reagent for detecting the expression level of ATP 6.
In order to achieve the purpose, the invention adopts the technical scheme that:
use of a reagent for detecting the expression level of ATP6 for the manufacture of a diagnostic kit for detecting fertility disorders in a patient.
As a preferred embodiment of the invention, the disease is a disease where the semen parameters are normal but the fertility is abnormal.
As a preferred embodiment of the present invention, the reagent is a real-time quantitative PCR detection reagent.
As a preferred embodiment of the invention, the real-time quantitative PCR detection reagent for detecting the expression level of ATP6 comprises a primer pair with the sequences shown as SEQ ID NO.1 and SEQ ID NO. 2.
As a preferred embodiment of the present invention, the diagnostic kit comprises a reagent for detecting the expression level of ACTB.
In a preferred embodiment of the present invention, the reagent for detecting the expression level of ACTB is a real-time quantitative PCR detection reagent.
As a preferred embodiment of the present invention, the real-time quantitative PCR detection reagent for detecting the expression level of ACTB comprises a primer pair having the sequences shown in SEQ ID NO.3 and SEQ ID NO. 4.
As a preferred embodiment of the invention, the reagent further comprises a purified semen reagent, a sperm RNA extraction reagent and a cDNA synthesis reagent.
As a preferred embodiment of the present invention, the diagnostic kit comprises instructions carrying: when the subject's sperm ATP6/ACTB <5, the subject is at potential risk of infertility even if the relevant semen parameters are normal.
As a preferred embodiment of the present invention, the step of using the reagent comprises:
(1) purifying the sperms by using a gradient density centrifugation method;
(2) extracting sperm RNA;
(3) cDNA synthesis was accomplished using a ThermocDNA first Strand Synthesis kit;
(4) fluorescence real-time quantitative PCR detection was performed using the SYbrGreen method.
As a preferred embodiment of the present invention, the method of the step (1) is: adding 0.5mL of 90% gradient liquid into a 5mL round-bottom centrifuge tube, covering 0.5mL of 45% gradient liquid on the centrifuge tube, carefully covering 1mL of liquefied semen on the gradient liquid, centrifuging for 20-30min at 600G, carefully absorbing the semen liquid layer and the 45% gradient liquid layer, removing cells at the junction of layers, adding one-time volume of HTF solution containing 5% HSA to wash the semen settled in the 90% layers, centrifuging for 20min at 600G, removing supernatant, and cracking the precipitate by using 500uLTrizol reagent; the sperm in step (1) excludes hemospermia sperm, because the erythrocyte has similar sedimentation coefficient with the sperm, and the abundance of the related gene can be influenced by excessive erythrocyte.
As a preferred embodiment of the present invention, the method used in the step (2) is a Trizol method or a centrifugal column method, and the Trizol method is: after the semen is purified, centrifuging to remove supernatant, adding 500uLTrizol reagent, blowing, beating, precipitating, resuspending and mixing uniformly, standing at room temperature for 5min to lyse cells, adding 100uL chloroform, shaking for 15 seconds, standing at room temperature for 10min, standing at 4 ℃, 12000g, centrifuging for 15min, layering liquid, taking upper aqueous phase, adding 250uL isopropanol, standing at room temperature for 10min, standing at 4 ℃, 12000g, centrifuging for 10min, removing supernatant, adding 500uL 75% ethanol (prepared by RNase-free water), shaking, suspending and precipitating, centrifuging for 5min at 8000g, removing supernatant, drying for several minutes to remove ethanol, adding 10-15uLRNase-free water, dissolving and precipitating, taking 1uL, and determining RNA concentration and quality by using nanodrop; the centrifugal column method comprises the following steps: adding RLT lysate to 350uL of upper aqueous phase obtained by the first centrifugation step of the Trizol method, adding 350uL 70% ethanol (prepared by using RNase-free water), blowing and uniformly mixing, adding to an RNeasy MiniElutespnapin column, closing a cover, centrifuging for 15 seconds, discarding waste liquid, adding 350uLRW1 liquid, closing the cover, centrifuging for 15 seconds, discarding waste liquid, repeating the steps once, replacing a new 2mL collecting tube, adding 500uLRPE buffer solution, closing the cover, centrifuging for 15 seconds, discarding waste liquid, adding 500uL 80% ethanol (prepared by using RNase-free water), closing the cover, centrifuging for 2min at 80000g, discarding waste liquid, opening the cover, centrifuging for 5min at full speed, discarding the collecting tube, replacing a 1.5mL collecting tube, adding 14uLRNase-free water, closing the cover, centrifuging for 1min, collecting eluent, and taking 1uL of eluent to determine the RNA concentration and the mass by using nanodrop.
As a preferred embodiment of the present invention, the method of the step (3) is: taking 0.5-5ug RNA, adding 1uL of primer (OligodT or RandomPrimer,100 mu M), supplementing 12uL with RNase-free water, carrying out water bath at 65 ℃ for 5min, immediately inserting ice for cooling for 2min, adding 4uL5X reaction buffer solution, 1uLRiboLockRNA inhibitor, 2uLdNTP (10mM) and 1 uLReverAidM-MuLV reverse transcriptase, mixing uniformly, incubating at 25 ℃ for 5min, then carrying out water bath at 42 ℃ for 1h, terminating the reaction in 70 ℃ water bath for 5min after the reaction is finished, storing the product at-20 ℃, and obtaining a reaction system which can be 2.
As a preferred embodiment of the present invention, the step (4) is to use ACTB as an internal reference; the method of the step (4) is as follows: fluorescent real-time quantitative PCR validation was performed using ABI 2 xsybgreeenmix (similar products from other companies may also be used), each reaction system (96 module 20uL) was formulated as follows: 10uL2 XSYbrGreenMux, 8uL double distilled water, 1uL primer (10 mu M of each upstream and downstream), 1uL template (about 100ng cDNA in each reaction system, which can be adjusted according to specific product quantity), reaction system equal proportion halving when 386 modules are used, 95 ℃ denaturation is set on a fluorescence quantitative PCR instrument for 15 seconds, 60 ℃ annealing/extension is set for 1min, a fluorescence signal is collected after the extension is finished, 40 cycles are totally carried out, the relative ACTB quantity of ATP6 is calculated by using the collected CT value, and the formula is calculated: m isATP6/mACTB＝2^(CTACTB-CTATP6) And when the calculation result is as follows:
ATP6/ACTB <5, indicates that the subject is potentially at risk of infertility even if the relevant semen parameters are normal.
The invention has the advantages that:
1. the invention provides a new application of ATP6, and proves that the expression level of ATP6 can be used as a reference index for evaluating the fertility of a person with normal semen parameters for the first time.
2. The sample is convenient, easily detects.
3. The invention can provide theoretical basis for treating abnormal fertility and has good application prospect.
Drawings
FIG. 1 is a graph showing the results of the experiment in example 3.
Detailed Description
The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.
EXAMPLE 1 preparation of diagnostic kit for detecting fertility disorders in patients
The manufacturing process and the operation flow of the diagnostic kit for detecting the fertility diseases of the patients are mainly based on the PCR technology.
A kind of sperm mRNA related to a patient with abnormal fertility is screened by technologies such as quantitative PCR and the like and is used as an index for predicting whether the patient has the diseases of normal semen parameters and abnormal fertility.
The kit comprises a batch of primers, wherein the primer sequences are as follows:
fluorescence real-time quantitative PCR detection was performed using SYBRGreen method, and the relative amount of ATP6 to ACTB was calculated using the collected CT values, using the formula: m isATP6/mACTB＝2^(CTACTB-CTATP6) If the calculation result is ATP6/ACTB<And 5, indicating that the subject has potential sterility risk even if the relevant semen parameters are normal.
EXAMPLE 2 method of Using diagnostic kit for detecting fertility disorders in patients
The diagnostic kit for detecting fertility disorders in a patient is used as follows:
(1) sperm were purified using gradient density centrifugation: adding 0.5mL of 90% gradient liquid into a 5mL round-bottom centrifuge tube, covering 0.5mL of 45% gradient liquid on the centrifuge tube, carefully covering 1mL of liquefied semen on the gradient liquid, centrifuging for 20-30min at 600G, carefully absorbing the semen liquid layer and the 45% gradient liquid layer, removing cells at the junction of layers, adding one-time volume of HTF solution containing 5% HSA to wash the semen settled in the 90% layers, centrifuging for 20min at 600G, removing supernatant, and cracking the precipitate by using 500uLTrizol reagent;
(2) extracting sperm RNA by adopting a Trizol method or a centrifugal column method: the Trizol method is as follows: after the semen is purified, centrifuging to remove supernatant, adding 500uLTrizol reagent, blowing, beating, precipitating, resuspending and mixing uniformly, standing at room temperature for 5min to lyse cells, adding 100uL chloroform, shaking for 15 seconds, standing at room temperature for 10min, standing at 4 ℃, 12000g, centrifuging for 15min, layering liquid, taking upper aqueous phase, adding 250uL isopropanol, standing at room temperature for 10min, standing at 4 ℃, 12000g, centrifuging for 10min, removing supernatant, adding 500uL 75% ethanol (prepared by RNase-free water), shaking, suspending and precipitating, centrifuging for 5min at 8000g, removing supernatant, drying for several minutes to remove ethanol, adding 10-15uLRNase-free water, dissolving and precipitating, taking 1uL, and determining RNA concentration and quality by using nanodrop; the centrifugal column method comprises the following steps: adding RLT lysate to 350uL of upper aqueous phase obtained by the first centrifugation step of the Trizol method, adding 350uL 70% ethanol (prepared by using RNase-free water), blowing and uniformly mixing, adding to an RNeasy MiniElutespnapin column, closing a cover, centrifuging for 15 seconds, discarding waste liquid, adding 350uLRW1 liquid, closing the cover, centrifuging for 15 seconds, discarding waste liquid, repeating the steps once, replacing a new 2mL collecting tube, adding 500uLRPE buffer solution, closing the cover, centrifuging for 15 seconds, discarding waste liquid, adding 500uL 80% ethanol (prepared by using RNase-free water), closing the cover, centrifuging for 2min at 80000g, discarding waste liquid, opening the cover, centrifuging for 5min at full speed, discarding the collecting tube, replacing a 1.5mL collecting tube, adding 14uLRNase-free water, closing the cover, centrifuging for 1min, collecting eluent, and taking 1uL of eluent to determine the RNA concentration and the mass by using nanodrop.
(3) cDNA synthesis was performed using a thermodna first strand synthesis kit: taking 0.5ug RNA, adding 1uL of primer (OligodT or RandomPrimer,100 mu M), supplementing 12uL with RNase-free water, carrying out water bath at 65 ℃ for 5min, immediately inserting ice for cooling for 2min, adding 4uL5X reaction buffer solution, 1uLRiboLockRNA inhibitor, 2uLdNTP (10mM) and 1 uLReverAidM-MuLV reverse transcriptase, mixing uniformly, incubating at 25 ℃ for 5min, then carrying out water bath at 42 ℃ for 1h, terminating the reaction in 70 ℃ water bath for 5min after the reaction is finished, storing the product at-20 ℃, and enabling the reaction system to be equal to 2.
(4) Fluorescence real-time quantitative PCR detection was performed using the SYbrGreen method: fluorescent real-time quantitative PCR validation was performed using ABI 2 xsybgreeenmix (similar products from other companies may also be used), each reaction system (96 module 20uL) was formulated as follows: 10uL2 XSYbrGreenMux, 8uL double distilled water, 1uL primer (10 mu M of each upstream and downstream), 1uL template (about 100ng cDNA in each reaction system, which can be adjusted according to specific product quantity), reaction system equal proportion halving when 386 modules are used, 95 ℃ denaturation is set on a fluorescence quantitative PCR instrument for 15 seconds, 60 ℃ annealing/extension is set for 1min, a fluorescence signal is collected after the extension is finished, 40 cycles are totally carried out, the relative ACTB quantity of ATP6 is calculated by using the collected CT value, and the formula is calculated: m isATP6/mACTB＝2^(CTACTB-CTATP6)。
The primer sequences used in the experimental procedure were:
example 3 clinical validation
The experiments in this example were all performed under the premise of the consent of the inventor, and signed with the consent form.
1 data of the experiment
Semen from adult males with normal parameters and normal fertility and from adult male patients with normal parameters and fertility defects, which were diagnosed by the hospital, were collected and divided into normal and patient groups, respectively.
2 method of experiment
Sperm cells were processed separately for normal and patient groups as follows:
(1) sperm were purified using gradient density centrifugation: adding 0.5mL of 90% gradient liquid into a 5mL round-bottom centrifuge tube, covering 0.5mL of 45% gradient liquid on the centrifuge tube, carefully covering 1mL of liquefied semen on the gradient liquid, centrifuging for 20-30min at 600G, carefully absorbing the semen liquid layer and the 45% gradient liquid layer, removing cells at the junction of layers, adding one-time volume of HTF solution containing 5% HSA to wash the semen settled in the 90% layers, centrifuging for 20min at 600G, removing supernatant, and cracking the precipitate by using 500uLTrizol reagent;
(2) extracting sperm RNA by using a Trizol method: after the semen is purified, centrifuging to remove supernatant, adding 500uLTrizol reagent, blowing, beating, precipitating, resuspending and mixing uniformly, standing at room temperature for 5min to lyse cells, adding 100uL chloroform, shaking for 15 seconds, standing at room temperature for 10min, standing at 4 ℃, 12000g, centrifuging for 15min, layering liquid, taking upper aqueous phase, adding 250uL isopropanol, standing at room temperature for 10min, standing at 4 ℃, 12000g, centrifuging for 10min, removing supernatant, adding 500uL 75% ethanol (prepared by RNase-free water), shaking, suspending and precipitating, centrifuging for 5min at 8000g, removing supernatant, drying for several minutes to remove ethanol, adding 10-15uLRNase-free water, dissolving and precipitating, taking 1uL, and determining RNA concentration and quality by using nanodrop;
(3) cDNA synthesis was performed using a thermodna first strand synthesis kit: taking 0.5ug RNA, adding 1uL of primer (OligodT or RandomPrimer,100 mu M), supplementing 12uL with RNase-free water, carrying out water bath at 65 ℃ for 5min, immediately inserting ice for cooling for 2min, adding 4uL5X reaction buffer solution, 1uLRiboLockRNA inhibitor, 2uLdNTP (10mM) and 1 uLReverAidM-MuLV reverse transcriptase, mixing uniformly, incubating at 25 ℃ for 5min, then carrying out water bath at 42 ℃ for 1h, terminating the reaction in 70 ℃ water bath for 5min after the reaction is finished, storing the product at-20 ℃, and enabling the reaction system to be equal to 2.
(4) Fluorescence real-time quantitative PCR detection was performed using the SYbrGreen method: ACTB was used as an internal control, and fluorescent real-time quantitative PCR was verified using 2XSYbrGreenMix, ABI, each reaction system (96 modules 20uL) was formulated as follows: 10uL of 2 XSYbrGreenMux, 8uL of double distilled water, 1uL of primers (10 mu M of each of the upstream and downstream), 1uL of template (about 100ng of cDNA in each reaction system, which can be adjusted according to specific product amount), and the equal ratio of the reaction systems when 386 modules are usedHalving, setting 95 ℃ denaturation for 15 seconds, 60 ℃ annealing/extension for 1min on a fluorescent quantitative PCR instrument, collecting a fluorescent signal after the extension is finished, and calculating the relative ACTB amount of ATP6 by using the collected CT value for 40 cycles, wherein the calculation formula is as follows: m isATP6/mACTB＝2^(CTACTB-CTATP6)。
The primer sequence is as follows:
sperm from the control group were treated identically to sperm from the experimental group.
3 results of the experiment
The results are shown in figure 1 and show that the patients group sperm have ATP6/ACTB <5, indicating that the subject is potentially at risk of infertility even if the relevant semen parameters are normal.
The ATP6/ACTB of the sperm in the normal group is more than or equal to 5, which indicates that the semen parameters are normal and the subject has no potential sterility risk.
4 conclusion of the experiment
It is shown by the above examples that ATP6 can be used as a marker for detecting diseases with normal semen parameters and abnormal fertility, and the ATP6 expression level can be used as a reference index for evaluating the fertility of a person with normal semen parameters.
The invention provides a new application of ATP6, and proves that the expression level of ATP6 can be used as a reference index for evaluating the fertility of a person with normal semen parameters for the first time; the sampling is convenient and the detection is easy; can provide theoretical basis for treating abnormal fertility and has good application prospect.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and additions can be made without departing from the principle of the present invention, and these should also be considered as the protection scope of the present invention.
SEQUENCE LISTING
<110> first-person hospital in Shanghai City
Application of <120> ATP6 expression level as patient fertility assessment and kit thereof
<130> /
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213> Artificial sequence
<400> 1
tacgcctaac cgctaacatt ac 22
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence
<400> 2
gcgacagcga tttctaggat ag 22
<210> 3
<211> 18
<212> DNA
<213> Artificial sequence
<400> 3
cctggcaccc agcacaat 18
<210> 4
<211> 18
<212> DNA
<213> Artificial sequence
<400> 4
gggccggact cgtcatac 18
Claims (2)
1. Use of a reagent for detecting the expression level of ATP6 in the manufacture of a diagnostic kit for detecting fertility disorders in a patient, wherein the fertility disorder is a disorder of normal semen parameters but abnormal fertility and the subject is a sperm of the semen, the diagnostic kit comprising instructions for carrying: when the subject's sperm ATP6/ACTB <5, the subject is at potential risk of infertility even if the relevant semen parameters are normal;
the reagent is a real-time quantitative PCR detection reagent, and the real-time quantitative PCR detection reagent for detecting the expression level of ATP6 comprises a primer pair with sequences shown as SEQ ID NO.1 and SEQ ID NO. 2; the diagnostic kit also comprises a reagent for detecting the expression level of the ACTB, the reagent for detecting the expression level of the ACTB is a real-time quantitative PCR detection reagent, and the real-time quantitative PCR detection reagent for detecting the expression level of the ACTB comprises a primer pair with sequences shown as SEQ ID NO.3 and SEQ ID NO. 4.
2. The use of claim 1, wherein the reagent further comprises a purified semen reagent, a sperm RNA extraction reagent, a cDNA synthesis reagent.
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| CN105039530A (en) * | 2015-07-03 | 2015-11-11 | 南京医科大学 | Mitochondria-related seminal plasma miRNAs taken as mankind severe asthenospermia markers, and applications thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105039530A (en) * | 2015-07-03 | 2015-11-11 | 南京医科大学 | Mitochondria-related seminal plasma miRNAs taken as mankind severe asthenospermia markers, and applications thereof |
Non-Patent Citations (4)
| Title |
|---|
| Biallelic Mutations in CFAP43 and CFAP44 Cause Male Infertility with Multiple Morphological Abnormalities of the Sperm Flagella;Shuyan Tang et,al.;《Am J Hum Genet.》;20170630;第100卷(第6期);854-864 * |
| 不育男性精子线粒体DNA突变与线粒体超微结构改变;王咏梅等;《解剖学报》;20010430;第32卷(第2期);第184-186页 * |
| 弱精子症患者精子线粒体 MTCYB、MTATP6基因的检测;冯春琼等;《中华男科学杂志》;20080430;第14卷(第4期);第321-323页 * |
| 梗阻性无精子症患者睾丸精子左旋肉碱培养后关键基因表达量的变化研究;师娟子等;《中华男科学杂志》;20101231;第16卷(第6期);摘要、第508页右栏第二段-最后一行、表1 * |
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