Primers, kits, methods and applications for identifying the sex of Trionychidae

Abstract

This application relates to the technical field of sex identification of Trionychidae, and specifically relates to primers, kits, methods and applications for identifying the sex of Trionychidae. Based on the target sequence shown in SEQ ID NO: 3, the primers, kits, methods and applications can accurately amplify the genomic DNA of Trionychidae, and can accurately identify the sex of Trionychidae according to the amplification products. Further, through the lateral flow strip chromatography reaction of the PCR amplification products, the sex of Trionychidae can be visually identified, with strong identification versatility and a wide identification range, and has very wide application value.

Description

Technical Field

[0001] This application relates to the technical field of sex identification of Trionychidae, and specifically relates to primers, kits, test strips, methods and applications for identifying the sex of Trionychidae. Background Art

[0002] Trionychidae refers to a family in the phylum Chordata, subphylum Vertebrata, class Reptilia, order Testudines. Trionychidae has 7 genera and 23 species, characterized by having skin instead of horny scutes on the outside. Trionychidae is centered in Asia, and can also be seen in Africa and North America, while only fossils are found in Oceania. Trionychidae is a group of semi-aquatic soft-shelled turtles. The body is flat, and the carapace is oval or ovate. The head is olive-shaped, the snout extends forward to form a pointed snout process, and the nostrils are located at the tip of the snout; the tympanic membrane is not obvious. There are fleshy soft lips around the jaws, there are no teeth on the jaw margin, and it is covered with a sharp horny sheath.

[0003] To achieve single-sex breeding and sex control breeding of Trionychidae, it is first necessary to determine the sex determination type of Trionychidae. There are two sex determination types in vertebrates, namely environmental sex determination (ESD) and genetic sex determination (GSD). In the ESD type, the sex of animals is affected by various environmental factors. For example, crocodiles produce females when incubated at low and high temperatures, while males are dominant when incubated at medium temperatures; painted turtles and giant side-necked turtles produce males when incubated at low temperatures and females when incubated at high temperatures. The GSD type is divided into two different heterogametic systems: XX / XY sex chromosomes and ZZ / ZW sex chromosomes. Trionychidae belongs to the ZZ / ZW type. The breeding cycle of Trionychidae is long, and it is possible to distinguish the sex by appearance only after 1 winter age under the ecological breeding mode. Therefore, developing an early sex molecular identification technology for Trionychidae to accurately identify the sex of newly hatched juvenile turtles helps to achieve single-sex pond breeding at the juvenile turtle stage, reduce costs and improve efficiency. Summary of the Invention

[0004] The inventors of this application designed primers and kits based on the conserved sequence (ZHBRAA6) of Trionychidae, which can perform PCR amplification on the genomic DNA of Trionychidae, and identify the sex of Trionychidae according to the PCR amplification products, with high discrimination efficiency and strong generality. In addition, the inventors of this application also constructed a lateral flow chromatography test strip. This test strip can perform chromatography detection on the amplification products used as samples, and visually and quickly identify the sex of Trionychidae through the T line and C line on the test strip.

[0005] In the first aspect, the embodiment discloses a primer set, including DNA molecules shown in SEQ ID NO: 1 and 2.

[0006] In a second aspect, the embodiments disclose a primer set, which 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, both the first label and the second label are selected from one of biotin, fluorescein isothiocyanate or digoxin, and the first label is different from the second label.

[0007] In a third aspect, the embodiments disclose a kit. The kit includes the primer set described in the first aspect and PCR amplification reagents.

[0008] 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).

[0009] 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).

[0010] In a fourth aspect, the embodiments disclose a method for identifying the sex of the Trionychidae, including extracting the genomic DNA of the individual Trionychidae to be tested; performing PCR amplification on it using the DNA molecules as shown in SEQ ID NO:1 and 2; performing electrophoresis detection on the product of this PCR amplification; and determining the sex of the individual Trionychidae to be tested according to the bands of this electrophoresis detection.

[0011] In some embodiments of this method, the reaction system for this PCR amplification is calculated based on 50 μL and includes: 1 μL of genomic DNA at 50 ng / μL, 2 μL of the DNA molecule as shown in SEQ ID NO:1 at 20 μmol / μL, 2 μL of the DNA molecule as 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.

[0012] In some embodiments of this method, the reaction steps of this PCR amplification include: 94°C for 5 min; 94°C for 30 s, 60°C for 30 s, 72°C for 100 s, 35 cycles; 72°C for 5 min.

[0013] In some embodiments of the method, the reaction system for PCR amplification, calculated as 50 μL, includes: 1 μL of genomic DNA at 50 ng / μL, 1 μL of the DNA molecule shown in SEQ ID NO: 1 at 10 μmol / μL, 1 μL of the DNA molecule shown in SEQ ID NO: 2 at 10 μmol / μL, 2.5 μL of magnesium acetate, 25 μL of RAA reaction buffer, and the balance of double-distilled water.

[0014] In some embodiments of the method, the reaction steps for PCR amplification include: reacting at 39 °C for 30 min.

[0015] In some embodiments of the method, if there is no electrophoresis band, the individual of the family Trionychidae to be tested is a male individual. If a target band of 213 bp appears, the individual of the family Trionychidae to be tested is a female individual.

[0016] In the 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 sequentially lapped and adhered on 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 described in the second aspect, the upstream primer is labeled with biotin and the downstream primer is labeled with digoxin.

[0017] 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.

[0018] 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 the goat anti-mouse antibody coated on the control line is 1 mg / mL, and the coating amount is 1 μL / cm.

[0019] The genomic DNA of the Trionychidae was amplified by PCR using biotin and digoxin primers, so that the positive amplification products carried double labels. At the same time, the gold-labeled conjugate pad on the colloidal gold test strip was coated with mouse anti-digoxin antibody, the detection line on the chromatographic membrane was coated with avidin that could specifically bind to biotin, and the control line was coated with goat anti-mouse antibody. During chromatography, the digoxin label of the positive amplification product bound to the gold-labeled complex on the gold-labeled conjugate pad, and then the biotin label carried thereon bound to the avidin on the detection line of the chromatographic membrane. The formed "sandwich" sandwich structure complex was captured by the detection line and thus showed color. The excess gold-labeled complex continued to chromatograph and bound to the coated goat anti-mouse antibody when reaching the control line, and thus was captured by the control line and showed color. Negative amplification products did not show color because they could not be captured by the detection line during chromatography due to the absence of double labels.

[0020] In a sixth aspect, the embodiments disclose a method for identifying the sex of the Trionychidae. The method includes: extracting genomic DNA from Trionychidae 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 identifying the sex of the Trionychidae according to the display of the detection line and the control line of the test strip.

[0021] In some embodiments of the method, if both the detection line and the control line show color, then the Trionychidae is female. If the detection line does not show color while the control line shows color, then the Trionychidae is male.

[0022] 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 described in the fourth or fifth aspect in identifying the sex of the Trionychidae. In some embodiments, the Trionychidae is selected from at least one of Pelodiscus sinensis, Trachemys scripta, Apalone spinifera, or Aspideretes swinhoei.

[0023] Compared with the prior art, the beneficial effects of the present application at least include:

[0024] Using the primers provided in the embodiments to perform PCR amplification on the genomic DNA of the Trionychidae, the sex of the Trionychidae can be directly identified according to the electrophoresis detection results of the PCR amplification products, and the identification process is simple and direct.

[0025] Furthermore, the embodiments also use labeled primers to perform PCR amplification on the genomic DNA of the Trionychidae, and the amplification products are detected by a lateral flow test strip, which can directly and simply visually display the results, and the identification results are simple and effective.

[0026] In addition, the primer set, kit, and method provided in the embodiments can identify the sex of Pelodiscus sinensis, Trachemys scripta, Apalone spinifera, or Aspideretes swinhoei, with strong identification versatility, wide identification range, and very wide application value.

[0027] The primer set, kit and method provided by the embodiment can combine the PCR amplification technology and the immunochromatography technology to achieve rapid detection of nucleic acid products. After PCR amplification, the product does not need to go through a complex gel electrophoresis experiment, and the sex identification of the Trionychidae can be achieved within 3 - 10 minutes.

[0028] The primer set, kit and method provided by the embodiment are easy to operate. It only needs to mix the nucleic acid product with a certain amount of PBS and then drop it into the sample well of the test strip, without any other operations.

[0029] The primer set, kit and method provided by the embodiment have a fast reaction speed. The sex identification of Pelodiscus sinensis can be achieved within 3 - 10 minutes, shortening the detection time.

[0030] The primer set, kit and method provided by the embodiment have low costs. The detection does not require complex instruments, just an ordinary PCR instrument, nor expensive special reagents, so the detection cost is low.

[0031] The primer set, kit and method provided by the embodiment have low requirements for operators. Since this technology does not require complex instruments, the operation process is simple, and the test results can be judged by the naked eye, anyone can be competent after self-study or simple training.

[0032] The primer set, kit and method provided by the embodiment have high safety. Special reagents are not needed, avoiding pollutants such as EB in gel electrophoresis, which are safe and harmless to operators and the test environment. Description of the Drawings

[0033] Figure 1 It is the electrophoresis detection result of general PCR amplification of Pelodiscus sinensis genomic DNA provided by the embodiment. Among them, the female code is ♀, and the male code is ♂.

[0034] Figure 2 It is the electrophoresis detection result of general PCR amplification of Apalone ferox, Apalone spinifera and Palea steindachneri genomic DNA provided by the embodiment. Among them, the female code is ♀, and the male code is ♂.

[0035] Figure 3 It is the electrophoresis detection result of RAA isothermal amplification of Pelodiscus sinensis genomic DNA provided by the embodiment. Among them, the female code is ♀, and the male code is ♂.

[0036] Figure 4 It is the electrophoresis detection result of RAA isothermal amplification of Apalone ferox, Apalone spinifera and Palea steindachneri genomic DNA provided by the embodiment. Among them, the female code is ♀, and the male code is ♂. Detailed Embodiment

[0037] To make the objectives, technical solutions and advantages of this application clearer, the following further elaborates on this application in combination with embodiments. It should be understood that the specific embodiments described herein are merely used to explain this application and are not used to limit this application. Reagents not specifically described in detail in this application are all conventional reagents and can be obtained from commercial channels; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0038] In this application, through screening, analysis, and experimental research, many genes with unsatisfactory inter-species specificity and many genes with unsatisfactory intra-species conservation were excluded. Finally, the conserved sequence of Trionychidae (ZHBRAA6, SEQ ID NO: 3, and the genomic location of this gene is ChrW: 7388446-7392658 (SEQ ID NO: 4)) was determined as the detection target sequence. Based on this target sequence, primers as shown in Table 1 were designed, which can effectively amplify ZHBRAA6 and achieve the identification of the gender of Trionychidae. The primer information is as follows:

[0039] Table 1 Primer Information Table

[0040]

[0041] For this reason, the embodiment provides a primer set, including DNA molecules as shown in SEQ ID NO: 1 and 2.

[0042] In some embodiments, the primer set includes a DNA molecule labeled with a first label as shown in SEQ ID NO: 1 and a DNA molecule labeled with a second label 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.

[0043] The embodiment discloses a kit, including this primer set and PCR amplification reagents.

[0044] In some embodiments of this kit, the PCR reagents include: dNTP mixture (the concentrations of ATP, GTP, CTP, and TTP are all 2.5 mmol / μL), 10×Ex Taq Buffer (Mg 2+ free), MgCl 2 (25 mM), and TaKaRa Ex Taq (5 U / μL).

[0045] In some embodiments of this 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).

[0046] The embodiment also discloses a method for identifying the sex of the Trionychidae. The method includes: extracting the genomic DNA of an individual of the Trionychidae to be tested; performing PCR amplification on it using the DNA molecules shown in SEQ ID NOs: 1 and 2; performing electrophoresis detection on the product of the PCR amplification; and determining the sex of the individual of the Trionychidae to be tested according to the bands detected by the electrophoresis.

[0047] In some embodiments of this method, the genomic DNA of an individual of the Trionychidae 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 a NanoDrop-2000 ultraviolet spectrophotometer, and the genomic DNA is stored at -20°C.

[0048] In some embodiments of this method, the reaction system for the PCR amplification includes, based on 50 μL: 1 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 a 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.

[0049] In some embodiments of this method, the reaction steps for the PCR amplification include: 5 min at 94°C; 30 s at 94°C, 30 s at 60°C, 100 s at 72°C, for 35 cycles; 5 min at 72°C.

[0050] In some embodiments of this method, the reaction system for the PCR amplification includes, based on 50 μL: 1 μL of genomic DNA at 50 ng / μL, 1 μL of the DNA molecule shown in SEQ ID NO: 1 at 10 μmol / μL, 1 μL of the DNA molecule shown in SEQ ID NO: 2 at 10 μmol / μL, 2.5 μL of magnesium acetate, 25 μL of an RAA reaction buffer (Jiangsu Qitian Gene Biotechnology Co., Ltd., China), and the balance of double-distilled water.

[0051] In some embodiments of this method, the reaction steps for the PCR amplification include: reacting at 39°C for 30 min.

[0052] Figure 2The electrophoresis results of the common PCR primers, kits and methods provided by the embodiments for identifying the sex of Chinese soft-shelled turtles are shown. As shown in the figure, no electrophoresis band was detected in male individuals, while a target band of 213 bp was detected in female individuals. Thus, it can be seen that the primers, kits and methods provided by the embodiments can accurately identify the sex of Chinese soft-shelled turtles.

[0053] Figure 3 The electrophoresis results of the primers, kits and methods provided by the embodiments for identifying the sex of Florida soft-shell turtles, spiny soft-shell turtles and wattle-necked soft-shell turtles by RAA amplification are shown. As shown in the figure, no electrophoresis band was detected in male individuals, while a target band of 213 bp was detected in female individuals. Thus, it can be seen that the primers, kits and methods provided by the embodiments can accurately identify the sex of Florida soft-shell turtles, spiny soft-shell turtles and wattle-necked soft-shell turtles.

[0054] In addition, the embodiments disclose a kit. The kit includes the PCR amplification kit, sample diluent and test strip provided by the above embodiments. The test strip is composed of a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad which are sequentially lapped and pasted on a backing. The conjugate pad is coated with 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 goat anti-mouse antibody. In the PCR amplification kit provided by the above embodiments, the upstream primer is labeled with biotin and the downstream primer is labeled with digoxin.

[0055] In some embodiments, the conjugate pad is coated with colloidal gold particles with 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.

[0056] 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.

[0057] The genomic DNA of the Trionychidae family is amplified by PCR using biotin and digoxin, so that the positive amplification products carry double labels. 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 develops 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 develops color. Negative amplification products do not develop color because there is no double-labeled substance during the chromatography process and cannot be captured by the detection line.

[0058] Based on this, the embodiment also discloses a method for identifying the gender of the Trionychidae family. The method includes: extracting genomic DNA from the tissues of the Trionychidae family; performing PCR amplification on the genomic DNA using a labeled primer set; loading the product of the PCR amplification onto the sample pad of a test strip; and identifying the gender of the Trionychidae family according to the display of the test 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.

[0059] In some embodiments, if both the test line and the control line are colored, then the Trionychidae family is female. If the test line is not colored and the control line is colored, then the Trionychidae family is male.

[0060] In the sixth aspect, the embodiment discloses 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 identifying the gender of the Trionychidae family. In some embodiments, the Trionychidae family is selected from at least one of Pelodiscus sinensis, Apalone mutica, Apalone spinifera, or Palea steindachneri.

[0061] 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 identifying the sex of turtles include: Extracting genomic DNA of the turtle family individuals to be tested; PCR amplification was performed using DNA molecules as shown in SEQ ID NOs: 1 and 2; Conducting electrophoresis detection on the PCR amplified product; The sex of the turtle individual to be tested is determined based on the bands detected by electrophoresis.

5. The method according to claim 4, wherein the PCR amplification reaction system comprises, in 50 μL: 1μL, 50ng / μL genomic DNA, 2μL, 20μmol / μL DNA molecule as shown in SEQ ID NO:1, 2μL, 20μmol / μL DNA molecule as shown in SEQ ID NO:2, 5μL 10×Ex Taq Buffer without magnesium ions, 3.3μL, 25mM MgCl2, 4μL mixed solution containing 2.5mmol / μL ATP, 2.5mmol / μL GTP, 2.5mmol / μL CTP and 2.5mmol / μL TTP, 0.25μL, 5U / μL TaKaRa Ex Taq and the remainder double distilled water.

6. The method according to claim 4, wherein the PCR amplification reaction system comprises, in 50 μL: 1 μL, 50 ng / μL of genomic DNA, 1 μL, 10 μmol / μL of the DNA molecule shown in SEQ ID NO: 1, 1 μL, 10 μmol / μL of the DNA molecule shown in SEQ ID NO: 2, 2.5 μL of magnesium acetate, 25 μL of RAA 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 identifying the sex of Trionycidae using the kit according to claim 7, comprising: Extraction of genomic DNA from Trionyx tissues; 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 Trionyx family is identified 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 Trionychidae is female; if the detection line is not colored, but the control line is colored, the Trionychidae 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 identifying the sex of Trionycis.

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