Primers, kit and method for rapidly detecting sex of wattle-necked soft-shelled turtles at pond head and application
By designing specific primers and kits, combined with PCR amplification and lateral flow chromatography technology, rapid and accurate detection of the gender of Shan Rui Turtle is achieved, solving the problem of low gender detection efficiency in the existing technology, reducing breeding costs, and improving breeding benefits.
Patent Information
- Application Number
- CN202510292441.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-12
- Publication Date
- 2025-05-30
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
During the breeding of Shanrui turtle, it is difficult for the existing technology to quickly and accurately detect the gender of the turtle, resulting in low single-sex breeding efficiency and increasing the breeding cost and disease risk.
Specific primers and kits were designed to detect the genomic DNA of Shan Ruibi using PCR amplification technology, and visually detect it through lateral flow chromatography test strips to achieve rapid and accurate gender identification.
This method can efficiently and easily distinguish the gender of Shan Rui Turtle, reduce breeding costs, improve breeding benefits, and is simple to operate and does not require complex instruments and expensive reagents.
Smart Images
Figure CN120060456A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the technical field of gender detection of Palea steindachneri, and specifically relates to primers, kits, methods and applications for rapid detection of the gender of Palea steindachneri in pond heads. Background Art
[0002] Palea steindachneri is an animal of the family Trionychidae and the genus Palea. It has a relatively large body size, is overall oval in shape, and its body surface is covered with soft leathery skin, with a dorsal and ventral carapace. Palea steindachneri mainly lives in rivers, lakes, reservoirs and mountain streams in mountainous areas, and likes to inhabit places with clear water, slow water flow, and many rocks or sediment at the bottom.
[0003] The growth rate of male Palea steindachneri is faster than that of female. Single-sex (male) farming can make the growth rate of the population relatively consistent, which is convenient for management and harvesting, can reach the market specification in a shorter time, and improve the farming efficiency. During the farming process, if male and female are mixed in farming, Palea steindachneri will mate and reproduce after reaching sexual maturity. This may lead to the energy consumption of the turtles in reproductive behaviors, affect the growth rate, and may also cause damage to the turtle body due to courtship, fighting and other behaviors, increasing the risk of disease infection. Single-sex farming can avoid these problems.
[0004] Therefore, the development of an early gender molecular identification technology for Palea steindachneri to accurately detect the gender of newly hatched juvenile turtles helps to achieve single-sex pond farming in the juvenile turtle stage, reduce costs and improve efficiency. Summary of the Invention
[0005] The inventors of this application designed primers and kits based on the conserved sequence (SRBRAA7) of Palea steindachneri, which can perform PCR amplification on the genomic DNA of Palea steindachneri, and detect the gender of Palea steindachneri according to the PCR amplification products, with high detection and discrimination efficiency and strong universality. In addition, the inventors of this application also constructed a lateral flow chromatographic test strip. This test strip can perform chromatographic detection on the amplification products as samples, and visually and quickly detect the gender of Palea steindachneri through the T line and C line on the test strip.
[0006] In the first aspect, the embodiment discloses a primer group, including DNA molecules shown in SEQ ID NO: 1 and 2.
[0007] In the second aspect, the embodiment discloses a primer group, the primer group includes a DNA molecule shown in SEQ ID NO: 1 labeled with a first label, and a DNA molecule shown in SEQ ID NO: 2 labeled with a second label; wherein, the first label and the second label are each selected from one of biotin, fluorescein isothiocyanate or digoxin, and the first label is different from the second label.
[0008] In a third aspect, the embodiments disclose a kit. The kit includes the primer set described in the first aspect and PCR amplification reagents.
[0009] In some embodiments, the PCR reagents include: dNTP mixture, 10×Ex Taq Buffer (Mg 2+ free), MgCl 2 (25 mM), and TaKaRa Ex Taq (5 U / μL).
[0010] In some embodiments, the PCR reagents are recombinase-mediated isothermal amplification reagents. The recombinase-mediated isothermal amplification reagents are from Jiangsu Qitian Gene Biotechnology Co., Ltd., China).
[0011] In a fourth aspect, the embodiments disclose a method for detecting the gender of soft-shelled turtles, including extracting genomic DNA of an individual soft-shelled turtle to be tested; performing an amplification reaction on it using the DNA molecules shown in SEQ ID NO: 1 and 2; performing electrophoresis detection on the amplification reaction product; and determining the gender of the individual soft-shelled turtle to be tested according to the bands detected by electrophoresis.
[0012] In some embodiments of this method, the amplification reaction system is 50 μL in total and includes: 5 μL of genomic DNA at 50 ng / μL, 2 μL of the DNA molecule shown in SEQ ID NO: 1 at 20 μmol / μL, 2 μL of the DNA molecule shown in SEQ ID NO: 2 at 20 μmol / μL, 5 μL of 10×Ex Taq Buffer (Mg 2+ free), 3.3 μL of MgCl 2 (25 mM), 4 μL of dNTP mixture (the concentrations of ATP, GTP, CTP, and TTP are all 2.5 mmol / μL), 0.25 μL of TaKaRa Ex Taq (5 U / μL), and the balance of double-distilled water.
[0013] In some embodiments of this method, the amplification reaction steps include: 94°C for 5 min; 94°C for 30 s, 60°C for 30 s, 72°C for 100 s, for 35 cycles; 72°C for 5 min.
[0014] In some embodiments of this method, the amplification reaction system is 50 μL in total and includes: 5 μL of genomic DNA at 50 ng / μL, 2 μL of the DNA molecule shown in SEQ ID NO: 1 at 10 μmol / μL, 2 μL of the DNA molecule shown in SEQ ID NO: 2 at 10 μmol / μL, 2.5 μL of 280 mM magnesium acetate, 25 μL of RAA reaction buffer, and the balance of double-distilled water.
[0015] In some embodiments of the method, the amplification reaction step includes: reacting at 39 °C for 30 min.
[0016] In some embodiments of the method, if there is no electrophoresis band, the individual soft-shelled turtle to be tested is a male individual. If a target band of 213 bp appears, the individual soft-shelled turtle to be tested is a female individual.
[0017] In a fifth aspect, an embodiment discloses a kit. The kit includes the kit described in the third aspect, a sample diluent, and a test strip. The test strip is composed of a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad that are sequentially overlapped and adhered to a backing. The conjugate pad is coated with colloidal gold particle-labeled mouse anti-digoxin antibody, and the nitrocellulose membrane has a detection line coated with avidin and a control line coated with goat anti-mouse antibody. In the kit described in the second aspect, the upstream primer is labeled with biotin and the downstream primer is labeled with digoxin.
[0018] In some embodiments, the colloidal gold particles coated on the conjugate pad have a particle size of 25 nm, 8.4 μg of mouse anti-digoxin antibody is labeled with 1 mL of colloidal gold particles, and the coating amount of the formed colloidal gold-antibody complex on the conjugate pad is 2 mL / 30 cm.
[0019] In some embodiments, the concentration of avidin coated on the detection line of the nitrocellulose membrane is 0.5 mg / mL, and the coating amount is 1 μL / cm; the concentration of goat anti-mouse antibody coated on the control line is 1 mg / mL, and the coating amount is 1 μL / cm.
[0020] Using biotin and digoxin to perform PCR amplification on the genomic DNA of soft-shelled turtles with a primer pair, so that the positive amplification product has a double label. At the same time, the conjugate pad of the colloidal gold test strip is coated with mouse anti-digoxin antibody, the detection line of the chromatographic membrane is coated with avidin that can specifically bind to biotin, and the control line is coated with goat anti-mouse antibody. During the chromatography process, the digoxin label of the positive amplification product binds to the gold-labeled complex on the conjugate pad, and then the biotin label carried thereon binds to the avidin on the detection line of the chromatographic membrane. The formed "sandwich" sandwich structure complex is captured by the detection line and thus shows color. The excess gold-labeled complex continues to chromatograph and binds to the coated goat anti-mouse antibody when it reaches the control line, and thus is captured by the control line and shows color. Negative amplification products cannot be captured by the detection line during the chromatography process due to the absence of double labels and thus do not show color.
[0021] In a sixth aspect, an embodiment discloses a method for detecting the gender of soft-shelled turtles. The method includes: extracting genomic DNA from soft-shelled turtle tissues; performing PCR amplification on the genomic DNA using the primer set described in the second aspect; loading the product of the PCR amplification onto the sample pad of the test strip; and detecting the gender of soft-shelled turtles according to the display of the detection line and the control line of the test strip.
[0022] In some embodiments of this method, if both the test line and the control line show color, then the soft-shelled turtle is female. If the test line does not show color while the control line shows color, then the soft-shelled turtle is male.
[0023] In a seventh aspect, the embodiments disclose the use of the primer set of the first aspect, the kit of the second or third aspect, and the method of the fourth or fifth aspect in detecting the sex of soft-shelled turtles.
[0024] Compared with the prior art, the beneficial effects of this application at least include:
[0025] Using the primers provided in the embodiments to perform PCR amplification on the genomic DNA of soft-shelled turtles, the sex of soft-shelled turtles can be directly detected according to the electrophoresis detection results of the PCR amplification products, and the detection process is simple and direct.
[0026] Furthermore, the embodiments also use labeled primers to perform PCR amplification on the genomic DNA of soft-shelled turtles, and the amplification products are detected by a lateral flow test strip, which can directly and simply visualize the results, and the detection results are simple and effective.
[0027] In addition, the primer set, kit and method provided in the embodiments can identify the sex of soft-shelled turtles, have strong identification versatility and a wide detection range, and have very wide application value.
[0028] The primer set, kit and method provided in the embodiments can combine PCR amplification technology and immunochromatography technology to achieve rapid detection of nucleic acid products. After PCR amplification, the products do not need to go through complex gel electrophoresis experiments, and the sex identification of soft-shelled turtles can be achieved within 3 - 10 minutes.
[0029] The primer set, kit and method provided in the embodiments are easy to operate. It only needs to mix the nucleic acid products with a certain amount of PBS and then drop them into the sample well of the test strip, without other operations.
[0030] The primer set, kit and method provided in the embodiments have a fast reaction speed, and the sex identification of soft-shelled turtles can be achieved within 3 - 10 minutes, shortening the detection time.
[0031] The primer set, kit and method provided in the embodiments have low costs. The detection does not require complex instruments, an ordinary PCR instrument is sufficient, nor does it require expensive special reagents, and the detection cost is low.
[0032] The primer set, kit and method provided in the embodiments have low requirements for operators. Since this technology does not require complex instruments, the operation process is simple, and the detection results are visually judged, anyone can be competent after self-study or simple training.
[0033] The primer sets, kits and methods provided by the embodiments are highly safe, do not require special reagents, avoid contaminants such as EB in gel electrophoresis, and are safe and harmless to operators and the test environment. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 It is the electrophoresis detection result of general PCR amplification of the genome DNA of the soft-shelled turtle provided by the embodiment. Among them, the female code is ♀, and the male code is ♂.
[0035] Figure 2 It is the schematic diagram for result interpretation of the disposable nucleic acid test strip provided by the embodiment. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0036] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application. The reagents not specifically described in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.
[0037] In the present application, through screening, analysis and experimental research, the conserved sequence of the soft-shelled turtle (SRBRAA, SEQ ID NO: 3) is used as the detection target sequence. Based on this target sequence, primers as shown in Table 1 are designed, which can effectively amplify SRBRAA and achieve the identification of the gender of the soft-shelled turtle. The primer information is as follows:
[0038] Table 1 Primer Information Table
[0039]
[0040] Therefore, the embodiment provides a primer set, which includes DNA molecules as shown in SEQ ID NO: 1 and 2.
[0041] In some embodiments of the primer set, the primer set includes a first-labeled DNA molecule as shown in SEQ ID NO: 1 and a second-labeled DNA molecule as shown in SEQ ID NO: 2; wherein, the first label and the second label are each selected from one of biotin, fluorescein isothiocyanate or digoxin, and the first label is different from the second label.
[0042] The embodiment discloses a kit, which includes the primer set and PCR amplification reagents.
[0043] In some embodiments of the kit, the PCR reagent includes: dNTP mixture (the concentrations of ATP, GTP, CTP and TTP are all 2.5 mmol / μL), 10×Ex Taq Buffer (Mg2+ free), MgCl 2 (25 mM), and TaKaRa Ex Taq (5 U / μL).
[0044] In some embodiments of the kit, the PCR amplification reagent is a recombinase-mediated isothermal amplification reagent. The recombinase-mediated isothermal amplification reagent is from Jiangsu Qitian Gene Biotechnology Co., Ltd., China).
[0045] The embodiment also discloses a method for detecting the gender of soft-shelled turtles. The method includes: extracting the genomic DNA of the individual soft-shelled turtle to be tested; performing an amplification reaction on it using the DNA molecules shown in SEQ ID NO: 1 and 2; performing electrophoresis detection on the amplification reaction product; and determining the gender of the individual soft-shelled turtle to be tested according to the bands detected by the electrophoresis.
[0046] In some embodiments of the method, the genomic DNA of the individual soft-shelled turtle to be tested is extracted with reference to the instruction manual of the Tiangen tissue genomic DNA extraction kit. The purity and concentration of the genomic DNA are detected by 1.0% agarose gel electrophoresis and NanoDrop-2000 ultraviolet spectrophotometer, and the genomic DNA is stored at -20°C.
[0047] In some embodiments of the method, the amplification reaction system is 50 μL in total and includes: 5 μL of genomic DNA at 50 ng / μL, 2 μL of the DNA molecule shown in SEQ ID NO: 1 at 20 μmol / μL, 2 μL of the DNA molecule shown in SEQ ID NO: 2 at 20 μmol / μL, 5 μL of 10×Ex Taq Buffer (Mg 2+ free), 3.3 μL of MgCl 2 (25 mM), 4 μL of dNTP mixture (the concentrations of ATP, GTP, CTP, and TTP are all 2.5 mmol / μL), 0.25 μL of TaKaRa Ex Taq (5 U / μL), and the balance of double-distilled water. In some embodiments of the method, the amplification reaction steps include: 94°C for 5 min; 94°C for 30 s, 60°C for 30 s, 72°C for 100 s, for 35 cycles; 72°C for 5 min. As Figure 1 shows the gel electrophoresis pattern of the PCR reaction product, where males show no band and females show a single band. This shows that. Using the primer pair shown in SEQ ID NO: 1 and 2 can effectively distinguish the gender of soft-shelled turtles.
[0048] In addition, the embodiments disclose a kit. The kit includes RAA reaction reagents, a sample diluent, and a test strip. The test strip is composed of a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad that are sequentially overlapped and adhered to a backing. The conjugate pad is coated with a mouse anti-digoxin antibody labeled with colloidal gold particles. The nitrocellulose membrane has a detection line coated with avidin and a control line coated with a goat anti-mouse antibody. In the kit for PCR amplification provided in the above embodiments, the upstream primer is labeled with biotin and the downstream primer is labeled with digoxin.
[0049] Among them, the above primers are labeled with biotin and digoxin, and the genomic DNA of the soft-shelled turtle is amplified by RAA using the labeled primer pair, so that the positive amplification product has a double label. The positive amplification product is diluted with the sample diluent and then loaded onto the test strip. Since the conjugate pad on the colloidal gold test strip is coated with a mouse anti-digoxin antibody, the detection line on the chromatographic membrane is coated with avidin that can specifically bind to biotin, and the control line is coated with a goat anti-mouse antibody. During the chromatography process, the digoxin label of the positive amplification product binds to the gold-labeled complex on the conjugate pad, and then the biotin label carried thereon binds to the avidin on the detection line of the chromatographic membrane. The formed "sandwich" sandwich structure complex is captured by the detection line and thus shows color. The excess gold-labeled complex continues to chromatograph and binds to the coated goat anti-mouse antibody when it reaches the control line, and thus is captured by the control line and shows color. The negative amplification product does not show color because there is no double-labeled substance and cannot be captured by the detection line during the chromatography process.
[0050] In some embodiments, the conjugate pad is coated with colloidal gold particles having a particle size of 25 nm, 8.4 μg of mouse anti-digoxin antibody is labeled with 1 mL of colloidal gold particles, and the coating amount of the formed colloidal gold-antibody complex on the conjugate pad is 2 mL / 30 cm.
[0051] In some embodiments, the concentration of avidin coated on the detection line of the nitrocellulose membrane is 0.5 mg / mL, and the coating amount is 1 μL / cm; the concentration of goat anti-mouse antibody coated on the control line is 1 mg / mL, and the coating amount is 1 μL / cm.
[0052] Based on this, the embodiments also disclose a method for detecting the gender of soft-shelled turtles. The method includes: extracting genomic DNA from soft-shelled turtle tissues; performing RAA amplification on the genomic DNA using a labeled primer set; adding the product of the RAA amplification to the sample pad of the test strip; and detecting the gender of the soft-shelled turtle according to the display of the detection line and the control line of the test strip. Among them, the primer set includes a first-labeled DNA molecule shown in SEQ ID NO:1 and a second-labeled DNA molecule shown in SEQ ID NO:2; wherein, the first label and the second label are each selected from one of biotin, fluorescein isothiocyanate, or digoxin, and the first label is different from the second label.
[0053] In some embodiments of the method, the reaction system for RAA amplification includes, based on 50 μL: 5 μL of genomic DNA at 50 ng / μL, 2 μL of a DNA molecule as shown in SEQ ID NO:1 at 10 μmol / μL, 2 μL of a DNA molecule as shown in SEQ ID NO:2 at 10 μmol / μL, 2.5 μL of 280 mM magnesium acetate, 25 μL of RAA reaction buffer (Jiangsu Qitian Gene Biotechnology Co., Ltd., China), and the balance of double-distilled water.
[0054] In some embodiments of the method, the reaction steps for RAA amplification include: reacting at 39 °C for 30 min.
[0055] In some embodiments, if both the test line and the control line are colored, then the soft-shelled turtle is female. If the test line is not colored while the control line is colored, then the soft-shelled turtle is male.
[0056] As Figure 2 shown, if the test strip shows a quality control line (C) and a test line (T), both being red bands, it is a positive result; a positive result indicates that the sample contains the nucleic acid fragment to be detected, and the quantity thereof ≥ the minimum detection amount of the test strip. When the concentration of the target nucleic acid product is low, the C line of the test strip is colored red, the T line is light red, or even light pink, and this result should also be judged as positive. When the concentration of the target nucleic acid product is high, the C line of the test strip is colored red, the T line is red, and this result should also be judged as positive.
[0057] As Figure 2 shown, if the quality control line (C line) of the test strip shows a red band and the test line (T line) has no band. A negative result indicates that the sample does not contain the target nucleic acid fragment, or the quantity thereof is lower than the minimum detection amount of the test strip.
[0058] As Figure 2 shown, if neither the quality control line (C line) nor the test line (T line) of the test strip shows a band, it indicates that the test strip or amplification reagent used may have been damaged, invalidated, or there was an error in the operation.
[0059] As described above, the above are only the preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed in the present application should be covered by the protection scope of the present application.
Claims
1. A primer set comprising the DNA molecules shown in SEQ ID NO: 1 and 2.
2. A primer set, comprising a first labeled DNA molecule as shown in SEQ ID NO: 1, and a second labeled DNA molecule as shown in SEQ ID NO: 2; wherein, The first label and the second label are both selected from one of biotin, fluorescein isothiocyanate or digoxigenin, and the first label is different from the second label.
3. A kit comprising the primer set as claimed in claim 1 or 2 and a PCR amplification reagent.
4. Methods for detecting the sex of mountain soft-shelled turtles include: Extracting genomic DNA of the tested mountain soft-shelled turtle individual; Amplification reaction was performed using DNA molecules as shown in SEQ ID NOs: 1 and 2; Conducting electrophoresis detection on the amplification reaction product; The sex of the tested soft-shelled turtle individual is determined based on the bands detected by electrophoresis.
5. The method according to claim 4, wherein the amplification reaction system comprises, in 50 μL: 5 μL, 50 ng / μL genomic DNA, 2 μL, 20 μmol / μL DNA molecule as shown in SEQ ID NO: 1, 2 μL, 20 μmol / μL DNA molecule as shown in SEQ ID NO: 2, 5 μL 10×ExTaqBuffer (Mg 2+ free), 3.3 μL MgCl2 (25 mM), 4 μL dNTP mixture (the concentrations of ATP, GTP, CTP and TTP were all 2.5 mmol / μL), 0.25 μL TaKaRa Ex Taq (5 U / μL) and the remainder double distilled water.
6. The method according to claim 4, wherein the amplification reaction system comprises, in 50 μL: 5 μL, 50 ng / μL of genomic DNA, 2 μL, 10 μmol / μL of the DNA molecule shown in SEQ ID NO: 1, 2 μL, 10 μmol / μL of the DNA molecule shown in SEQ ID NO: 2, 2.5 μL of magnesium acetate, 25 μL RRAA reaction buffer, and the balance of double distilled water.
7. A test kit, comprising the test kit according to claim 3, a sample diluent and a test strip, wherein the test strip is composed of a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad which are overlapped and adhered on a backing in sequence, the conjugation pad is coated with mouse anti-digoxigenin antibody labeled with colloidal gold particles, the nitrocellulose membrane is provided with a detection line coated with avidin and a control line coated with goat anti-mouse antibody, and in the test kit, the upstream primer is labeled with biotin, and the downstream primer is labeled with digoxigenin.
8. A method for detecting the sex of a soft-shelled turtle using the kit according to claim 7, comprising: Extract genomic DNA from the tissues of the mountain softshell turtle; Performing PCR amplification on the genomic DNA using the primer set described in claim 2; Adding the PCR amplified product to the sample pad of the test strip; The sex of the mountain soft-shelled turtle is detected according to the display of the detection line and the display of the control line of the test strip.
9. The method according to claim 8, if both the detection line and the control line are colored, the mountain soft-shelled turtle is female; if the detection line is not colored and the control line is colored, the mountain soft-shelled turtle is male.
10. Use of the primer set according to claim 1 or 2, the kit according to claim 3 or 7, and the method according to any one of claims 4, 5, 6, 8, and 9 in detecting the sex of Trionyx sutchuenensis.