CN112458167A - Product for detecting sperm quality and use method thereof - Google Patents
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Abstract
The invention relates to a product for detecting sperm quality and a using method thereof, belonging to the technical field of medicines. The invention provides a product for detecting sperm quality, which takes hsa _ piR _016118, hsa _ piR _017507 and/or hsa _ piR _006215 as piRNA markers related to sperm quality, wherein the expression level of hsa _ piR _016118 has strong correlation with normal sperm motility, the expression levels of hsa _ piR _017507 and hsa _ piR _006215 have strong correlation with normal sperm morphology, and seminal plasma is present in all mature males and is sufficient in spermatic plasma even in oligozoospermia males, so that the product can be used for accurately detecting male patients with sufficient sperms and oligospermia patients, and has a wide application range.
Description
Technical Field
The invention relates to a product for detecting sperm quality and a use method thereof, belonging to the technical field of chemistry.
Background
According to investigation, the infertility rate in China currently exceeds 15% on average, about 2000 ten thousand pairs of nationwide infertile couples exist, the infertility caused by the reproductive health reasons of men accounts for about 30%, and compared with the prior art, the proportion of men is increased, and the problem of male infertility accounts for more and more serious social problems. However, after the birth control policy of China is comprehensively implemented, the requirements of people on good prenatal and postnatal care are higher and higher. Therefore, it is important to accurately measure the quality of male sperm to ensure the quality of fertility.
The quality of male sperm mainly includes two aspects of vitality and morphology. At present, the quality of male sperms is mainly detected by the following method in domestic clinic: the sperm motility is judged by counting the number of the forward motile sperms, then the Shorr method is used for judging whether the sperm morphology is normal, and finally the sperm quality of the male is judged by integrating the results of the two aspects of the motility and the morphology. However, this method requires a sufficient amount of sperm in the male subject, and if the subject is an oligozoospermia patient, it is difficult to make an accurate and thorough diagnosis. Therefore, it is urgently needed to find an accurate and comprehensive sperm quality detection method suitable for patients with sufficient sperm quantity and patients with oligospermia.
Disclosure of Invention
In order to solve the above problems, the present invention provides a product for detecting sperm quality, comprising hsa _ piR _016118, a specific primer for amplifying hsa _ piR _016118, and/or a reverse transcription primer for reverse transcribing hsa _ piR _016118 into cDNA;
the product comprises hsa _ piR _017507, a specific primer for amplifying hsa _ piR _017507 and/or a reverse transcription primer for reverse transcribing hsa _ piR _017507 into cDNA;
and/or the product comprises hsa _ piR _006215, a specific primer for amplifying hsa _ piR _006215 and/or a reverse transcription primer for reverse transcribing hsa _ piR _006215 into cDNA.
In one embodiment of the invention, the nucleotide sequence of hsa _ piR _016118 is set forth in SEQ ID NO 1; the nucleotide sequence of hsa _ piR _017507 is shown as SEQ ID NO. 2; the nucleotide sequence of hsa _ piR _006215 is shown as SEQ ID NO. 3.
In one embodiment of the invention, the nucleotide sequences of the specific primers used for amplification of hsa _ piR _016118 are shown as SEQ ID NO. 4 and SEQ ID NO. 5; the nucleotide sequence of the specific primer for amplifying hsa-piR-017507 is shown as SEQ ID NO. 6 and SEQ ID NO. 7; the nucleotide sequences of the specific primers for amplifying hsa-piR-006215 are shown as SEQ ID NO. 8 and SEQ ID NO. 9. Since piRNA is a very small RNA, which is reverse transcribed by PolyT after adding a polyA tail to the piRNA before reverse transcription, AAAAAAAAAA is used as the right primer and the left primer is specific to each piRNA when amplifying hsa-piR-016118, hsa-piR-017507, and hsa-piR-006215 by PCR.
In one embodiment of the invention, the nucleotide sequence of the reverse transcription primer for reverse transcription of hsa _ piR _016118 into cDNA is set forth in SEQ ID NO. 10; the nucleotide sequence of the reverse transcription primer for reverse transcribing hsa-piR-017507 into cDNA is shown as SEQ ID NO. 10; the nucleotide sequence of the reverse transcription primer for reverse transcribing hsa-piR-006215 into cDNA is shown in SEQ ID NO. 10.
In one embodiment of the invention, the product comprises a reverse transcription primer for reverse transcribing hsa _ piR _016118 to cDNA;
the product comprises a reverse transcription primer for reverse transcribing hsa _ piR _017507 into cDNA;
and/or the product comprises a reverse transcription primer for reverse transcribing hsa _ piR _006215 into cDNA.
In one embodiment of the invention, the product is a detection reagent or a detection kit.
In one embodiment of the invention, the sperm quality refers to the motility and morphology of the sperm.
The invention also provides a using method of the product, which comprises the steps of taking seminal plasma, extracting hsa _ piR _016118 in the seminal plasma, and then carrying out reverse transcription on the extracted hsa _ piR _016118 into cDNA by using a reverse transcription primer for carrying out reverse transcription on hsa _ piR _016118 into cDNA;
the using method comprises the steps of taking seminal plasma, firstly extracting hsa _ piR _017507 from the seminal plasma, and then carrying out reverse transcription on the extracted hsa _ piR _017507 into cDNA by using a reverse transcription primer for carrying out reverse transcription on hsa _ piR _017507 into the cDNA;
and/or the using method is that seminal plasma is taken, hsa _ piR _006215 in the seminal plasma is extracted firstly, and then the extracted hsa _ piR _006215 is subjected to reverse transcription into cDNA by using a reverse transcription primer for carrying out reverse transcription of hsa _ piR _006215 into cDNA.
In one embodiment of the invention, the using method is seminal plasma taking, firstly, an miRNA extraction kit is used for extracting hsa _ piR _016118 from the seminal plasma, then, a reverse transcription primer for reverse transcribing hsa _ piR _016118 into cDNA is used for reverse transcribing the extracted hsa _ piR _016118 into cDNA, and finally, the expression level of hsa _ piR _016118 in the seminal plasma is detected by RT-qPCR (reverse transcription-quantitative polymerase chain reaction) according to the cDNA obtained by reverse transcription;
the using method comprises the steps of taking seminal plasma, extracting hsa _ piR _017507 in the seminal plasma by using a miRNA extraction kit, then carrying out reverse transcription on the extracted hsa _ piR _017507 into cDNA by using a reverse transcription primer for carrying out reverse transcription on hsa _ piR _017507 into the cDNA, and finally detecting the expression quantity of hsa _ piR _017507 in the seminal plasma by RT-qPCR (reverse transcription-quantitative polymerase chain reaction) according to the cDNA obtained by the reverse transcription;
and/or the using method is to take seminal plasma, firstly extract hsa _ piR _006215 in the seminal plasma by using a miRNA extraction kit, then reverse transcribe the extracted hsa _ piR _006215 into cDNA by using a reverse transcription primer for reverse transcribing hsa _ piR _006215 into cDNA, and finally detect the expression level of hsa _ piR _006215 in the seminal plasma by RT-qPCR according to the cDNA obtained by reverse transcription.
In one embodiment of the invention, the using method is seminal plasma taking, firstly, the miRNA extraction kit is used for extracting hsa _ piR _016118 in the seminal plasma, then, the reverse transcription primer for reverse transcribing hsa _ piR _016118 into cDNA is used for reverse transcribing the extracted hsa _ piR _016118 into cDNA, and then, the expression quantity of hsa _ piR _016118 relative to the internal reference U6 in the seminal plasma is detected by RT-qPCR (2) according to the cDNA obtained by reverse transcriptionCt,U6-Ct,016118) Finally, the sperm quality is judged according to the expression quantity of hsa _ piR _016118 relative to the internal reference U6, the sperms of the male with the non-asthenospermia and the non-teratospermia correspond to the seminal plasma with the expression quantity of hsa _ piR _016118 being less than 0.86529154, the sperms of the male with the asthenospermia correspond to the sperms of the male with the non-asthenospermia and the expression quantity of hsa _ piR _016118 being more than 0.86529154, and the sperms of the male with the teratospermia correspond to the sperms of the male with the expression quantity of hsa _ piR _016118 being more than 5;
the application method comprises taking seminal plasma, and makingExtracting hsa _ piR _017507 in seminal plasma by using a miRNA extraction kit, then carrying out reverse transcription on the extracted hsa _ piR _017507 into cDNA by using a reverse transcription primer for carrying out reverse transcription on hsa _ piR _017507 into cDNA, and detecting the expression amount of hsa _ piR _017507 in seminal plasma relative to internal reference U6 by RT-qPCR (2)Ct,U6-Ct,016118) Finally, the sperm quality is judged according to the expression quantity of hsa _ piR _017507 relative to the internal reference U6, wherein the seminal plasma of hsa _ piR _017507, the expression quantity of which is more than 0.02405454, corresponds to the sperms of the male with non-asthenospermia and non-teratospermia, the seminal plasma of hsa _ piR _017507, the expression quantity of which is more than 0.02405454, corresponds to the sperms of the male with non-asthenospermia, and the expression quantity of hsa _ piR _017507, the expression quantity of which is more than 0.023850807, corresponds to the sperms of the male with non-teratospermia;
and/or the using method comprises the steps of taking seminal plasma, extracting hsa _ piR _006215 in the seminal plasma by using a miRNA extraction kit, then carrying out reverse transcription on the extracted hsa _ piR _006215 into cDNA by using a reverse transcription primer for carrying out reverse transcription on the hsa _ piR _006215 into the cDNA, and detecting the expression quantity of the hsa _ piR _006215 in the seminal plasma relative to the internal reference U6 by RT-qPCR (2)Ct,U6-Ct,016118) And finally, judging the sperm quality according to the expression level of hsa _ piR _006215 relative to the internal reference U6, wherein the seminal plasma of hsa _ piR _006215 with the expression level larger than 0.089511359 corresponds to the sperms of the male with non-asthenospermia and non-teratospermia, the seminal plasma of hsa _ piR _006215 with the expression level larger than 0.089511359 corresponds to the sperms of the male with non-asthenospermia, and the seminal plasma of hsa _ piR _006215 with the expression level larger than 0.090479887 corresponds to the sperms of the male with non-teratospermia. U6 has left and right primers, and the right primer is its reverse transcription primer, wherein the nucleotide sequence of the left primer is shown in SEQ ID NO. 11, the nucleotide sequence of the right primer is shown in SEQ ID NO. 12, and the nucleotide sequence of U6 is shown in SEQ ID NO. 13.
The invention also provides the application of the product or the method in the aspect of detecting the quality of the sperms.
In one embodiment of the invention, the sperm quality refers to the motility and morphology of the sperm.
The technical scheme of the invention has the following advantages:
the invention provides a product for detecting sperm quality, which takes hsa _ piR _016118, hsa _ piR _017507 and/or hsa _ piR _006215 as piRNA markers related to sperm quality, wherein the expression level of hsa _ piR _016118 has strong correlation with normal sperm motility, the correlation is as high as-0.94 (p =0.02), the expression level of hsa _ piR _017507 has strong correlation with normal sperm morphology, the correlation is as high as-0.604 (p = 0.049), the expression level of hsa _ piR _006215 has strong correlation with normal sperm morphology and is as high as-0.586 (p = 0.045), therefore, the quality of male sperm detected by using the product has the advantage of accurate detection result, and when the product is used for detecting the male sperm quality, only hsa _ piR _016118, hsa _ piR _006215 or hsa _ 638 _ 006215/8 is taken firstly, then the quality of male sperm in the male seminal plasma is detected, therefore, the product has the advantages of short period and simple steps when used for detecting the male sperm quality, in addition, seminal plasma is owned by all mature males, and even oligospermia males have enough seminal plasma, so the product can be used for accurately detecting male patients with enough sperms and also can be used for accurately detecting oligospermia patients, and the application range is wide.
Drawings
FIG. 1: number of differentially expressed piRNAs in seminal plasma of four subjects.
FIGS. 2 to 9: expression of different piRNAs in seminal plasma of subjects in four groups.
FIGS. 10 to 11: aspenozolsemia ROC profile.
FIGS. 12 to 13: teratozoospermia (teratozospermia) ROC graph.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The following examples do not show specific experimental procedures or conditions, and can be performed according to the procedures or conditions of the conventional experimental procedures described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
Experimental example 1: correlation experiments between different piRNAs and male sperm quality
1.1 Experimental materials
1.1.1 Primary reagents and consumables
1) Xylene, paraformaldehyde, potassium chloride, potassium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate anhydrous ethanol: chemical agents of the national drug group, ltd;
2) sperm staining solution kit: nanjing Xindi biological pharmaceutical Co., Ltd;
3) piRNAs extraction kit: beijing Aidlab, Inc.;
4) reverse transcription kit: beijing Aidlab, Inc.;
5) fluorescence quantitative PCR kit: beijing Aidlab, Inc.;
1.1.2 Main instruments and Equipment
1) Pressure steam sterilization pot: shanghai Shenan medical instruments factory;
2) electronic balance, PH meter: sartorius, germany;
3) a magnetic stirrer: shanghai Spire Instrument Co., Ltd;
4) a water purifier: millipore, USA;
5) a desk centrifuge: eppendorf corporation, USA;
6) fluorescence microscopy: olympus corporation, japan;
7) automatic sperm detection analyzer: beijing Cuikaga Co;
8) gradient PCR instrument: eppendorf corporation, USA;
9) fluorescent quantitative PCR instrument (via 7): life Technologies, USA;
1.2 Experimental methods
1.2.1 Collection of samples
The semen is collected in a semen collection cup by a subject through masturbation 4-6 days after abstinence before semen collection.
1.2.2 semen analysis
1.2.2.1 sperm motility assay
1) Liquefying the collected semen sample in a water bath kettle at 37 ℃ for 30 min;
2) gently blowing, beating and uniformly mixing, and pouring 3 mu L of liquefied semen into a sperm counting plate;
3) placing on an automatic sperm analyzer for automatic detection.
1.2.2.2 sperm morphology assay: shorr method
1) After the semen sample is liquefied, the semen sample is washed for 2 times by using normal saline to prepare a sperm suspension, smeared and naturally dried.
2) Dripping the dyeing solution A on the coating, dyeing for 1min, and washing off the redundant dyeing solution under running water;
3) dripping hydrochloric acid ethanol on the smear, standing for 15s at room temperature, and washing off the redundant staining solution under running water;
4) dripping the dyeing solution B on the coating, dyeing for 2min, and washing the redundant dyeing solution under running water;
5) and (5) performing microscopic examination after air drying.
1.2.3 extraction of plasma piRNAs
1) Adding 750 μ L lysine buffer into 250 μ L seminal plasma, vortex, shaking, mixing, and incubating at room temperature for 5 min;
2) adding 200 μ L chloroform (chloroform), shaking vigorously for 15s, and standing at room temperature for 2 min;
3) after centrifugation at 12000rpm for 10min at 4 ℃, the sample will be divided into three layers: a colorless aqueous phase, an intermediate layer and a lower organic phase. The piRNAs are present in the aqueous phase. The supernatant (approximately 500. mu.L) was carefully transferred to a new 1.5mL EP tube, 750. mu.L of room temperature absolute ethanol was added, and vortexed to mix. If the precipitate appears, blowing, beating and uniformly mixing the mixture immediately without centrifugation, and immediately carrying out the next step;
4) adding the mixture into an adsorption column in a collecting pipe twice, centrifuging at 12000r for 60s, and discarding waste liquid in the collecting pipe;
5) adding 700 mu L of Wash south 1, centrifuging at 12000r for 30s, and discarding waste liquid in a collecting pipe;
6) adding 500 mu L of Wash Solution 2/3, centrifuging at 12000r for 30s, and discarding the waste liquid. Repeating the steps for one time;
7) putting the adsorption column back into an empty collection tube, centrifuging for 2min at 13000r, and removing rinsing liquid as much as possible;
8) taking out the adsorption column, placing into an RNase free centrifuge tube, adding 30-40 μ L preheated RNase free water at the middle part of the adsorption membrane according to the expected RNA yield, standing at room temperature for 1min, and centrifuging at 12000r for 1 min. The eluent is added back to the adsorption column for repeated elution.
1.2.4 reverse transcription of piRNAs
Preparing a reverse transcription reaction system on ice (finally adding E.coli Poly (A) Polymerase and TRUEscript H RTase);
Total RNA 2μg
2×miRT Reaction Mix 10μL
E.coli Poly(A) Polymerase(5U/μL) 0.3μL
TRUEscript H RTase(200U/μD) 0.8μL
RNase free H2O to final volume 20μL
gently blowing, mixing, placing in a PCR instrument, reacting at 42 deg.C for 60min, heating at 85 deg.C for 5s to inactivate E.coli Poly (A) Polymerase and TRUEScript H RTase;
the synthesized cDNA was stored at-20 ℃.
1.2.5 real-time fluorescent quantitative PCR
when in use, the 2 XmiRNA qPCR Mix is gently and uniformly mixed by pouring up and down to avoid foaming, and is used after slight centrifugation. If the reagents are not mixed evenly, the reaction performance is reduced;
preparing a reaction solution on ice according to the following components;
2×miRNA qPCR Mix (With Sybr Green) 10μL
Forward primer (10μM ) 0.5μL
Reverse primer(10μM) 0.5μL
0.5. mu.L of first strand cDNA of piRNA
500×ROX 0.5μL
ddH2O to final volume 20μL
Reaction cycle conditions
PCR cycle (two-step method)
94℃ 2min
Dissociation Stage
1.3 statistical treatment
Data were entered into the SPSS16.0 system for statistical analysis. Data comparisons between the two groups used the T-Test, and differences were statistically significant when P was < 0.05.
1.4 results of the experiment
Results of PiRNA sequencing of seminal plasma of 1.4.119 subjects
In order to obtain the difference in the molecular level of male infertility patients, 19 subjects' seminal plasma was collected and divided into 4 groups according to the results of their seminal fluid analysis. In the normal (B _ noma) group, 5 cases, both abnormal in viability and morphology (B _ aboo) group, abnormal in viability, 4 cases, normal in morphology (V _ aboo) group, and 19 cases, in total, of 5 cases, abnormal in morphology and normal in viability (M _ aboo) group, the plasma was subjected to piRNA sequencing. The statistics of the subjects on the age, total sperm motility, percentage of morphologically normal sperm, and the like are shown in Table 1.
TABLE 1 basic conditions of age, total sperm motility, percentage of morphologically normal sperm, etc. of the subjects
As shown in fig. 1: the subject's seminal plasma piRNA sequencing results showed significant differences in piRNAs in four seminal plasma groups, 963 piRNAs were up-regulated compared to the normal (B _ noma) group, 155 piRNAs were down-regulated, and activity was abnormal, 3936 piRNAs were up-regulated compared to the normal (B _ noma) group, 389 piRNAs were down-regulated, 2561 piRNAs were up-regulated compared to the normal (B _ noma) group, and 355 piRNAs were down-regulated compared to the normal (B _ noma) group.
All detected up-regulated piRNAs are subjected to correlation analysis and summary with sphere morphology and sphere visibility respectively, and the analysis results are shown in tables 2-5.
As shown in tables 2-5: the pi RNAs related to the sphere morphology are hsa _ piR _001258, hsa _ piR _004842, hsa _ piR _006215, hsa _ piR _014997 and hsa _ piR _017507, and the pi RNAs related to the sphere visibility are hsa _ piR _000478, hsa _ piR _012901, hsa _ piR _016118 and hsa _ piR _ 016271.
TABLE 2 correlation of piRNAs with morphologically normal sperm
Note: "-" indicates P < 0.05.
TABLE 3 correlation of piRNAs with normal viable sperm
Note: "-" indicates P < 0.05.
TABLE 4 piRNAs associated with morphologically normal sperm
TABLE 5 piRNAs associated with normal viable sperm
Note: "-" indicates P < 0.05.
Seminal plasma validation results of 1.4.280 subjects
To verify the results of seminal plasma sequencing, 80 clinical subjects were selected, their seminal fluid collected, sperm function tested, and divided into 4 groups based on their sperm motility and morphology. 20 cases of the normal group (normozoospermia), 10 cases of the oligoasthenozoospermia group (oligoasthenospermia), 20 cases of the oligoasthenospermia group (oligoasthenospermia), 19 cases of the oligoasthenospermia group (astenospermia) and 11 cases of the teratozoospermia group (teratozoospermia). RT-qPCR verification of statistically significant piRNAs in tables 4-5 was performed.
Extracting piRNAs in seminal plasma of the 80 subjects, carrying out reverse transcription to form cDNA, detecting the expression condition of each piRNAs in different groups by using RT-qPCR, further verifying the result of seminal plasma sequencing, and drawing the obtained result by using GraphPad Prism 7 statistical analysis (see particularly FIGS. 2-9);
wherein, the nucleotide sequence of hsa _ piR _016118 is shown as SEQ ID NO. 1, the nucleotide sequence of the specific primer for amplifying hsa _ piR _016118 is shown as SEQ ID NO. 4 and SEQ ID NO. 5, and the nucleotide sequence of the reverse transcription primer for reverse transcribing hsa _ piR _016118 into cDNA is shown as SEQ ID NO. 10;
the nucleotide sequence of hsa _ piR _017507 is shown as SEQ ID NO. 2, the nucleotide sequence of a specific primer for amplifying hsa _ piR _017507 is shown as SEQ ID NO. 6 and SEQ ID NO. 7, and the nucleotide sequence of a reverse transcription primer for reverse transcribing hsa _ piR _017507 into cDNA is shown as SEQ ID NO. 10;
the nucleotide sequence of hsa _ piR _006215 is shown as SEQ ID NO. 3, the nucleotide sequence of the specific primer for amplifying hsa _ piR _006215 is shown as SEQ ID NO. 8 and SEQ ID NO. 9, and the nucleotide sequence of the reverse transcription primer for reverse transcribing hsa _ piR _006215 into cDNA is shown as SEQ ID NO. 10.
As shown in FIGS. 2-9, the expression levels of hsa _ piR _016118 in the oligozoospermia group, the asthenospermia group and the teratozoospermia group are all increased, the expression levels of hsa _ piR _000478 in the oligozoospermia group and the teratozoospermia group are increased, the expression level of hsa _ piR _017507 in the oligozoospermia group is remarkably reduced, and the expression level of hsa _ piR _006215 in the oligozoospermia group and the teratozoospermia group is remarkably reduced.
The receiver operator characteristic curve-ROC curve (receiver operator characteristic curve) is a curve drawn by using a true positive rate (sensitivity) as an ordinate and a false positive rate (1-specificity) as an abscissa. In order to judge whether the piRNAs can be used as the index or the auxiliary diagnostic index of the clinical diagnosis of the total sperm motility and the sperm morphology, the results of the piRNAs with statistical significance detected by RT-qPCR and the percentage of the sperms with normal sperm motility and morphology detected by four groups of subjects through an automatic sperm analysis system are used for drawing an ROC curve through SPSS (see the figures 10-13 in particular), and the corresponding area under the ROC curve (AUC) is calculated (see the tables 6-9 in particular).
Combining the results of RT-qPCR verification of 80 cases of seminal plasma in FIGS. 2-12, analyzing the ROC curve and the area under the curve in FIGS. 10-13, it can be found that AUC of hsa _ piR _016118 in the asthenospermia group and the teratospermia group are 0.911 and 0.991, respectively, P value <0.05, AUC of hsa _ piR _017507 in the asthenospermia group and the teratospermia group are 0.983 and 0.977, respectively, P value <0.05, AUC of hsa _ piR _006215 in the asthenospermia group and the teratospermia group are 0.967 and 0.959, respectively, and P value <0.05, therefore, it can be found that hsa _ piR _016118, hsa _ piR _017507 and hsa _ piR _006215 have the best diagnostic value and are more diagnostic value for male infertility with abnormal sperm motility and morphologic abnormality.
Furthermore, as can be seen from FIGS. 10-13, seminal plasma whose expression level of hsa _ piR _016118 is less than 0.86529154 corresponds to sperm of male with non-asthenospermia and non-teratospermia, seminal plasma whose expression level of hsa _ piR _016118 is greater than 0.86529154 corresponds to sperm of male with asthenospermia, and seminal plasma whose expression level of hsa _ piR _016118 is greater than 5.37 corresponds to sperm of male with teratospermia;
seminal plasma with expression level of hsa _ piR _017507 greater than 0.02405454 corresponds to sperm of male with non-asthenospermia and non-teratospermia, seminal plasma with expression level of hsa _ piR _017507 greater than 0.02405454 corresponds to sperm of male with non-asthenospermia, seminal plasma with expression level of hsa _ piR _017507 greater than 0.023850807 corresponds to sperm of male with non-teratospermia;
seminal plasma with expression level of hsa _ piR _006215 greater than 0.089511359 corresponds to sperm of male with non-asthenospermia and non-teratospermia, seminal plasma with expression level of hsa _ piR _006215 greater than 0.089511359 corresponds to sperm of male with non-asthenospermia, and seminal plasma with expression level of hsa _ piR _006215 greater than 0.090479887 corresponds to sperm of male with non-teratospermia.
TABLE 6 area under the curve of piRNA in the oligoteratocarcinoma group (olgiasteneteraospermia)
TABLE 7 area under the curve of piRNA in the oligoasthenospermia (oligoasthenospermia) group
TABLE 8 area under the curve of the piRNA in the group of asthenospermia (astenozopermia)
TABLE 9 area under the curve of the group of picoRNAs with teratozoosperms (teratozospermia)
Experimental example 2: product for detecting sperm quality
The embodiment provides a product for detecting sperm quality, which comprises hsa _ piR _016118, a specific primer for amplifying hsa _ piR _016118 and/or a reverse transcription primer for reverse transcribing hsa _ piR _016118 into cDNA;
comprises hsa _ piR _017507, a specific primer for amplifying hsa _ piR _017507 and/or a reverse transcription primer for reverse transcribing hsa _ piR _017507 into cDNA;
and/or comprises hsa _ piR _006215, a specific primer for amplifying hsa _ piR _006215 and/or a reverse transcription primer for reverse transcribing hsa _ piR _006215 into cDNA;
wherein, the nucleotide sequence of hsa-piR-016118 is shown as SEQ ID NO: 1; the nucleotide sequence of hsa-piR-017507 is shown as SEQ ID NO: 2; the nucleotide sequence of hsa _ piR _006215 is shown as SEQ ID NO 3; the nucleotide sequence of the specific primer for amplifying hsa-piR-016118 is shown as SEQ ID NO. 4 and SEQ ID NO. 5; the nucleotide sequences of the specific primers for amplifying hsa-piR-017507 are shown as SEQ ID NO. 6 and SEQ ID NO. 7; the nucleotide sequences of the specific primers for amplifying hsa-piR-006215 are shown as SEQ ID NO. 8 and SEQ ID NO. 9; the nucleotide sequence of the reverse transcription primer for reverse transcribing hsa _ piR _016118 into cDNA is shown as SEQ ID NO. 10; the nucleotide sequence of the reverse transcription primer for reverse transcribing hsa _ piR _017507 into cDNA is shown as SEQ ID NO. 10; the nucleotide sequence of the reverse transcription primer used to reverse transcribe hsa-piR-006215 into cDNA is shown in SEQ ID NO: 10.
Experimental example 3: application method of product for detecting sperm quality
This example provides the use of the product for sperm quality detection of example 2, the use being:
taking seminal plasma, extracting hsa _ piR _016118 in the seminal plasma by using a miRNA extraction kit, then carrying out reverse transcription on the extracted hsa _ piR _016118 into cDNA by using a reverse transcription primer for carrying out reverse transcription on hsa _ piR _016118 into the cDNA, and then detecting the expression quantity of hsa _ piR _016118 in the seminal plasma relative to an internal reference U6 by RT-qPCR (2)Ct,U6-Ct,016118) And finally, obtaining hsa _ piR _0161 according to detection18 relative to the expression level of the internal reference U6, judging the sperm quality, wherein the sperms of the male with the non-asthenospermia and the non-teratospermia correspond to the sperms of the male with the expression level of hsa _ piR _016118 being less than 0.86529154, the sperms of the male with the non-asthenospermia and the expression level of hsa _ piR _016118 being more than 0.86529154, and the sperms of the male with the teratospermia correspond to the sperms of the male with the expression level of hsa _ piR _016118 being more than 5.37;
taking seminal plasma, extracting hsa _ piR _017507 in the seminal plasma by using a miRNA extraction kit, then carrying out reverse transcription on the extracted hsa _ piR _017507 into cDNA by using a reverse transcription primer for carrying out reverse transcription on hsa _ piR _017507 into the cDNA, and then detecting the expression quantity of hsa _ piR _017507 in the seminal plasma relative to an internal reference U6 by RT-qPCR (2)Ct,U6-Ct,016118) Finally, the sperm quality is judged according to the expression quantity of hsa _ piR _017507 relative to the internal reference U6, wherein the seminal plasma of hsa _ piR _017507, the expression quantity of which is more than 0.02405454, corresponds to the sperms of the male with non-asthenospermia and non-teratospermia, the seminal plasma of hsa _ piR _017507, the expression quantity of which is more than 0.02405454, corresponds to the sperms of the male with non-asthenospermia, and the expression quantity of hsa _ piR _017507, the expression quantity of which is more than 0.023850807, corresponds to the sperms of the male with non-teratospermia;
and/or taking seminal plasma, firstly extracting hsa _ piR _006215 in the seminal plasma by using a miRNA extraction kit, then carrying out reverse transcription on the extracted hsa _ piR _006215 into cDNA by using a reverse transcription primer for carrying out reverse transcription on hsa _ piR _006215 into the cDNA, and then detecting the expression quantity of hsa _ piR _006215 in the seminal plasma relative to the internal reference U6 by RT-qPCR (2)Ct,U6-Ct,016118) And finally, judging the sperm quality according to the expression level of hsa _ piR _006215 relative to the internal reference U6, wherein the seminal plasma of hsa _ piR _006215 with the expression level larger than 0.089511359 corresponds to the sperms of the male with non-asthenospermia and non-teratospermia, the seminal plasma of hsa _ piR _006215 with the expression level larger than 0.089511359 corresponds to the sperms of the male with non-asthenospermia, and the seminal plasma of hsa _ piR _006215 with the expression level larger than 0.090479887 corresponds to the sperms of the male with non-teratospermia.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
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Claims (7)
1. A product for use in detecting sperm quality, said product comprising hsa _ piR _016118, a specific primer for amplifying hsa _ piR _016118 and/or a reverse transcription primer for reverse transcribing hsa _ piR _016118 to cDNA;
the product comprises hsa _ piR _017507, a specific primer for amplifying hsa _ piR _017507 and/or a reverse transcription primer for reverse transcribing hsa _ piR _017507 into cDNA;
and/or the product comprises hsa _ piR _006215, a specific primer for amplifying hsa _ piR _006215 and/or a reverse transcription primer for reverse transcribing hsa _ piR _006215 to cDNA;
the nucleotide sequence of hsa _ piR _016118 is shown as SEQ ID NO: 1;
the nucleotide sequence of hsa _ piR _017507 is shown as SEQ ID NO. 2;
the nucleotide sequence of hsa _ piR _006215 is shown as SEQ ID NO. 3.
2. The product of claim 1, wherein the nucleotide sequence of the specific primer for amplifying hsa _ piR _016118 is shown in SEQ ID No. 4 and SEQ ID No. 5; the nucleotide sequence of the specific primer for amplifying hsa-piR-017507 is shown as SEQ ID NO. 6 and SEQ ID NO. 7; the nucleotide sequences of the specific primers for amplifying hsa-piR-006215 are shown as SEQ ID NO. 8 and SEQ ID NO. 9.
3. The product of claim 1, wherein the nucleotide sequence of the reverse transcription primer for reverse transcribing hsa _ piR _016118 into cDNA is set forth in SEQ ID No. 10; the nucleotide sequence of the reverse transcription primer for reverse transcribing hsa-piR-017507 into cDNA is shown as SEQ ID NO. 10; the nucleotide sequence of the reverse transcription primer for reverse transcribing hsa-piR-006215 into cDNA is shown in SEQ ID NO. 10.
4. The product of any one of claims 1 to 3, wherein the product is a detection reagent or a detection kit.
5. The product of any one of claims 1-3, wherein sperm quality is sperm motility and morphology.
6. The use of the product of any of claims 1-3, wherein the use is taking seminal plasma, first extracting hsa _ piR _016118 from the seminal plasma, and then reverse transcribing the extracted hsa _ piR _016118 into cDNA using reverse transcription primers for reverse transcribing hsa _ piR _016118 into cDNA;
the using method comprises the steps of taking seminal plasma, firstly extracting hsa _ piR _017507 from the seminal plasma, and then carrying out reverse transcription on the extracted hsa _ piR _017507 into cDNA by using a reverse transcription primer for carrying out reverse transcription on hsa _ piR _017507 into the cDNA;
and/or the using method is that seminal plasma is taken, hsa _ piR _006215 in the seminal plasma is extracted firstly, and then the extracted hsa _ piR _006215 is subjected to reverse transcription into cDNA by using a reverse transcription primer for carrying out reverse transcription of hsa _ piR _006215 into cDNA.
7. The use of claim 6, wherein the use comprises taking seminal plasma, extracting hsa _ piR _016118 from the seminal plasma, performing reverse transcription of the extracted hsa _ piR _016118 into cDNA by using a reverse transcription primer for reverse transcription of hsa _ piR _016118 into cDNA, and detecting the expression level of hsa _ piR _016118 in the seminal plasma by RT-qPCR based on the reverse transcription of the cDNA;
the using method comprises the steps of taking seminal plasma, firstly extracting hsa _ piR _017507 from the seminal plasma, then carrying out reverse transcription on the extracted hsa _ piR _017507 into cDNA by using a reverse transcription primer for carrying out reverse transcription on hsa _ piR _017507 into the cDNA, and finally detecting the expression quantity of hsa _ piR _017507 in the seminal plasma by RT-qPCR (reverse transcription-quantitative polymerase chain reaction) according to the cDNA obtained by reverse transcription;
and/or the using method comprises the steps of taking seminal plasma, extracting hsa _ piR _006215 from the seminal plasma, then carrying out reverse transcription on the extracted hsa _ piR _006215 into cDNA by using a reverse transcription primer for carrying out reverse transcription on hsa _ piR _006215 into the cDNA, and finally detecting the expression level of hsa _ piR _006215 in the seminal plasma by RT-qPCR (reverse transcription-quantitative polymerase chain reaction).
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| CN117385009A (en) * | 2023-12-04 | 2024-01-12 | 湖南工程学院 | Probe set and method for detecting piRNA based on rolling circle transcription and CRISPR-Cas13a cascade shear |
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