DNA fragments, primer set and kit for genetic sex identification of acipenser sinensis, and uses

Abstract

Provided are DNA fragments, a primer set and a kit for genetic sex identification of Acipenser sinensis, and uses. In a first aspect, provided is a use of a DNA fragment common to both male and female Acipenser sinensis and a female Acipenser sinensis-specific DNA fragment in genetic sex identification of Acipenser sinensis, wherein the nucleotide sequence of the DNA fragment common to both male and female Acipenser sinensis is as shown in SEQ ID NO: 1, and the nucleotide sequence of the female Acipenser sinensis-specific DNA fragment is as shown in SEQ ID NO: 2. The two DNA fragments provided are used as target amplification fragments, and genetic sex identification is carried out on Acipenser sinensis by means of environmental DNA samples, thereby realizing non-contact genetic sex identification and achieving good stability and accuracy.

Description

DNA fragments, primer sets, kits and applications for genetic sex identification of Chinese sturgeon

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on February 29, 2024, with application number 202410231404.5 and application name “DNA fragments, primer sets, kits and applications for genetic sex identification of Chinese sturgeon”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application relates to a DNA fragment, a primer set, a kit and applications for genetic sex identification of Chinese sturgeon, and relates to the technical field of genetic sex identification of Chinese sturgeon. Background Art

[0003] The Chinese sturgeon (Acipenser sinensis) is a typical anadromous fish. During its breeding season, from September to November each year, mature individuals migrate upstream to the upper reaches of the Yangtze River to spawn. The newly born juveniles migrate downstream to the Yangtze River estuary, where they fatten and then migrate to the ocean to grow, returning to the Yangtze River to reproduce again after reaching sexual maturity. Due to hydropower construction and other human activities, the Chinese sturgeon population has declined sharply. To conserve this species, several domestic research institutions have implemented various conservation measures, including ex situ conservation and stocking enhancement.

[0004] However, there is no obvious difference in the external appearance of male and female Chinese sturgeons. There is no nuptial color during the reproductive period. Both male and female individuals have no external genitalia and no secondary sexual characteristics such as beads, making it difficult to determine their gender.

[0005] Early sex identification methods, including ultrasound, endoscopy, and molecular testing, have been developed. However, these methods require the fish to be older or may damage the fish. For example, ultrasound testing of Chinese sturgeons requires them to be over 5 years old, endoscopy requires an incision in the lower abdomen and has a high accuracy rate for fish over 5 years old, and molecular testing, while capable of identifying fish of any age, requires tissue samples.

[0006] Organisms release their own DNA into their environments through various channels, including skin, mucus, urine, feces, blood, sperm, eggs, and decaying corpses. This DNA is called environmental DNA. Unlike DNA in biological tissues, environmental DNA exists in different ways and is susceptible to varying degrees of degradation. Therefore, the target sequence should be relatively short. However, existing target sequences are mostly used for species identification, and there have been no reports of genetic sex determination of the Chinese sturgeon using environmental DNA samples.

[0007] Summary of the Invention

[0008] The present application provides a DNA fragment, a primer set, a kit and applications for genetic sex identification of Chinese sturgeon.

[0009] In the first aspect of the present application, a common DNA fragment between male and female Chinese sturgeons and a DNA fragment specific to female Chinese sturgeons are provided for use in genetic sex identification of Chinese sturgeons. The nucleotide sequence of the common DNA fragment between male and female Chinese sturgeons is shown in SEQ ID NO: 1, and the nucleotide sequence of the DNA fragment specific to female Chinese sturgeons is shown in SEQ ID NO: 2.

[0010] Based on the genomic information and differential sites of male and female Chinese sturgeons, this application provides a DNA fragment common to both male and female Chinese sturgeons (whose nucleotide sequence is shown in SEQ ID NO: 1). This DNA fragment can be stably and efficiently amplified in both male and female Chinese sturgeons of all ages, and can be used as a target amplification sequence to determine whether environmental DNA contains Chinese sturgeon DNA. This application also provides a DNA fragment unique only to female Chinese sturgeons (whose nucleotide sequence is shown in SEQ ID NO: 2). This fragment is unique only to female Chinese sturgeons and not present in male Chinese sturgeons. It can be used as a target amplification sequence to determine the genetic sex of Chinese sturgeons and improve detection accuracy.

[0011] Based on the DNA fragment shared by male and female Chinese sturgeons and the DNA fragment unique to female Chinese sturgeons provided in the first aspect of the present application, the second aspect of the present application provides a primer set for amplifying the DNA fragment shared by male and female Chinese sturgeons and the DNA fragment unique to female Chinese sturgeons, comprising a first primer and a second primer for amplifying the DNA fragment shared by male and female Chinese sturgeons, and a third primer and a fourth primer for amplifying the DNA fragment unique to female Chinese sturgeons;

[0012] The nucleotide sequence of the first primer is shown in SEQ ID NO: 3;

[0013] The nucleotide sequence of the second primer is shown in SEQ ID NO: 4;

[0014] The nucleotide sequence of the third primer is shown in SEQ ID NO: 5;

[0015] The nucleotide sequence of the fourth primer is shown in SEQ ID NO: 6.

[0016] It should be noted that the first primer provided in this application is a downstream primer for amplifying a DNA fragment common to male and female Chinese sturgeons, and the second primer is an upstream primer for a DNA fragment common to male and female Chinese sturgeons; the third primer is a downstream primer for amplifying a DNA fragment unique to female Chinese sturgeons, and the fourth primer is an upstream primer for amplifying a DNA fragment unique to female Chinese sturgeons.

[0017] The third aspect of the present application provides a kit for genetic sex identification of Chinese sturgeon, comprising the primer set provided in the second aspect above.

[0018] The kit as described above further comprises a negative control substance.

[0019] The fourth aspect of the present application provides the use of the primer set provided in the second aspect or the kit provided in the third aspect in the genetic sex identification of Chinese sturgeon.

[0020] A fifth aspect of the present application provides a method for genetic sex identification of Chinese sturgeon, comprising the following steps:

[0021] Collect DNA samples of Chinese sturgeon to be tested;

[0022] According to the DNA fragment shared by male and female Chinese sturgeons and the DNA fragment unique to female Chinese sturgeons provided in the first aspect above, a first primer and a second primer for amplifying the DNA fragment shared by male and female Chinese sturgeons, as well as a third primer and a fourth primer for amplifying the DNA fragment unique to female Chinese sturgeons are designed;

[0023] Performing a first real-time fluorescence quantitative PCR test on the DNA sample using the first primer and the second primer, and performing a second real-time fluorescence quantitative PCR test on the DNA sample using the third primer and the fourth primer;

[0024] The genetic sex of the Chinese sturgeon to be tested is determined based on the results of the first real-time fluorescence quantitative PCR test and the second real-time fluorescence quantitative PCR test.

[0025] In a specific embodiment, the above method includes the following steps:

[0026] Step 1: Collect DNA samples of Chinese sturgeon to be tested.

[0027] First, collect a DNA sample from a Chinese sturgeon. This DNA sample is an environmental DNA sample. It is understood that when the environmental DNA sample comes from a water sample in which Chinese sturgeons live, it is necessary to limit the presence of a single individual Chinese sturgeon in the water sample. For example, the individual Chinese sturgeon to be tested can be temporarily placed in a separate tank. Once Chinese sturgeon DNA is present in the water, the water sample is collected and DNA extraction is performed. For wild Chinese sturgeons, mucus, feces, and other sources of environmental DNA can be collected.

[0028] Based on the obtained samples to be tested, DNA extraction can be performed using conventional technical means in the field. In a specific embodiment, taking the water sample in which the Chinese sturgeon lives as an example, the DNA extraction process includes the following steps: First, the collected water sample is filtered using a filter membrane, and the filtration can be performed using a vacuum filtration device. Secondly, the blank area around the filter membrane is cut off, and after cutting it into 4 sectors of equal area along the center line, it is added to a 50ml centrifuge tube containing 3ml SLX reaction solution and 500mg glass beads, mixed at maximum speed on a vortex instrument, and then the centrifuge tube is placed in an ultrasonic instrument for ultrasonic treatment, with intermittent vortexing during the process, so that all the substances on the filter membrane are eluted into the SLX reaction solution, and the DNA on the filter membrane is obtained to the maximum extent. Then, a DNA extraction kit is used to extract DNA, and the extracted DNA is stored at -20℃ for future use.

[0029] In addition, during ultrasonic treatment, appropriate heating can be performed, with the temperature not exceeding 50°C, which helps shorten the elution time of the substance on the filter membrane.

[0030] Step 2: Based on the DNA fragment shared by male and female Chinese sturgeons and the DNA fragment unique to female Chinese sturgeons provided in the first aspect above, design a first primer and a second primer for amplifying the DNA fragment shared by male and female Chinese sturgeons, as well as a third primer and a fourth primer for amplifying the DNA fragment unique to female Chinese sturgeons.

[0031] In a specific embodiment, the primers provided in the second aspect of the present application can be used to amplify subsequent DNA fragments.

[0032] Step 3: Perform a first real-time fluorescence quantitative PCR test on the DNA sample using the first primer and the second primer, and perform a second real-time fluorescence quantitative PCR test on the DNA sample using the third primer and the fourth primer.

[0033] The present application does not limit the order of the first real-time fluorescence quantitative PCR detection and the second real-time fluorescence quantitative PCR detection, that is, the first primer and the second primer can be used to perform real-time fluorescence quantitative PCR detection on the DNA sample first, and then the third primer and the fourth primer can be used to perform real-time fluorescence quantitative PCR detection on the DNA sample; alternatively, the third primer and the fourth primer can be used to perform real-time fluorescence quantitative PCR detection on the DNA sample first, and then the first primer and the second primer can be used to perform real-time fluorescence quantitative PCR detection on the DNA sample.

[0034] The fluorescent quantitative PCR detection process can be carried out according to conventional technical means in the field. This application mainly explains the judgment of the test results: when the Ct values ​​of the first fluorescent quantitative PCR test result and the second fluorescent quantitative PCR test result are both ≤36, the genetic sex of the Chinese sturgeon to be tested is determined to be female; when the Ct value of the second fluorescent quantitative PCR test result is greater than 36 or displays Undetermined, and the Ct value of the first fluorescent quantitative PCR test result is ≤36, the genetic sex of the Chinese sturgeon to be tested is determined to be male; when the Ct values ​​of the first fluorescent quantitative PCR test result and the second fluorescent quantitative PCR test result are greater than 36 or display Undetermined, it is determined that the DNA sample does not include DNA derived from the Chinese sturgeon.

[0035] It is understood that when the DNA sample is subjected to real-time fluorescence quantitative PCR, the negative control is subjected to fluorescence quantitative PCR using the same method. The negative control can be sterile water.

[0036] In summary, the common DNA fragments of male and female Chinese sturgeons and the unique DNA fragments of female Chinese sturgeons provided in this application can be used to detect and identify the genetic sex of Chinese sturgeons. Not only will it not cause any damage to the Chinese sturgeons, it will effectively avoid the impact of Chinese sturgeon breeding during the identification process. Moreover, the shorter target sequence helps prevent the degradation of environmental DNA samples and cause detection failure. It can realize the rapid and accurate identification of the genetic sex of individual Chinese sturgeons of different age groups, and is suitable for Chinese sturgeons throughout their life cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1a is a fluorescence PCR amplification curve obtained by performing real-time fluorescence quantitative PCR on female Chinese sturgeon samples using the common primer pair Co-Nuc-FM for both males and females in Example 1 of the present application;

[0038] FIG1b is a melting curve obtained by real-time fluorescence quantitative PCR detection of female Chinese sturgeon samples using the common primer pair Co-Nuc-FM for both males and females in Example 1 of the present application;

[0039] FIG2a is a fluorescence PCR amplification curve obtained by performing real-time fluorescence quantitative PCR on male Chinese sturgeon samples using the common primer pair Co-Nuc-FM for both males and females in Example 1 of the present application;

[0040] FIG2 b is a melting curve obtained by real-time fluorescence quantitative PCR detection of male Chinese sturgeon samples using the common primer pair Co-Nuc-FM for both males and females in Example 1 of the present application;

[0041] FIG3a is a fluorescence PCR amplification curve obtained by performing real-time fluorescence quantitative PCR on female Chinese sturgeon samples using the female-specific primer pair Spec-Nuc-F in Example 1 of the present application;

[0042] FIG3 b is a melting curve obtained by real-time fluorescence quantitative PCR detection of female Chinese sturgeon samples using the female-specific primer pair Spec-Nuc-F in Example 1 of the present application;

[0043] FIG4a is a fluorescence PCR amplification curve obtained by performing real-time fluorescence quantitative PCR on male Chinese sturgeon samples using the female-specific primer pair Spec-Nuc-F in Example 1 of the present application;

[0044] FIG4 b is a melting curve obtained by performing real-time fluorescence quantitative PCR on male Chinese sturgeon samples using the female-specific primer pair Spec-Nuc-F in Example 1 of the present application;

[0045] FIG5a is a fluorescence PCR amplification curve obtained by performing real-time fluorescence quantitative PCR on female Chinese sturgeon samples using the common primer pair Co-Nuc-FM for both males and females in Example 2 of the present application;

[0046] FIG5b is a melting curve obtained by real-time fluorescence quantitative PCR detection of female Chinese sturgeon samples using the common primer pair Co-Nuc-FM for both males and females in Example 2 of the present application;

[0047] FIG6a is a fluorescence PCR amplification curve obtained by performing real-time fluorescence quantitative PCR on male Chinese sturgeon samples using the common primer pair Co-Nuc-FM for both males and females in Example 2 of the present application;

[0048] FIG6 b is a melting curve obtained by real-time fluorescence quantitative PCR detection of male Chinese sturgeon samples using the common primer pair Co-Nuc-FM for both males and females in Example 2 of the present application;

[0049] FIG7a is a fluorescence PCR amplification curve obtained by performing real-time fluorescence quantitative PCR on a negative control sample using the common primer pair Co-Nuc-FM in Example 2 of the present application;

[0050] FIG7 b is a melting curve obtained by performing real-time fluorescence quantitative PCR on a negative control sample using the male and female common primer pair Co-Nuc-FM in Example 2 of the present application;

[0051] FIG8a is a fluorescence PCR amplification curve obtained by performing real-time fluorescence quantitative PCR on female Chinese sturgeon samples using the female-specific primer pair Spec-Nuc-F in Example 2 of the present application;

[0052] FIG8b is a melting curve obtained by real-time fluorescence quantitative PCR detection of female Chinese sturgeon samples using the female-specific primer pair Spec-Nuc-F in Example 2 of the present application;

[0053] FIG9a is a fluorescence PCR amplification curve obtained by performing real-time fluorescence quantitative PCR on male Chinese sturgeon samples using the female-specific primer pair Spec-Nuc-F in Example 2 of the present application;

[0054] FIG9 b is a melting curve obtained by real-time fluorescence quantitative PCR detection of male Chinese sturgeon samples using the female-specific primer pair Spec-Nuc-F in Example 2 of the present application;

[0055] FIG10 a is a fluorescence PCR amplification curve obtained by performing real-time fluorescence quantitative PCR on a negative control sample using the female-specific primer pair Spec-Nuc-F in Example 2 of the present application;

[0056] FIG10 b is a melting curve obtained by performing real-time fluorescence quantitative PCR on a negative control sample using the female-specific primer pair Spec-Nuc-F in Example 2 of the present application;

[0057] FIG11a is a fluorescence PCR amplification curve obtained by performing real-time fluorescence quantitative PCR on a 2-fold diluted water sample using the female-specific primer pair Spec-Nuc-F in Example 3 of the present application;

[0058] FIG11b is a melting curve obtained by performing real-time fluorescence quantitative PCR on a 2-fold diluted water sample using the female-specific primer pair Spec-Nuc-F in Example 3 of the present application;

[0059] FIG12 is a diagram showing the sensitivity detection of the female-specific primer pair Spec-Nuc-F in Example 3 of the present application. DETAILED DESCRIPTION

[0060] To make the purpose, technical solutions, and advantages of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0061] Example 1 Verification of Genetic Sex Specificity of Female-Specific Primer Pairs

[0062] Step 1: Select three male and three female Chinese sturgeons of known genetic sex, cut their fin rays and extract DNA.

[0063] Step 2: Fluorescent quantitative PCR was performed on female and male Chinese sturgeons using the primer pair Co-Nuc-FM, which amplifies a DNA fragment common to both male and female Chinese sturgeons, and the primer pair Spec-Nuc-F, which amplifies a DNA fragment unique to female Chinese sturgeons. The amplification system consisted of 20 μL of 2×Q3 SYBR qPCR Master Mix (Universal), 10 μL of 2×Q3 SYBR qPCR Master Mix (Universal), 0.4 μL of each upstream and downstream primer, 1 μL of DNA template, and 8.2 μL of ddH2O. The reaction conditions were: initial denaturation at 95°C for 30 s; denaturation at 95°C for 10 s; annealing at 56°C for 30 s; extension at 72°C for 30 s; and 40 cycles.

[0064] The PCR amplification results are shown in Figures 1a-4b, respectively. The primer pair Co-Nuc-FM, which amplifies the DNA fragment shared by both sexes, had Ct values ​​≤ 36 in both female and male Chinese sturgeon samples, indicating positive results. The primer pair amplifying the DNA fragment unique to female Chinese sturgeon had Ct values ​​≤ 36 in female samples, indicating positive results; however, the Ct values ​​in male samples were > 36 or undetermined, indicating negative results. This confirms that the shared primer pair has good amplification efficiency in both male and female Chinese sturgeons, while the female-specific primer pair only amplified positive results in female Chinese sturgeons, demonstrating good genetic sex specificity.

[0065] Example 2 Application of two primer pairs in individual Chinese sturgeon water samples

[0066] Step 1: Select one female and one male Chinese sturgeon of about one year old from the Chinese sturgeon breeding pond and place them in separate aquariums for temporary breeding. Collect 2 L of water sample after 12 hours. Use 0.22 μm filter membrane for vacuum filtration, and use one filter membrane for every liter of water. Place the filtered filter membrane in a centrifuge tube and store it at -20℃ for later use. Wear gloves during the entire operation and use tweezers to transfer the filter membrane. Rinse the filter with sterile water before filtering different samples to prevent cross contamination. Set up a sterile water negative control.

[0067] Step 2: Cut off the blank area around each filter membrane and cut it into 4 sectors of equal area along the center line. Add 3 ml of SLX reaction solution and 500 mg of glass beads into a 50 ml centrifuge tube. Mix at the maximum speed on a vortexer. Then place the centrifuge tube in an ultrasonic instrument to heat and sonicate, vortexing intermittently. DNA was extracted from the membrane using the Water DNA Kit (Omega, USA) and stored at -20°C for later use.

[0068] Step 3. Fluorescent quantitative PCR was performed on the water samples and negative samples using the primer pair Co-Nuc-FM, which amplifies a DNA fragment shared by both male and female Chinese sturgeons. The amplification system consisted of 20 μL of 2×Q3 SYBR qPCR Master Mix (Universal), 10 μL of 2×Q3 SYBR qPCR Master Mix (Universal), 0.4 μL of each upstream and downstream primer, 1 μL of DNA template, and 8.2 μL of ddH2O. The reaction conditions were: initial denaturation at 95°C for 30 s; denaturation at 95°C for 10 s; annealing at 56°C for 30 s; extension at 72°C for 30 s; and 40 cycles.

[0069] The amplification results are shown in Figures 5a-7b. The Ct values ​​of the common primer pair Co-Nuc-FM in both water samples were ≤36, indicating that the results were positive, indicating the presence of Chinese sturgeon in the collected water. The Ct values ​​of the negative controls were all >36 or Undetermined, indicating negative results.

[0070] Step 4: Use the primer pair Spec-Nuc-F that amplifies the female Chinese sturgeon-specific DNA fragment to perform fluorescent quantitative PCR on the above water samples and negative samples. The amplification system and conditions are the same as step 3.

[0071] The amplification results are shown in Figures 8a-10b. The Ct values ​​of the female-specific primer pair Spec-Nuc-F in the water samples of female Chinese sturgeons were all ≤36, indicating a positive result, indicating that the genetic sex of this Chinese sturgeon was female. The Ct values ​​in the water samples of male Chinese sturgeons were >36 or Undetermined, indicating a negative result, indicating that the genetic sex of this Chinese sturgeon was male. The genetic sex identification results of the water samples using the two sets of primer pairs were consistent with the actual genetic sex of the Chinese sturgeons, confirming that the genetic sex of individual Chinese sturgeons can be successfully identified through water samples using these two sets of primer pairs.

[0072] Example 3 Sensitivity Detection of Primer Pairs

[0073] According to the above two examples, both primer pairs can stably perform amplification reactions in positive samples. This example tests the sensitivity of the female-specific primers.

[0074] The DNA from the water sample in the pool was diluted two-fold in a series, with the first dilution being 1 / 2 of the initial concentration, the second dilution being 1 / 4, and so on to produce four dilutions. PCR amplification was performed according to the amplification system and reaction conditions used for the water sample test in Example 2. The amplification results are shown in Figures 11a-11b.

[0075] According to Figures 11a-11b, the initial DNA concentration in the water sample was 2.1 ng / μl. When diluted 8-fold, the Ct value was close to 36, and after dilution to 16-fold, the Ct value was ≤36. Based on the initial DNA concentration, the DNA concentration corresponding to different dilution factors was converted by 2-fold decrease, and the linear relationship between the Ct value and DNA concentration was plotted. According to Figure 12, the lowest effective water sample concentration that can be detected by the female-specific primer pair is approximately 0.25 ng / μl.

[0076] In summary, the common DNA fragments of male and female Chinese sturgeons and the unique DNA fragments of female Chinese sturgeons provided in this application can be used to detect and identify the genetic sex of Chinese sturgeons. Not only will it not cause any damage to the Chinese sturgeons, it will effectively avoid the impact of Chinese sturgeon breeding during the identification process. Moreover, the shorter target sequence helps prevent the degradation of environmental DNA samples and cause detection failure. It can realize the rapid and accurate identification of the genetic sex of individual Chinese sturgeons of different age groups, and is suitable for Chinese sturgeons throughout their life cycle.

[0077] In the description of this application specification, the terms "first" and "second" are used to distinguish similar objects and are not necessarily used to describe a specific order or precedence. It should be understood that the terms used in this manner are interchangeable where appropriate, so that the embodiments of the present application described herein can be implemented in orders other than those illustrated or described herein.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application.

Claims

1. An application of a DNA fragment common to both male and female Chinese sturgeons and a DNA fragment unique to female Chinese sturgeons in genetic sex identification of Chinese sturgeons, wherein: The nucleotide sequence of the DNA fragment shared by male and female Chinese sturgeons is shown in SEQ ID NO: 1, and the nucleotide sequence of the DNA fragment unique to female Chinese sturgeons is shown in SEQ ID NO:

2.

2. A primer set for amplifying a DNA fragment common to both male and female Chinese sturgeons and a DNA fragment unique to female Chinese sturgeons, wherein: The method comprises a first primer and a second primer for amplifying a DNA fragment common to male and female Chinese sturgeons, and a third primer and a fourth primer for amplifying a DNA fragment unique to female Chinese sturgeons; The nucleotide sequence of the first primer is shown in SEQ ID NO: 3; The nucleotide sequence of the second primer is shown in SEQ ID NO: 4; The nucleotide sequence of the third primer is shown in SEQ ID NO: 5; The nucleotide sequence of the fourth primer is shown in SEQ ID NO:

6.

3. A kit for genetic sex identification of Chinese sturgeon, wherein: Comprising the primer set according to claim 2.

4. The kit according to claim 3, wherein The kit also includes a negative control.

5. Use of the primer set according to claim 2 or the kit according to any one of claims 3 to 4 in genetic sex identification of Chinese sturgeon.

6. A method for identifying the genetic sex of a Chinese sturgeon, wherein: The steps include: Collect DNA samples of Chinese sturgeon to be tested; The DNA fragment common to male and female Chinese sturgeons and the DNA fragment specific to female Chinese sturgeons according to claim 1, a first primer and a second primer designed to amplify the DNA fragment common to male and female Chinese sturgeons, and a third primer and a fourth primer designed to amplify the DNA fragment specific to female Chinese sturgeons; Performing a first real-time fluorescence quantitative PCR test on the DNA sample using the first primer and the second primer, and performing a second real-time fluorescence quantitative PCR test on the DNA sample using the third primer and the fourth primer; The genetic sex of the Chinese sturgeon to be tested is determined based on the results of the first real-time fluorescence quantitative PCR test and the second real-time fluorescence quantitative PCR test.

7. The method according to claim 6, wherein: The DNA sample is an environmental DNA sample.

8. The method according to claim 6 or 7, wherein: The nucleotide sequence of the first primer is shown in SEQ ID NO: 3; the nucleotide sequence of the second primer is shown in SEQ ID NO:

4.

9. The method according to claim 6 or 7, wherein: The nucleotide sequence of the third primer is shown in SEQ ID NO: 5; the nucleotide sequence of the fourth primer is shown in SEQ ID NO:

6.

10. The method according to any one of claims 6 to 9, wherein: Determine the genetic sex of the Chinese sturgeon to be tested based on the results of the first real-time fluorescence quantitative PCR test and the second real-time fluorescence quantitative PCR test, specifically including: When the Ct values ​​of the first fluorescence quantitative PCR test result and the second fluorescence quantitative PCR test result are both ≤36, the genetic sex of the Chinese sturgeon to be tested is determined to be female; when the Ct value of the second fluorescence quantitative PCR test result is greater than 36 or displays Undetermined, and the Ct value of the first fluorescence quantitative PCR test result is ≤36, the genetic sex of the Chinese sturgeon to be tested is determined to be male; when the Ct values ​​of the first fluorescence quantitative PCR test result and the second fluorescence quantitative PCR test result are greater than 36 or displays Undetermined, it is determined that the DNA sample does not include DNA derived from the Chinese sturgeon.

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