Primers, kits, methods and applications for identifying the sex of Chinese soft-shelled turtle

Abstract

The present application relates to the technical field of sex identification of Chinese soft-shelled turtle, and specifically to primers, kits, methods and applications for sex identification of Chinese soft-shelled turtle. The primers, kits, methods and applications are based on the target sequence shown in SEQ ID NO:3, and can accurately amplify Chinese soft-shelled turtle genomic DNA, and can accurately identify the sex of the soft-shelled turtle based on the amplified product. The PCR amplification product is further subjected to lateral flow test strip chromatography reaction, which can realize visual sex identification of Chinese soft-shelled turtle, has strong identification versatility, a wide identification range, and has very wide application value.

Description

Technical Field

[0001] The present application relates to the technical field of sex identification of Chinese soft-shelled turtle, and specifically to primers, kits, test strips, methods and applications for sex identification of Chinese soft-shelled turtle. Background Art

[0002] The Chinese soft-shell turtle (Pelodiscus sinensis) is a reptile of the family Testudinidae and genus Testudinidae. The Chinese soft-shell turtle has a flat, oval body, with a tortoise shell on its back and abdomen, and soft leathery skin on its limbs without keratinous scales. The head is thick and slightly triangular at the front. The snout is elongated and tubular, with a long fleshy snout, about the same size as the eyes. There are no teeth in the mouth, the neck is slender, flexible, and the vision is sharp. The plastron is grayish white or yellowish white, flat and smooth. The tail is short, the limbs are flat, the hind limbs are more developed than the forelimbs, and all the limbs can be retracted into the shell. The carapace is dark green or yellowish brown, surrounded by thick connective tissue, commonly known as the "skirt".

[0003] In order to realize the monosexual breeding and sex-controlled breeding of Chinese soft-shelled turtles, it is first necessary to determine the sex determination type of Chinese soft-shelled turtles. There are two types of sex determination in vertebrates, namely environmental sex determination (ESD) and genotypic sex determination (GSD). In the ESD type, the sex of animals is affected by various environmental factors. For example, crocodiles produce females when hatched at low and high temperatures, while males are dominant when hatched at medium temperatures; painted turtles and giant side-necked turtles produce males when hatched at low temperatures, while females are hatched at high temperatures. The GSD type is divided into two different heterogametic systems: XX / XY sex chromosomes and ZZ / ZW sex chromosomes. Chinese soft-shelled turtles belong to the ZZ / ZW type. The breeding cycle of Chinese soft-shelled turtles is long, and it takes one winter in ecological breeding mode to identify sex by appearance. Therefore, the development of early sex molecular identification technology for Chinese soft-shelled turtles can accurately identify the sex of newly hatched turtles, which will help to achieve monosexual pond breeding during the turtle stage, reduce costs and improve efficiency. Summary of the invention

[0004] The inventors of the present application designed primers and kits based on the conserved sequence of Chinese soft-shelled turtle (ZHBRAA2), which can perform PCR amplification on the genomic DNA of Chinese soft-shelled turtle, and identify the sex of Chinese soft-shelled turtle based on the PCR amplification product, with high identification efficiency and strong versatility. In addition, the inventors of the present application will also construct a lateral flow chromatography test strip. The test strip can perform chromatography detection on the amplification product as a sample, and visualize and quickly identify the sex of Chinese soft-shelled turtle through the T line and C line on the test strip.

[0005] In a first aspect, the embodiments disclose a primer set comprising DNA molecules as shown in SEQ ID NOs: 1 and 2.

[0006] In a second aspect, an embodiment discloses 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 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.

[0007] In a third aspect, the embodiment discloses a kit, which includes the primer set and PCR amplification reagent described in the first aspect.

[0008] In some embodiments, the PCR reagents include: dNTP mixture, 10×Ex TaqBuffer (Mg 2+ free), MgCl2 (25mM) and TaKaRa Ex Taq (5U / μL).

[0009] In some embodiments, the PCR reagent is a recombinase-mediated isothermal amplification reagent. The recombinase-mediated isothermal amplification reagent is from Jiangsu Qitian Gene Biotechnology Co., Ltd., China).

[0010] In a fourth aspect, an embodiment discloses a method for identifying the sex of a Chinese soft-shelled turtle, comprising extracting genomic DNA of a Chinese soft-shelled turtle individual to be tested; performing PCR amplification on the DNA molecules shown in SEQ ID NOs: 1 and 2; performing electrophoresis detection on the PCR amplification products; and determining the sex of the Chinese soft-shelled turtle individual to be tested based on the bands detected by the electrophoresis.

[0011] In some embodiments of the method, the PCR amplification reaction system includes, in 50 μL, 1 μL, 50 ng / μL genomic DNA, 2 μL, 20 μmol / μL of the DNA molecule shown in SEQ ID NO: 1, 2 μL, 20 μmol / μL of the DNA molecule shown in SEQ ID NO: 2, 5 μL 10×Ex Taq Buffer (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 TaKaRaEx Taq (5 U / μL) and the remainder double distilled water.

[0012] In some embodiments of the method, the reaction steps of the 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 this method, the PCR amplification reaction system includes, in 50 μL, 1 μL, 50 ng / μL of genomic DNA, 1 μL, 10 μmol / μL of a DNA molecule as shown in SEQ ID NO: 1, 1 μL, 10 μmol / μL of a DNA molecule as shown in SEQ ID NO: 2, 2.5 μL of magnesium acetate, 25 μL RRAA reaction buffer, and the remainder of double distilled water.

[0014] In some embodiments of the method, the PCR amplification reaction step includes: reacting at 39° C. for 30 minutes.

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

[0016] In the fifth aspect, the embodiment discloses a test kit. The test kit comprises the test kit described in the third aspect, a sample diluent and a test strip. The test strip is composed of a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad which are overlapped and pasted on a backing in sequence. The conjugation pad is coated with mouse anti-digoxigenin antibody labeled with colloidal gold particles, and the nitrocellulose membrane has a detection line coated with avidin and a control line coated with sheep anti-mouse antibody. In the test kit described in the second aspect, the upstream primer is labeled with biotin, and the downstream primer is labeled with digoxigenin.

[0017] In some embodiments, the colloidal gold particles coated on the binding pad have a particle size of 25 nm, 1 mL of colloidal gold particles labels 8.4 μg of mouse anti-digoxigenin antibody, and the coating amount of the formed colloidal gold-antibody complex on the binding pad is 2 mL / 30 cm.

[0018] In some embodiments, the concentration of avidin coated on the detection line on the nitrocellulose membrane is 0.5 mg / mL, and the coating volume is 1 μL / cm; the concentration of goat anti-mouse antibody coated on the control line is 1 mg / mL, and the coating volume is 1 μL / cm.

[0019] The genomic DNA of Chinese soft-shell turtle is PCR amplified using biotin and digoxin as primers, so that the positive amplification product is double-labeled. At the same time, the gold label binding pad on 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 quality control line is coated with goat anti-mouse antibody. During the chromatography process, the digoxin label of the positive amplification product is combined with the gold label complex on the gold label binding pad, and then the biotin label carried is combined with the avidin on the detection line of the chromatographic membrane. The "sandwich" sandwich structure complex formed is captured by the detection line to develop color, and the excess gold label complex continues to chromatograph, and when it reaches the control line, it is combined with the coated goat anti-mouse antibody, so that it is captured by the control line and develops color. The negative amplification product cannot be captured by the detection line during the chromatography process due to the absence of double labels, so it does not develop color.

[0020] In the sixth aspect, the embodiment discloses a method for identifying the sex of a Chinese soft-shelled turtle. The method comprises: extracting genomic DNA from a Chinese soft-shelled turtle tissue; performing PCR amplification on the genomic DNA using the primer set described in the second aspect; adding the PCR amplification product to a sample pad of a test strip; and identifying the sex of a Chinese soft-shelled turtle according to the display of the detection line and the display of the control line of the test strip.

[0021] In some embodiments of the method, if both the detection line and the control line are colored, the Chinese soft-shelled turtle is female. If the detection line is not colored, but the control line is colored, the Chinese soft-shelled turtle is male.

[0022] In the 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 identifying the sex of the Chinese soft-shelled turtle.

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

[0024] By using the primers provided in the embodiment to perform PCR amplification on the genomic DNA of the Chinese soft-shelled turtle, the sex of the Chinese soft-shelled turtle can be directly identified based on the electrophoresis detection results of the PCR amplification products, and the identification process is simple and direct.

[0025] Furthermore, the embodiment also uses labeled primers to perform PCR amplification on the genomic DNA of Chinese soft-shell turtle, and the amplified product is tested by lateral flow test strips, which can directly and easily visualize the results, and the identification results are simple and effective.

[0026] The primer set, kit and method provided in the embodiment can combine PCR amplification technology with immunochromatography technology to realize rapid detection of nucleic acid products. After PCR amplification, the product does not need to pass complex gel electrophoresis experiments, and the sex identification of Chinese soft-shelled turtle can be realized within 3-10 minutes.

[0027] The primer set, kit and method provided in the embodiment are simple to operate. It is only necessary to mix the nucleic acid product with a certain amount of PBS and then drip it into the sample hole of the test strip, and no other operation is required.

[0028] The primer set, kit and method provided in the embodiment have a fast reaction speed, and the sex identification of Chinese soft-shelled turtle can be achieved within 3-10 minutes, thus shortening the detection time.

[0029] The primer set, kit and method provided in the embodiment have low cost, and detection does not require complicated instruments, but only a common PCR instrument, and no expensive special reagents are required, so the detection cost is low.

[0030] The primer sets, kits and methods provided in the embodiments have low requirements on operators. Since the technology does not require complex instruments, the operation process is simple, and the detection results can be interpreted by naked eyes, anyone can perform the operation after self-study or simple training.

[0031] The primer set, kit and method provided in the embodiment are highly safe, do not require the use of special reagents, avoid pollutants such as EB in gel electrophoresis, and are safe and harmless to operators and the experimental environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The electrophoresis test results of the general PCR amplification of the Chinese soft-shelled turtle genomic DNA provided in the embodiment. Among them, the female code is ♀, and the male code is ♂.

[0033] Figure 2 The electrophoresis test results of RAA isothermal amplification of Chinese soft-shelled turtle genomic DNA provided in the example. Among them, the female code is ♀, and the male code is ♂. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0035] In this application, after screening, analysis and experimental research, it was determined that the conserved sequence of Chinese soft-shelled turtle (ZHBRAA2, SEQ ID NO: 3, the genomic location of the gene (SEQ ID NO: 4) is> ChrW: 6241923-6246272) was used as the detection target sequence. Based on the target sequence, the primers shown in Table 1 were designed to effectively amplify ZHBRAA2 and identify the sex of Chinese soft-shelled turtle. The primer information is as follows:

[0036] Table 1 Primer information

[0037]

[0038] To this end, the embodiment provides a primer set including the DNA molecules shown in SEQ ID NO: 1 and 2.

[0039] In some embodiments, 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 both selected from one of biotin, fluorescein isothiocyanate or digoxigenin, and the first label is different from the second label.

[0040] The embodiment discloses a kit, including the primer set and a PCR amplification reagent.

[0041] In some embodiments of the kit, the PCR reagents include: dNTP mixture (the concentration of ATP, GTP, CTP and TTP are all 2.5mmol / μL), 10×Ex Taq Buffer (Mg 2+ free), MgCl2 (25mM) and TaKaRaEx Taq (5U / μL).

[0042] 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).

[0043] The embodiment also discloses a method for identifying the sex of a Chinese soft-shelled turtle. The method comprises: extracting genomic DNA of a Chinese soft-shelled turtle individual to be tested; performing PCR amplification on the DNA molecules shown in SEQ ID NOs: 1 and 2; performing electrophoresis detection on the PCR amplification product; and determining the sex of the Chinese soft-shelled turtle individual to be tested based on the bands detected by the electrophoresis.

[0044] In some embodiments of the method, the genomic DNA of the Chinese soft-shelled turtle individual to be tested is extracted according to the instructions 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 UV spectrophotometer, and the genomic DNA is stored at -20°C.

[0045] In some embodiments of the method, the PCR amplification reaction system includes, in 50 μL, 1 μL, 50 ng / μL genomic DNA, 2 μL, 20 μmol / μL of a DNA molecule as shown in SEQ ID NO: 1, 2 μL, 20 μmol / μL of a DNA molecule as shown in SEQ ID NO: 2, 5 μL 10×Ex Taq Buffer (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.

[0046] In some embodiments of the method, the reaction steps of the 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.

[0047] In some embodiments of the method, the PCR amplification reaction system includes, in 50 μL, 1 μL, 50 ng / μL of genomic DNA, 1 μL, 10 μmol / μL of a DNA molecule as shown in SEQ ID NO: 1, 1 μL, 10 μmol / μL of a DNA molecule as shown in SEQ ID NO: 2, 2.5 μL of magnesium acetate, 25 μL RAA reaction buffer (Jiangsu Qitian Gene Biotechnology Co., Ltd., China), and the remainder of double distilled water.

[0048] In some embodiments of the method, the PCR amplification reaction step includes: reacting at 39° C. for 30 minutes.

[0049] Figure 1 The electrophoresis results of the common PCR primers, kits and methods provided in the examples for identifying the sex of Chinese soft-shelled turtles are shown. As shown in the figure, no electrophoresis band is detected in male individuals, while a target band of 213 bp is detected in female individuals. It can be seen that the primers, kits and methods provided in the examples can accurately identify the sex of Chinese soft-shelled turtles.

[0050] Figure 2 The electrophoresis results of using the primers, kits and methods for RAA amplification provided in the embodiment to identify the sex of pearl softshell turtle, horned softshell turtle and mountain softshell turtle are shown. As shown in the figure, no electrophoresis band is detected in the male individual, while a target band of 213 bp is detected in the female individual. It can be seen that the primers, kits and methods provided in the embodiment can accurately identify the sex of pearl softshell turtle, horned softshell turtle and mountain softshell turtle.

[0051] In addition, the embodiment discloses a test kit. The test kit includes the test kit for PCR amplification provided in the above embodiment, a sample diluent and a test strip. The test strip is composed of a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad which are overlapped and pasted on a backing in sequence. The conjugation pad is coated with mouse anti-digoxigenin antibody labeled with colloidal gold particles, and the nitrocellulose membrane has a detection line coated with avidin and a control line coated with sheep anti-mouse antibody. In the test kit for PCR amplification provided in the above embodiment, the upstream primer is labeled with biotin, and the downstream primer is labeled with digoxigenin.

[0052] In some embodiments, the colloidal gold particles coated on the binding pad have a particle size of 25 nm, 1 mL of colloidal gold particles labels 8.4 μg of mouse anti-digoxigenin antibody, and the coating amount of the formed colloidal gold-antibody complex on the binding pad is 2 mL / 30 cm.

[0053] In some embodiments, the concentration of avidin coated on the detection line on the nitrocellulose membrane is 0.5 mg / mL, and the coating volume is 1 μL / cm; the concentration of goat anti-mouse antibody coated on the control line is 1 mg / mL, and the coating volume is 1 μL / cm.

[0054] The genomic DNA of Chinese soft-shell turtle is PCR amplified using biotin and digoxin as primers, so that the positive amplification product is double-labeled. At the same time, the gold label binding pad on 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 quality control line is coated with goat anti-mouse antibody. During the chromatography process, the digoxin label of the positive amplification product is combined with the gold label complex on the gold label binding pad, and then the biotin label carried is combined with the avidin on the detection line of the chromatographic membrane. The "sandwich" sandwich structure complex formed is captured by the detection line to develop color, and the excess gold label complex continues to chromatograph, and when it reaches the control line, it is combined with the coated goat anti-mouse antibody, so that it is captured by the control line and develops color. The negative amplification product cannot be captured by the detection line during the chromatography process due to the absence of double labels, so it does not develop color.

[0055] Based on this, the embodiment also discloses a method for identifying the sex of a Chinese soft-shelled turtle. The method comprises: extracting genomic DNA from a Chinese soft-shelled turtle tissue; performing PCR amplification on the genomic DNA using a labeled primer set; applying the PCR amplification product to a sample pad of a test strip; and identifying the sex of a Chinese soft-shelled turtle according to the display of the detection line and the display of the control line of the test strip. The primer set comprises 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; the first label and the second label are both selected from one of biotin, fluorescein isothiocyanate or digoxin, and the first label is different from the second label.

[0056] In some embodiments, if both the detection line and the control line are colored, the Chinese soft-shelled turtle is female. If the detection line is not colored, but the control line is colored, the Chinese soft-shelled turtle is male.

[0057] In a sixth 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 identifying the sex of the Chinese soft-shelled turtle.

[0058] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within 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 Chinese soft-shelled turtle include: Extracting genomic DNA of the Chinese soft-shelled turtle individual 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; Determine the sex of the Chinese soft-shelled turtle individual to be tested according to the target band detected by electrophoresis; If there is no electrophoresis band, the Chinese soft-shelled turtle to be tested is a male individual; If the target band of 213 bp appears, the Chinese soft-shelled turtle individual to be tested is a female individual.

5. The method according to claim 4, wherein the PCR amplification reaction system comprises, in 50 μL: 1 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×Ex Taq Buffer without magnesium ions, 3.3 μL 25 mM MgCl2, 4 μL dNTP mixture containing 2.5 mmol / μL ATP, 2.5 mmol / μL GTP, 2.5 mmol / μL CTP and 2.5 mmol / μL TTP, 0.25 μL, 5 U / 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 kit comprising a sample diluent, a test strip, a primer set and a PCR amplification reagent, wherein the primer set comprises a biotin-labeled DNA molecule as shown in SEQ ID NO: 1 and a digoxigenin-labeled DNA molecule as shown in SEQ ID NO: 2, the test strip comprises a sample pad, a conjugation pad, a nitrocellulose membrane and an absorbent pad which are sequentially overlapped and adhered to a backing, the conjugation pad is coated with mouse anti-digoxigenin 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.

8. A method for identifying the sex of a Chinese soft-shell turtle using the kit according to claim 7, comprising: Extract genomic DNA from Chinese soft-shell turtle tissues; Performing PCR amplification on the genomic DNA using the primer set; Adding the PCR amplified product to the sample pad of the test strip; The sex of the Chinese soft-shelled turtle is identified according to the display of the detection line and the control line of the test strip; if the detection line and the control line are both colored, the Chinese soft-shelled turtle is female; if the detection line is not colored, but the control line is colored, the Chinese soft-shelled turtle is male.

9. Use of the primer set of claim 1 in identifying the sex of a Chinese soft-shell turtle, the step of identifying the sex of a Chinese soft-shell turtle comprising: Extracting genomic DNA of the Chinese soft-shelled turtle individual 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; Determine the sex of the Chinese soft-shelled turtle individual to be tested according to the target band detected by electrophoresis; If there is no electrophoresis band, the Chinese soft-shelled turtle to be tested is a male individual; If the target band of 213 bp appears, the Chinese soft-shelled turtle individual to be tested is a female individual.

10. Use of the method according to claim 8 in identifying the sex of Chinese soft-shell turtle.

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