Raman spectrum-based fast measurement method for motile sperm deoxyribonucleic acid (DNA)
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A technology of motile sperm and Raman spectroscopy, applied in the fields of Raman scattering, material excitation analysis, etc.
Inactive Publication Date: 2014-07-23
FUJIAN NORMAL UNIV
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However, the current study has not seen the microscopic Raman spectroscopy study of a single motile sperm
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Embodiment 1
[0028] (1) Semen liquefaction
[0029] Collect a 2ml sample of semen in a sterile test tube, and let it stand for 20 minutes at room temperature to liquefy the semen naturally.
[0030] (2) Separation and preparation of motile sperm in semen.
[0031] Use a pipette to suck up the liquefied semen, add it to a 1.5ml centrifuge tube containing a density gradient centrifugal fluid (Puresperm 40 / 80), centrifuge at 1000g for 10 minutes, aspirate and discard the upper semen, keep the sperm at the bottom of the centrifuge tube, and add 5ml Resuspend in PBS buffer, centrifuge again at 1000g for 10 minutes, wash, aspirate the supernatant, and then add 1ml of PBS buffer to resuspend to prepare sperm suspension, and incubate in a 37°C incubator for 30 minutes.
[0032] (3) "Fixed" treatment of motile sperm
[0033] First, paste a 2×4cm aluminum alloy metal bottom plate on the petri dish, add PBS buffer to the petri dish, use the elongated glass capillary to suck a small amount of the sperm suspens...
Embodiment 2
[0040] (1) Semen liquefaction
[0041] Collect a 2ml sample of semen in a sterile test tube, and let it stand for 20 minutes at room temperature to liquefy the semen naturally.
[0042] (2) "Fixed" treatment of motile sperm
[0043] First, stick a Ramchip substrate made of stainless steel with a smooth surface on a Petri dish of 60mm×15mm size; add a certain amount of PBS buffer to make the liquid surface immerse the upper surface of the substrate to a depth of 2-3mm; the long-necked glass pipette uses capillary Principle: Take a small amount of sperm suspension after centrifugation and resuspension; finally, immerse the glass tube vertically into the Petri dish filled with PBS solution, so that the tip of the straw is 2-5mm away from the bottom Ramchip base and kept for 3 minutes ; The sperm will swim out with the head down and touch the metal bottom plate to form an adsorption state where the head is "fixed" and the tail is violently swinging.
[0044] (3) Measurement of Raman spec...
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Abstract
The invention relates to a raman spectrum-based fast measurement method for a motile sperm deoxyribonucleic acid (DNA). The invention adopts the following technical scheme: collecting a semen sample by a test tube; naturally liquefying semen in a room-temperature environment; absorbing the liquefied semen and adding to a centrifugal tube containing density gradient centrifugate; absorbing upper seminal plasma after centrifuging, discarding, then adding a PBS buffer solution and centrifugally washing again; absorbing supernatant, discarding, adding the PBS buffer solution to prepare a sperm suspension, and incubating; attaching a metal bottom plate to a culture dish; adding the PBS buffer solution, and absorbing the incubated sperm suspension, soaking in the culture dish, wherein the sperm swims outside and contacts the metal bottom plate; setting a spectrum response range, integrating time and the excitation light wavelength, so as to obtain raman spectrum data from the head part of the sperm. The motile sperm swimming out from the pointed-end part of a sucker can be attached to the metal bottom plate by operation of a thin tube pipette, and the damage condition of the sperm DNA is evaluated by measuring the sperm of which the head is attached to the metal bottom plate.
Description
Technical field [0001] The present invention involves a Raman spectrum fast measurement method for active sperm DNA. Background technique [0002] In recent years, infertility patients have increased year by year.According to the Statistics of the World Health Organization, about 10 to 15%of the world's couple encountered infertility.Among the married people in China, the ratio of infertility reached 7%to 10%, showing a rapid rise.Among them, infertility caused by male factors accounted for nearly 40%.Clinically, routine semen examination mainly uses computer -assisted evaluation, analyzes the concentration, vitality, and morphology of semen (usually called CASA).Studies have shown that if the proportion of sperm with poor quality in men's semen exceeds a certain range, it means that the probability of natural conception is low, and supplementary reproductive technology is required.Since the birth of the earliest auxiliary reproductive technology (commonly known as the first -gen...
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