Probe method-based kit and method for tracking behavior trajectory of acipenser dabryanus dumeril released upstream of yangtze river

Abstract

Disclosed are a probe method-based kit and method for tracking a behavior trajectory of Acipenser dabryanus Dumeril released upstream of the Yangtze River. By analyzing mitochondrial DNA sequences of Acipenser dabryanus Dumeril and common fish species in the Yangtze River basin, a pair of specific primers ND5-F4 / ND5-R4 capable of specifically amplifying mitochondrial DNAs of Acipenser dabryanus Dumeril and a Taqman fluorescent probe ND5 Probe 4 are designed and screened. A kit composed of the specific primers, the Taqman fluorescent probe, and a ddPCR master mix is used to carry out ddPCR testing on environmental DNA samples collected before and after release, so that a behavior trajectory of the released Acipenser dabryanus Dumeril can be tracked and monitored, achieving "non-contact" tracking of the behavior trajectory of the released Acipenser dabryanus Dumeril, avoiding damage to fish bodies in the tracking process, leading to reduced tracking costs and improved accuracy of overall tracking.

Description

Probe-based tracking kit and method for tracking the behavior of Yangtze sturgeons released into the upper reaches of the Yangtze River

[0001] This application claims priority to a Chinese patent application filed with the Patent Office of China on October 8, 2023, with application number 202311293132.3 and application name “Probe-based method for tracking the behavioral trajectory of Yangtze sturgeon released into the upper reaches of the Yangtze River and tracking method”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application belongs to the technical field of fish monitoring and tracking, and specifically relates to a probe-based behavior trajectory tracking kit and tracking method for Yangtze sturgeons released into the upper reaches of the Yangtze River. Background Art

[0003] The Yangtze sturgeon (Acipenser dabryanus Dumeril), also known as sturgeon and sandbar, is a sedentary freshwater fish that inhabits the fast-flowing, rocky riverbeds of the upper Yangtze River. Along with the Chinese sturgeon and the Chinese white sturgeon, it is a Yangtze sturgeon native to the region. While smaller and lighter than the other two species, the Yangtze sturgeon faces the same challenges, with its population size declining. On July 21, 2022, the International Union for Conservation of Nature and Natural Resources (IUCN) released an update to its Red List of Threatened Species, declaring the Chinese white sturgeon extinct, the Yangtze sturgeon extinct in the wild, and the Chinese sturgeon remaining critically endangered. Wei Qiwei, the only Chinese member of the IUCN Sturgeon Specialist Group and Chief Scientist at the Chinese Academy of Fishery Sciences, stated that, unlike the Chinese white sturgeon, the Yangtze and Chinese sturgeons have achieved captive conservation. To protect the wild population of Yangtze sturgeon, local governments and relevant research institutions have conducted annual wild sturgeon stocking and release activities since 2018. However, to date, no naturally bred Yangtze sturgeon juveniles have been found throughout the entire Yangtze River. Whether released Yangtze sturgeon can establish a stable population and allow natural reproduction is crucial for the restoration of wild sturgeon populations. Investigating the behavioral patterns of released Yangtze sturgeon can help us further understand their behavior in natural waters, laying the foundation for research into their preferred habitats and subsequent habitat restoration, among other strategies for restoring the Yangtze sturgeon population.

[0004] Traditional fish detection methods rely on catching fish in nets to survey their distribution. While intuitive and effective, these methods are inherently harmful to fish populations and are time-consuming and labor-intensive. Currently, several techniques are used to track and mark released Yangtze sturgeons: PIT tagging, sonar tagging, and T-tags. Sonar tagging can record the movements of Yangtze sturgeons in the Yangtze River; however, this requires the installation of multiple sonar receivers along the river, which is costly. PIT tagging, T-tags, and DNA tagging require marking fish. Because the proportion of tagged fish in the released population is relatively small, tracking tagged fish to infer the overall fish population often results in missed detections, resulting in limited usable data and hindering a comprehensive understanding of the behavior and distribution preferences of released fish. Furthermore, tagging can harm fish, and sonar devices can interfere with the transmission and reception of information by aquatic organisms, causing abnormalities in their movements and, in severe cases, death. Therefore, it is necessary to develop a tracking method that does not harm the fish or disrupt the normal life activities of other aquatic organisms to reveal the movements of released Yangtze sturgeons.

[0005] Summary of the Invention

[0006] In response to the above technical problems, the present application provides a probe-based behavioral trajectory tracking kit and tracking method for released Yangtze sturgeons in the upper reaches of the Yangtze River. By setting reasonable sampling points, DNA in the water environment is collected, and then by designing specific primer pairs and Taqman fluorescent probes, the concentration of the collected water environment DNA is detected, thereby realizing "non-contact" tracking of the behavioral trajectory of released Yangtze sturgeons.

[0007] In order to achieve the above-mentioned purpose, the present application provides a probe-based method for tracking the behavioral trajectory of Yangtze sturgeons released into the upper reaches of the Yangtze River, wherein the kit comprises a specific primer pair, a Taqman fluorescent probe, a DNA template, a ddPCR premix and ddH2O.

[0008] Preferably, the specific primer pair includes an upstream primer ND5-F4, whose sequence is SEQ ID NO: 1; a downstream primer ND5-R4, whose sequence is SEQ ID NO: 2; and a Taqman fluorescent probe whose sequence is SEQ ID NO: 3.

[0009] The 5' end of the Taqman fluorescent probe is connected to the fluorescent group FAM, and the 3' end is connected to the quenching group BHQ.

[0010] Preferably, the ddPCR premix comprises 2×supermix.

[0011] Preferably, the DNA template is water sample DNA, and the concentration of Yangtze sturgeon DNA contained in the water sample is ≥0.05 copies / μL.

[0012] The present application also provides a method for tracking the behavioral trajectory of Yangtze sturgeons released into the upper reaches of the Yangtze River based on a probe method, comprising the following steps:

[0013] (1) Set up a sampling section in the study waters and set up sampling points on the sampling section. Collect 2L of water at each sampling point every day from 1 day before release to 5 days after release.

[0014] (2) vacuum filtering the collected water using a mixed cellulose filter membrane within 24 hours, and preserving the filtered mixed cellulose filter membrane;

[0015] (3) Extract DNA from the mixed cellulose filter and store at -20°C for future use;

[0016] (4) Using the DNA obtained in step (3) as a template, the tracking kit is used to perform ddPCR detection to determine the concentration of the target sequence in each collected water body, thereby determining the behavioral trajectory of the Yangtze sturgeon.

[0017] Preferably, the interval of the sampling sections in step (1) is 2-3 km, and the number of sampling points is 2-5.

[0018] Preferably, the pore size of the mixed cellulose filter membrane in step (2) is 0.22 μm.

[0019] Preferably, the storage method in step (2) is storage at -20°C or storage in Longmire's buffer at room temperature.

[0020] Preferably, the DNA extraction method in step (3) is to use a Water DNA Kit (Omega) for extraction.

[0021] Preferably, the amplification system for the ddPCR detection in step (4) is 8.5 μL of DNA template, 0.5 μL of forward and reverse primers, 0.5 μL of probe, and 10 μL of 2×ddPCR premix; the amplification program is 95°C pre-denaturation for 10 min; 94°C denaturation for 30 s, 60°C annealing for 40 s, 72°C extension for 30 s, 40 cycles; 98°C enzyme inactivation for 10 min, and holding at 4°C.

[0022] The beneficial effects of this application are:

[0023] 1. Through the analysis of the mitochondrial DNA sequences of the Yangtze sturgeon and common fish in the Yangtze River basin, the specific primer pair ND5-F4 / ND5-R4 and Taqman fluorescent probe ND5 Probe4 were designed and screened to specifically detect the target DNA fragment of the Yangtze sturgeon. This specific primer pair and Taqman fluorescent probe can be used to specifically, stably and effectively amplify the target DNA fragment of the Yangtze sturgeon.

[0024] 2. The screened specific primer pair ND5-F4 / ND5-R4 and Taqman fluorescent probe ND5Probe4 can successfully detect the concentration of DNA fragments of the Yangtze sturgeon in aquaculture water and released water. Even if the concentration of Yangtze sturgeon DNA in the water sample is low, as long as it is ≥0.05 copies / μL, it can still be detected using ddPCR technology with high sensitivity.

[0025] 3. Provided is a method for tracking the behavioral trajectory of artificially propagated and released Yangtze sturgeon, which is mainly based on the detection of water environmental DNA concentration using the screened specific primer pair ND5-F4 / ND5-R4 and the Taqman fluorescent probe ND5 Probe4, so as to determine the main location and behavioral trajectory of the Yangtze sturgeon after release. Compared with traditional fishing methods and commonly used sonar monitoring methods, it is a "non-contact" tracking method, which does not harm the fish body and saves time and effort. At the same time, it does not require a large amount of expensive equipment, avoiding the missed detection caused by the limited number of markers and equipment deployment, and thus the inability to fully understand the behavioral trajectory of the target fish. Overall, the method for tracking the behavioral trajectory of the Yangtze sturgeon released in the upper Yangtze River based on the probe method of this application will not cause any damage to the original aquatic organisms and target fish in the investigated waters. It not only focuses on the behavioral trajectory of the marked fish, but also can track the behavioral trajectory of the entire released Yangtze sturgeon population. The research results are more representative than individual marking and tracking. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG1 is a fluorescence quantitative amplification curve of various primer pairs and probes thereof in Example 1;

[0027] FIG2 is a correlation curve between dilution factor and ddPCR detection sensitivity in Example 3;

[0028] FIG3 shows the environmental DNA concentration of Yangtze sturgeon at various sections one day before release (October 23) in Example 4;

[0029] FIG4 shows the environmental DNA concentration of Yangtze sturgeon at various sections on the first day after release (October 25) in Example 4;

[0030] FIG5 shows the environmental DNA concentration of Yangtze sturgeon at various sections on the second day after release (October 26) in Example 4;

[0031] FIG6 shows the environmental DNA concentration of Yangtze sturgeon at various sections on the third day after release (October 27) in Example 4;

[0032] FIG7 shows the environmental DNA concentration of Yangtze sturgeon at various sections on the fourth day (October 28) after release in Example 4;

[0033] FIG8 shows the environmental DNA concentration of Yangtze sturgeon at various sections on the fifth day (October 29) after release in Example 4;

[0034] FIG9 is a location distribution diagram of fixed sonar signal detection sites in Example 5;

[0035] Figure 10 is a sonar signal location distribution map of the released Yangtze sturgeon in 2021 in Example 5; the red solid circles in the figure represent fixed sonar signal monitoring stations, i.e., sonar monitoring sections; the line segments of different colors represent different Yangtze sturgeon individuals marked by sonar; the hollow circles represent the frequency of the Yangtze sturgeon appearing in a certain monitoring section. The larger the circle, the more times the Yangtze sturgeon was monitored at this station and the longer it stayed;

[0036] Figure 11 is a sonar signal location distribution map of the released Yangtze sturgeon in 2020 in Example 5; the red solid circles in the figure represent fixed sonar signal monitoring stations, that is, sonar monitoring sections; line segments of different colors represent different Yangtze sturgeon individuals marked by sonar; hollow circles represent the frequency of the Yangtze sturgeon appearing in a certain monitoring section. The larger the circle, the more times the Yangtze sturgeon was monitored at this station and the longer it stayed. DETAILED DESCRIPTION

[0037] The technical solution of the present application is further explained below with reference to the accompanying drawings and specific embodiments. It is worth noting that the following embodiments are only preferred embodiments of the present application and should not be construed as limiting the present application. The scope of protection of the present application shall be based on the contents of the claims. Any modifications or substitutions made to the technical solution of the present application by those skilled in the art without creative work shall fall within the scope of protection of the present application.

[0038] Water DNA Kit was purchased from Omega.

[0039] ddPCR premix was purchased from Biorad.

[0040] Example 1 Design and screening of specific primers

[0041] Based on the Yangtze sturgeon mitochondrial sequence, the sequence positions with significant differences were determined by software comparison and primer pairs and probes were designed. Then, real-time fluorescence quantitative PCR technology was used for detection and the designed primer pairs and probes were screened. The specific steps include:

[0042] (1) Based on the fish species list of the Upper Yangtze River National Nature Reserve, a representative fish mitochondrial sequence was downloaded for each subfamily, totaling 35 fish mitochondrial sequences. After alignment using DNAMAN software, 7 pairs of specific primers were designed at the sequence positions with large differences in 16S, COI, and ND5 loci. One probe was designed within the primer pair sequence interval, as shown in Table 1.

[0043] (2) The fin rays or muscle tissues of common fishes from the upper reaches of the Yangtze River were cut and the fish DNA was extracted. The DNA concentrations of the fishes were detected by qubit. The concentrations of DNA of the fishes were as follows: 59.4 ng / μL for mullet; 23.2 ng / μL for bonito; 25ng / μL; red-eyed trout 21.8ng / μL; small mullet 110ng / μL; scorpionfish 34.4ng / μL; round-mouthed copperfish 42.4ng / μL; copperfish 34.5ng / μL; spotted sand loach 26.2ng / μL; high-bodied minnow 36.6ng / μL; blacktail red 77.8ng / μL; loach 63.2ng / μL; carp 120ng / μL; white-margined 38.8ng / μL; snakehead 248ng / μL; yellow catfish 45.8ng / μL; Chinese spiny barb 60.4ng / μL; longnose catfish 20.4ng / μL; Yangtze sturgeon 90.1ng / μL;

[0044] Table 1 Primer and probe sequences

[0045] (3) Using the primer pairs and probes designed in step (1), the fish DNA obtained in step (2) was subjected to real-time fluorescence quantitative PCR detection to screen and determine the primer pairs and probes that can successfully amplify the target sequence; the amplification system was 20 μL: 10 μL of 2×SuperReal Premix, 0.6 μL of upstream and downstream primers, 0.4 μL of probe, 0.6 μL of DNA template, and 6.8 μL of ddH2O; the reaction conditions were: pre-denaturation at 95°C for 15 min; denaturation at 95°C for 1 s; annealing / extension at 60°C for 30 s; and 40 cycles.

[0046] The results are shown in Figure 1. The designed primer pair ND5-F4 / ND5-R4 and its probe ND5Probe4 have strong specificity. The fluorescence quantitative PCR amplification curve shows that the target sequence was not amplified in the DNA of other fish except the Yangtze sturgeon. This primer pair and probe combination can successfully separate the Yangtze sturgeon from other fish.

[0047] Example 2: Validation of primer pairs and probes for water sample detection

[0048] Yangtze sturgeon aquaculture water was collected and ddPCR detection was performed using the specific primer pair and probe screened in Example 1 to determine whether the primer and probe could be successfully used for detection in water samples and verify their effectiveness. The water sample DNA was diluted 10-fold to preliminarily verify the stability of the primer and probe. The specific steps included:

[0049] (1) A 2-liter water sample was collected from a Yangtze sturgeon aquaculture pond with a diameter of 4 m, a height of 1 m, a breeding size of 30 cm, and a population of approximately 100 sturgeons. The water sample was vacuum filtered using a 0.22 μm mixed cellulose filter membrane to obtain a mixed cellulose filter membrane containing a water DNA sample.

[0050] (2) Adoption The DNA sample on the filter membrane obtained in step (1) was extracted using the Water DNA Kit, and its concentration was detected using qubit to determine the initial concentration to be 6 ng / μL, and stored at -20°C for later use;

[0051] (3) The DNA sample with an initial concentration of 6 ng / μL obtained in step (2) was diluted 10-fold, 100-fold, and 1000-fold with ultrapure water as a DNA template for ddPCR, and ultrapure water was used as a DNA template for the negative control;

[0052] (4) The DNA template obtained in step (3) was used to prepare a ddPCR amplification system, and then the detection was performed using a Bio-Rad QX200TM droplet digital PCR instrument. The ddPCR amplification system is shown in Table 2, the amplification program is shown in Table 3, and the detection results are shown in Table 4. Each DNA template was tested in parallel three times.

[0053] Table 2 ddPCR amplification system

[0054] Table 3 ddPCR amplification procedure

[0055] Table 4 ddPCR test results

[0056] The results are shown in Table 4. Using ND5-F4 and ND5-R4 as the primer pair and ND5 Probe4 as the Taqman fluorescent probe, the DNA concentration of Yangtze sturgeon can be successfully detected in aquaculture water. After 10-fold dilution, the DNA detection concentration of the sample decreased proportionally with the dilution factor, demonstrating the effectiveness and stability of the primer pair and probe.

[0057] Example 3: Sensitivity Detection of Primer Pairs and Probes

[0058] The sensitivity detection of the screened Yangtze sturgeon-specific primer pair ND5-F4 / ND5-R4 and its Taqman fluorescent probe ND5 Probe4 includes the following steps:

[0059] (1) The Yangtze sturgeon water sample DNA to be tested was diluted using the two-fold dilution method, with the first dilution being 1 / 2 of the initial concentration, the second dilution being 1 / 4, and so on to produce 10 dilution solutions;

[0060] (2) Using the 10 dilutions obtained in step (1) as DNA templates, the 10 dilutions were detected using a Bio-Rad QX200™ droplet digital PCR instrument to obtain the detection concentrations. The detection method was the same as in Example 2.

[0061] (3) Perform logarithmic transformation on the detection concentration obtained in step (2), perform correlation analysis, and draw a correlation curve.

[0062] The dilution multiples and sample ddPCR detection concentrations are shown in Table 5: the ddPCR detection concentration of the water sample to be tested is 112.2 copies / μL, the minimum detection concentration after dilution to 1024 times is 0.05 copies / μL, and no DNA can be detected after dilution to 2048 times.

[0063] Table 5 ddPCR test results of serially diluted water sample DNA

[0064] The dilution factor and ddPCR detection concentration showed a linear relationship after logarithmic transformation, as shown in Figure 2 (R 2 =0.989, P <0.01). In summary, this primer pair and probe have high sensitivity for detecting Yangtze sturgeon in water samples, and concentrations above 0.05 copies / μL can be detected by ddPCR.

[0065] Example 4: Tracking the Behavior of Released Yangtze River Sturgeons

[0066] On October 24, 2021, approximately 35,000 Yangtze sturgeons were released opposite the mouth of the Minjiang River (between sections 12 and 13). The behavioral trajectory of the artificially released Yangtze sturgeons was tracked using the specific primer pair ND5-F4 / ND5-R4 and the Taqman fluorescent probe ND5 Probe4 screened in the above example, specifically including the following steps:

[0067] (1) According to literature research, the Yangtze sturgeon has been concentrated in the lower Jinsha River and the upper Yangtze River in the river section from Xiangjiaba to Lizhuang. A section was set up every 2-3 km in this section (Table 5), and three sampling points were set up on the left, middle, and right sides of each section.

[0068] (2) According to the release time of the Yangtze River sturgeon in 2021 (October 24), 2L of water was collected from the sampling points set in step (1) as samples; the sampling time was one day before the release (October 23) and five days after the release (October 25-October 29);

[0069] Table 5 Latitude and longitude of the cross section

[0070] Note: MJ is the release location.

[0071] (3) The collected water samples were vacuum filtered through a mixed cellulose filter membrane with a pore size of 0.22 μm within 24 h. The filtered mixed cellulose filter membrane was placed in a centrifuge tube and stored at -20°C. One filter membrane was used for each liter of water. Gloves were worn during the entire operation. Tweezers were used to transfer the filter membrane. The filter was rinsed with sterile water before filtering samples from different sections to prevent cross contamination.

[0072] (4) DNA was extracted from the mixed cellulose filter membrane containing water DNA from each section and each sampling point obtained in step (3) using the EZNA Water DNA Kit and stored at -20°C, with ultrapure water used as a negative control DNA template;

[0073] (5) ddPCR was performed using the specific primers ND5-F4 / ND5-R4 and Taqman fluorescent probe ND5 Probe4 determined in Example 3 to detect the concentration of the target fragment in each sample.

[0074] Figures 3-8 show that the concentration of environmental DNA of Yangtze sturgeon was much lower before release than after release, with relatively high concentrations primarily occurring within 10 km downstream of Xiangjiaba Dam. Sampling results from the first day after release (October 25) showed higher concentrations in the first four sections, indicating that the released Yangtze sturgeon were primarily distributed within the 10 km downstream from Xiangjiaba Dam. On the second day after release (October 26), high concentrations of environmental DNA samples were concentrated in sections 7 to 10. On the third day after release (October 27), high concentrations of environmental DNA samples were primarily found in section 12, the Minjiang River estuary, and sections 18-20, indicating that the released Yangtze sturgeon were primarily distributed in sections 12 to 20. On the fourth day after release (October 28), concentrations in all sections, with the exception of sections 3 and 21, remained similar to pre-release levels. On the fifth day after release (October 29), the concentrations and distribution of detected Yangtze sturgeon environmental DNA remained similar to those before release. The test results show that the released Yangtze River sturgeon have obvious movement trends. The movement trends of the released Yangtze River sturgeon based on this year's sampling are summarized as follows:

[0075] 1. In the area 5km to 10km downstream from Xiangjiaba Dam, samples with relatively high concentrations were collected both before and after the release, suggesting that Yangtze sturgeons have always been distributed there.

[0076] 2. After the release of the Yangtze sturgeon, the concentration of environmental DNA in the water increased rapidly. Five days later, the concentration was similar to that before release. Based on the detected concentration trend, it can be seen that the activity range of the Yangtze sturgeon was relatively concentrated in the early stage of release. Over time, the distribution range of the Yangtze sturgeon expanded, so the concentration values ​​obtained with a fixed sampling volume showed a continuous downward trend.

[0077] 3. According to the environmental DNA concentration test results 5 days after release and the location of the release point (between sections 12 and 13), after release from the release point, most of the Yangtze sturgeons first swam upward toward Xiangjiaba, and then began to swim downward.

[0078] Example 5 Sonar monitoring of the behavior of Yangtze sturgeon after release

[0079] To support the results of environmental DNA detection based on the probe method in Example 4, 26 juvenile Yangtze sturgeons tagged with V7 and V16 sonar markers were released in Shuifu on November 12 of the same year. The behavioral trajectories of the Yangtze sturgeons after release were detected using multiple fixed sonar signal monitoring stations (Figure 9).

[0080] The results are shown in Figure 10. Sonar signals were successfully received from 22 of the 26 tagged juvenile Yangtze sturgeons. From the time of release until April 2022 (the following April), the released juvenile Yangtze sturgeons were primarily concentrated in the river section from below the dam to the Nanxi Maritime Affairs Department (the correspondence between water sampling sections and sonar monitoring sections is as follows: Section 1 is approximately at Shuifu, Section 12 is approximately at the Yibin Fisheries Administration, Yanpingba is located between Sections 15 and 16, and Section 19 is approximately at the Water Police Department). The Shuifu section received the most signals and the juveniles stayed the longest, indicating that Yangtze sturgeons have consistently been present in the Shuifu section (i.e., the section below the dam), consistent with the results of environmental DNA monitoring, which inferred that Yangtze sturgeons have been distributed below the dam.

[0081] Although the accuracy of sonar and environmental DNA monitoring methods differs, and sonar monitoring is not as sensitive as environmental DNA, nor can environmental DNA track Yangtze sturgeon for the long periods of time that sonar can, comparing the results of the two monitoring methods can help determine the movement trends of Yangtze sturgeon. After the tagging and release on November 12, signals were primarily detected near Shuifu for the first two days (November 13 and 14). On November 16 and 17, signals were primarily detected in Shuifu, followed by the mouth of Fuxi River. This is consistent with the environmental DNA monitoring results, which showed that Yangtze sturgeon were primarily distributed in the river downstream of the dam on the first day after release, and were primarily distributed in the river downstream of the dam and sections 18-21 (approximately 45-65 km below the dam) five days later. The number and frequency of sonar signals received also decreased over time, consistent with the decreasing concentration of environmental DNA detected, indicating that after release, juveniles initially maintain a relatively concentrated distribution pattern before gradually dispersing.

[0082] The two monitoring methods provide corroborative evidence that released Yangtze sturgeons have consistently been distributed in the Shuifu section of the river downstream of the dam. Initially, their range was relatively concentrated, but their distribution expanded over time. Because the sonar-tagged release site for the Yangtze sturgeons that year was located in Shuifu, downstream of the dam, the results in Figure 10 cannot be used to conclude that the released Yangtze sturgeons first migrated upstream to Xiangjiaba and then downstream. However, based on sonar tracking results (Figure 11) for Yangtze sturgeons tagged and released between Shuifu and Xuzhou districts in 2020, 9 of the 25 juvenile signals successfully received after the release on October 24th were first detected in Shuifu, downstream of the dam, before being detected at downstream stations. This confirms the upward and then downward movement trend observed in environmental DNA monitoring for some juveniles.

Claims

1. A probe-based tracking kit for tracking the behavior of Yangtze sturgeons released into the upper reaches of the Yangtze River, wherein: The kit comprises a specific primer pair, a Taqman fluorescent probe, a DNA template, a ddPCR premix and ddH2O.

2. A probe-based tracking kit for tracking the behavior of Yangtze sturgeons released into the upper reaches of the Yangtze River according to claim 1, wherein: The specific primer pair includes an upstream primer ND5-F4, whose sequence is SEQ ID NO: 1; a downstream primer ND5-R4, whose sequence is SEQ ID NO: 2; and a Taqman fluorescent probe whose sequence is SEQ ID NO:

3.

3. A probe-based behavior tracking kit for releasing Yangtze sturgeon in the upper reaches of the Yangtze River according to claim 1, wherein: The 5' end of the Taqman fluorescent probe is connected to a fluorescent group FAM, and the 3' end is connected to a quenching group BHQ.

4. A probe-based behavior tracking kit for Yangtze sturgeon released in the upper reaches of the Yangtze River according to claim 1, wherein: The ddPCR premix includes 2×supermix.

5. A probe-based behavior tracking kit for releasing Yangtze sturgeon in the upper reaches of the Yangtze River according to claim 1, wherein: The DNA template is a water sample DNA, and the concentration of Yangtze sturgeon DNA contained in the water sample is ≥0.05 copies / μL.

6. A method for tracking the behavior trajectory of Yangtze sturgeons released into the upper reaches of the Yangtze River based on a probe method, wherein: The following steps are involved: (1) Set up a sampling section in the study waters and set up sampling points on the sampling section. Collect 2L of water at each sampling point every day 1 day before and 5 days after release; (2) vacuum filtering the collected water using a mixed cellulose filter membrane within 24 hours, and preserving the filtered mixed cellulose filter membrane; (3) extracting DNA from the mixed cellulose filter and storing it at -20°C for future use; (4) Using the DNA obtained in step (3) as a template, perform ddPCR detection using the tracking kit described in any one of claims 1 to 3 to determine the concentration of the target sequence in each collected water body, thereby determining the behavioral trajectory of the Yangtze sturgeon.

7. A method for tracking the behavior trajectory of Yangtze sturgeons released into the upper reaches of the Yangtze River based on a probe method according to claim 6, wherein: The interval of the sampling sections described in step (1) is 2-3 km, and the number of sampling points is 2-5.

8. The method for tracking the behavior trajectory of Yangtze sturgeons released into the upper reaches of the Yangtze River based on the probe method according to claim 6, wherein: The pore size of the mixed cellulose filter membrane described in step (2) is 0.22 μm.

9. The method for tracking the behavior trajectory of Yangtze sturgeons released into the upper reaches of the Yangtze River based on the probe method according to claim 6, wherein: The storage method in step (2) is to store at -20°C or in Longmire's buffer at room temperature.

10. The method for tracking the behavior trajectory of Yangtze sturgeons released into the upper reaches of the Yangtze River based on the probe method according to claim 6, wherein: The DNA extraction method described in step (3) is to use a Water DNA Kit for extraction.

11. The method for tracking the behavior trajectory of Yangtze sturgeons released into the upper reaches of the Yangtze River based on the probe method according to claim 6, wherein: The amplification system of the ddPCR detection described in step (4) is 8.5 μL of DNA template, 0.5 μL of forward and reverse primers, 0.5 μL of probe, and 10 μL of 2×ddPCR premix; the amplification program is 95°C pre-denaturation for 10 min; 94°C denaturation for 30 s, 60°C annealing for 40 s, 72°C extension for 30 s, 40 cycles; 98°C enzyme inactivation for 10 min, and 4°C maintenance.

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