High-sensitivity Chinese sturgeon environment DNA specific detection kit and detection method thereof

Abstract

The invention discloses a high-sensitivity Chinese sturgeon environment DNA (deoxyribonucleic acid) specific detection kit and a detection method thereof. The high-sensitivity Chinese sturgeon environment DNA specific detection kit is characterized by comprising a primer group and a probe for Chinese sturgeon detection, and the primer group comprises an upstream primer and a downstream primer which are matched with each other; comprising the following steps: step 1, extracting environmental DNA in a water body sample; 2, carrying out a PCR amplification reaction on the extracted environment DNA; step 3, carrying out comprehensive analysis on the result of the PCR amplification reaction; wherein if the amplification curve after the PCR amplification reaction is S-shaped, the detection result of the water body sample is positive, and otherwise, the detection result of the water body sample is negative. The method has the advantages that only Chinese sturgeons are specifically detected, other related sturgeons cannot be detected wrongly, the problem of wrong detection is effectively solved, the influence of other related sturgeons in the detection work is prevented, meanwhile, the detection line amplification field is short, the method is suitable for short-segment environment DNA detection, and the false negative detection effect is effectively avoided.

Description

Technical Field

[0001] This invention relates to the field of aquatic organism detection technology, specifically to a highly sensitive environmental DNA-specific detection kit for Chinese sturgeon and its detection method. Background Technology

[0002] The Chinese sturgeon (Acipenser sinensis) is a national treasure-level fish in my country, a typical long-distance migratory species that lives between rivers and the sea. It grows and develops in seawater and spawns and reproduces in freshwater, making it one of the largest fish species globally capable of transitioning from marine to freshwater ecosystems. In recent years, the Chinese sturgeon population has declined sharply. It has been listed as a Class I protected animal in China and is now on the endangered species list. The IUCN Red List of Threatened Species lists it as CR (Critically Endangered). The rarity of this endangered species makes the Chinese sturgeon difficult to observe. Traditional fishing survey methods may harm its population and are also subject to legal restrictions, making them unsuitable for monitoring and surveying Chinese sturgeon resources. In recent years, environmental DNA technology has been widely used in aquatic ecological monitoring. Environmental DNA consists of DNA fragments found in water, derived from the tissues and excrement of organisms. Species can be monitored by detecting this DNA in the water. This technology does not require the collection of live organisms, thus avoiding the impact and harm on endangered species. It also has the potential to detect species in very small numbers, making it an environmentally friendly monitoring technology that does not damage living organisms.

[0003] Currently, Chinese patent CN 117535421 B discloses a kit and method for tracking the behavioral trajectory of Yangtze sturgeon released into the upper reaches of the Yangtze River based on a probe method. Similarly, Chinese patent CN 118185921 A discloses a method for enriching and extracting environmental DNA from Chinese sturgeon in low-salinity waters.

[0004] While environmental DNA can be used to monitor Chinese sturgeon, some technical challenges remain: 1. The Chinese sturgeon has many close relatives, such as the Yangtze sturgeon (Acipenser dabryanus), whose DNA sequences are very similar to those of the Chinese sturgeon. In the wild, when DNA from these closely related sturgeon species mixes with the DNA of the Chinese sturgeon, it can easily cause false positives in detection. 2. At the same time, due to the low density of wild Chinese sturgeon, the concentration of their environmental DNA is even lower, and the DNA will degrade over time, further shortening the environmental DNA of Chinese sturgeon, thus causing false negatives.

[0005] Therefore, short-segment, highly sensitive detection systems are needed to achieve comprehensive field monitoring of Chinese sturgeon. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a highly sensitive environmental DNA-specific detection kit and method for Chinese sturgeon. This kit specifically detects only Chinese sturgeon without falsely detecting other closely related sturgeon species, effectively solving the problem of false detection of closely related sturgeon species with DNA extremely similar to that of Chinese sturgeon. It also prevents the environmental DNA detection of Chinese sturgeon from being affected by other closely related sturgeon species. Furthermore, the detection line has a short amplification domain of only 102 bp, making it suitable for detecting short fragments of environmental DNA and effectively avoiding false negatives.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a highly sensitive environmental DNA-specific detection kit for Chinese sturgeon, comprising a primer set and a probe for detecting Chinese sturgeon. The primer set includes a compatible upstream primer and a downstream primer, the sequences of which are as follows: Upstream primer: 5'-AATCACGGCCGTGCTTCTC-3'; Downstream primer: 5'-GCTGGGTCAAAGAAGGTGGT-3'; Probe: 5'-FAM-CCAGTGCTGGCTGCGGGGAT-TAMRA-3'.

[0008] By using the COI domains of various sturgeon mitochondrial DNAs, primer sets and probes for detecting Chinese sturgeon were developed, preventing erroneous amplification of closely related sturgeon species other than Chinese sturgeon. This resulted in the specific detection of Chinese sturgeon only, effectively solving the problem of erroneous detection of closely related sturgeon species with DNA that are extremely similar to Chinese sturgeon. Furthermore, the amplification domain is only 102 bp, making it suitable for the detection of short environmental DNA fragments.

[0009] On the other hand, the present invention provides the following technical solution: a detection method based on a high-sensitivity Chinese sturgeon environmental DNA specific detection kit, comprising using the above-mentioned high-sensitivity Chinese sturgeon environmental DNA specific detection kit to detect Chinese sturgeon environmental DNA.

[0010] The present invention is further configured to include the following steps: Step 1: Extract environmental DNA from water samples; Step 2: Perform PCR amplification on the extracted environmental DNA; Step 3: Perform a comprehensive analysis of the PCR amplification results; If the PCR amplification curve is S-shaped, the water sample test result is positive, indicating the presence of Chinese sturgeon environmental DNA in the water sample; conversely, if the water sample test result is negative, the water sample does not contain Chinese sturgeon environmental DNA.

[0011] If the environmental DNA of Chinese sturgeon is detected in the water sample, it indicates that Chinese sturgeon are present in the target river and are in their activity area; otherwise, there are no Chinese sturgeon in the target river and they are not in their activity area.

[0012] The present invention is further configured such that the extraction of environmental DNA from water samples includes the following steps: Step 1: Collect water samples from the target river area; Step 2: Seal and preserve the collected water samples; Step 3: Vacuum filter the membrane and retain the filter membrane containing the target DNA vector after vacuum filtration. The filter membrane needs to be stored below -20℃. Step 4: Use a DNA extraction kit to extract environmental DNA from the filter membrane containing the target DNA vector. The environmental DNA needs to be stored below -20°C.

[0013] The present invention is further configured such that the water sample is stored at room temperature and immediately vacuum filtered using a filter membrane.

[0014] The ambient temperature setting reduces the temperature requirements, making it easier for staff to carry out their work.

[0015] The present invention is further configured such that: the water sample is stored at low temperature, a reagent to prevent DNA degradation is added to the water sample, and vacuum filtration is completed using a filter membrane within 24 hours, while the storage temperature at low temperature is not higher than 4°C.

[0016] Low temperatures can slow down the degradation rate of environmental DNA, thereby extending the time required for water samples to be filtered, facilitating the work of staff, and ensuring the reliability of test results.

[0017] The present invention is further configured such that the PCR amplification reaction system includes 10 µL of PCR reaction premix, 1.8 µL of upstream primer, 1.8 µL of downstream primer, 0.25 µL of probe, 2 µL of DNA template, and 4.15 µL of ddH2O, and the concentrations of upstream primer, downstream primer, and probe are all set to 10 µM.

[0018] The present invention is further configured such that the PCR amplification reaction conditions are: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 15 s, 60℃ annealing extension for 1 min, and 55 cycles.

[0019] The annealing extension temperature was set to 60℃, which is the optimal operating temperature for Taq enzyme to exhibit exonuclease activity, ensuring the reliability of the detection results.

[0020] In summary, the present invention has the following beneficial effects: By using the COI domains of various sturgeon mitochondrial DNAs, primer sets and probes for detecting Chinese sturgeon were developed, preventing erroneous amplification of closely related sturgeon species other than Chinese sturgeon. This resulted in the specific detection of Chinese sturgeon only, effectively solving the problem of erroneous detection of closely related sturgeon species with DNA that are extremely similar to Chinese sturgeon. Furthermore, the amplification domain is only 102 bp, making it suitable for the detection of short environmental DNA fragments. Attached Figure Description

[0021] Figure 1 This is a sequence diagram of the upstream primer, downstream primer, and probe.

[0022] Figure 2 This is a comparison chart showing the results of primer validity verification for this invention.

[0023] Figure 3 This is a comparison chart of the probe effectiveness verification results in this invention.

[0024] Figure 4 This is an amplification curve of the Chinese sturgeon standard sample of this invention.

[0025] Figure 5 This is a quantitative standard curve diagram of environmental DNA from the Chinese sturgeon, as presented in this invention.

[0026] Figure 6 A schematic diagram of fitting PCR results to a generalized linear regression model (GLM).

[0027] Figure 7 This is a graph used to verify the relationship between environmental DNA concentration and biomass density in aquaculture experiments.

[0028] Figure 8 This is a schematic diagram of sampling sites and sampling results during a field survey. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the accompanying drawings.

[0030] like Figures 1 to 5 As shown, the present invention provides the following technical solution: a highly sensitive environmental DNA-specific detection kit for Chinese sturgeon, comprising a primer set and a probe for detecting Chinese sturgeon. The primer set includes a compatible upstream primer and a downstream primer, the sequences of which are as follows: Upstream primer: 5'-AATCACGGCCGTGCTTCTC-3'; Downstream primer: 5'-GCTGGGTCAAAGAAGGTGGT-3'; Probe: 5'-FAM-CCAGTGCTGGCTGCGGGGAT-TAMRA-3'.

[0031] By using the COI domains of various sturgeon mitochondrial DNAs, primer sets and probes for detecting Chinese sturgeon were developed, preventing erroneous amplification of closely related sturgeon species other than Chinese sturgeon. This resulted in the specific detection of Chinese sturgeon only, effectively solving the problem of erroneous detection of closely related sturgeon species with DNA that are extremely similar to Chinese sturgeon. Furthermore, the amplification domain is only 102 bp, making it suitable for the detection of short environmental DNA fragments.

[0032] like Figure 1 As shown, this patent utilizes the COI domain of 15 sturgeon mitochondrial DNA species to develop upstream primers, downstream primers, and probes specific to the Chinese sturgeon. The base differences in the probes prevent erroneous amplification of sturgeon species other than the Chinese sturgeon. The probes developed in this patent are perfectly matched to the Chinese sturgeon, while also differing from the Yangtze sturgeon by one base, and from at least two bases other 13 closely related sturgeon species. Therefore, this ensures that the Chinese sturgeon environmental DNA specific detection kit can accurately detect the presence of Chinese sturgeon in water samples, and the detection results are not affected by other closely related sturgeon species. The closely related sturgeon species selected during the development of this patent are: Yangtze sturgeon (Acipenser dabryanus), Siberian sturgeon (Acipenser baerii), lake sturgeon (Acipenser fulvescens), Russian sturgeon (Acipenser gueldenstaedtii), medium-snout sturgeon (Acipenser medirostris), Mikadoi sturgeon (Acipenser mikadoi), peach sturgeon (Acipenser persicus), naked sturgeon (Acipenser nudiventris), sharp-snout sturgeon (Acipenser oxyrinchus), small sturgeon (Acipenser ruthenus), Schrencki sturgeon (Acipenser schrenckii), European sturgeon (Acipenser sturio), high-headed sturgeon (Acipenser transmontanus), and short-snout sturgeon (Acipenser brevirostrum).

[0033] The upstream and downstream primers were also matched with the Chinese sturgeon, and the results of primer validity verification were as follows: Figure 2 As shown, the probe was successfully amplified on DNA samples from tissue samples of three different Chinese sturgeon individuals; the results of the probe's effectiveness verification are as follows. Figure 3As shown, the DNA of three different individuals of Chinese sturgeon was successfully identified, while effectively avoiding the identification of Yangtze sturgeon DNA. On the other hand, the present invention provides the following technical solution: a detection method based on a high-sensitivity Chinese sturgeon environmental DNA specific detection kit, comprising using the above-mentioned high-sensitivity Chinese sturgeon environmental DNA specific detection kit to detect Chinese sturgeon environmental DNA.

[0034] The detection method includes the following steps: Step 1: Extracting Environmental DNA from Water Samples; Extracting environmental DNA from water samples includes the following steps: 1) Collecting water samples from the target river area; 2) Sealing and preserving the collected water samples; 3) Vacuum filtration using a filter membrane, and retaining the filter membrane containing the target DNA vector after vacuum filtration; 4) Extracting environmental DNA from the filter membrane containing the target DNA vector using a DNA extraction kit; the DNA extraction kit can be purchased directly; if the water sample is stored at room temperature, it needs to be vacuum filtered immediately using a filter membrane; if stored at low temperature, vacuum filtration needs to be completed within 24 hours, and the storage temperature at low temperature should not exceed 4°C. At the same time, it is necessary to add a DNA degradation prevention reagent to the water sample. The DNA degradation prevention reagent can be any one or more of benzalkonium chloride, EDTA, Tris-HCl buffer, SDS (sodium dodecyl sulfate), and CTAB (hexadecyltrimethylammonium bromide). The filter membrane obtained by vacuum filtration should be stored below -20℃ before environmental DNA extraction, and the extracted environmental DNA should also be stored below -20℃. Low temperature can slow down the degradation rate of environmental DNA, which is convenient for staff to carry out the work and ensures the reliability of the test results. Sealed storage can also slow down the degradation rate of environmental DNA and ensure the reliability of the test results.

[0035] Step 2: Perform PCR amplification on the extracted environmental DNA; The reaction system for the above PCR amplification reaction includes 10 µL of PCR reaction premix, 1.8 µL of upstream primer, 1.8 µL of downstream primer, 0.25 µL of probe, 2 µL of DNA template, and 4.15 µL of ddH2O. The concentrations of the upstream primer, downstream primer, and probe are all set to 10 µM, as shown in Table 1 below.

[0036] Table 1 PCR reaction system

[0037] Meanwhile, the reaction conditions for the above PCR amplification reaction are: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 15 s, 60℃ annealing extension for 1 min, and 55 cycles; the annealing extension temperature is set to 60℃, which is the optimal working temperature for Taq enzyme to have exonuclease activity, to ensure the reliability of the detection results, as shown in Table 2 below.

[0038] Table 2 PCR Amplification Reaction Conditions and Procedures

[0039] Step 3: Perform a comprehensive analysis of the PCR amplification results. If the PCR amplification curve is S-shaped, the water sample test result is positive, indicating the presence of Chinese sturgeon environmental DNA in the water sample. Conversely, if the water sample test result is negative, it indicates the absence of Chinese sturgeon environmental DNA in the water sample. The detection of Chinese sturgeon environmental DNA in the water sample indicates the presence of Chinese sturgeon in the target river area and that it is within its activity range. Conversely, the absence of Chinese sturgeon in the target river area and that it is not within its activity range indicates the absence of Chinese sturgeon in the target river area.

[0040] LOD Calculation: The 95% limit of detection (LOD) for a single reaction was determined using Chinese sturgeon standard samples. PCR experiments were performed using 0, 1, 2, 3, 5, 8, 13, and 21 copies of the standard samples, with each standard sample repeated 6 times. PCR amplification reactions were strictly performed according to the above requirements. A generalized linear regression (GLM) model was used to fit the PCR results. Figure 6 As shown, the single-reaction LOD reached 5.15 copies of DNA, which is close to the theoretical limit of the detection method.

[0041] Aquaculture Experiment: Five Chinese sturgeon rearing ponds were set up, each containing 1, 2, 2, 3, and 4 individual Chinese sturgeon in sequence. The weight of the Chinese sturgeon in each pond was measured, and the biomass density of the Chinese sturgeon was calculated based on the pond volume. The detection method of this patent was used to detect the concentration of environmental DNA in the Chinese sturgeon in each pond. A linear regression model (LM) was used to fit the relationship between the environmental DNA concentration and the biomass density of the Chinese sturgeon. Figure 7 As shown, the detected environmental DNA concentration of Chinese sturgeon was significantly positively correlated with its biomass density. For every 1 g / L increase in biomass density, the environmental DNA concentration increased by 12452 copies / L (p = 0.0208). The intercept of the model was 5293 (p = 0.7139), and the adjusted R-squared of the model reached 0.8263, indicating that it can reflect the biomass density (g / L) of Chinese sturgeon as (environmental DNA concentration (copies / L) + 5293) / 12452.

[0042] Field surveys: such as Figure 8As shown in Table 3, a field survey of Chinese sturgeon was conducted in the nearshore area of ​​Taizhou in 2024. Twelve water samples were collected and tested according to the detection method. Environmental DNA of Chinese sturgeon was successfully detected at station S4, and the concentration of Chinese sturgeon environmental DNA was 275 copies / L. Based on the model of environmental DNA concentration and biomass density, the biomass density of Chinese sturgeon at this location was 0.447 g / L.

[0043] Table 3. Field Survey Sample Collection Locations

[0044] In summary, the present invention has the following beneficial effects: By using the COI domains of various sturgeon mitochondrial DNAs, primer sets and probes for detecting Chinese sturgeon were developed, preventing erroneous amplification of closely related sturgeon species other than Chinese sturgeon. This resulted in the specific detection of Chinese sturgeon only, effectively solving the problem of erroneous detection of closely related sturgeon species with DNA that are extremely similar to Chinese sturgeon. Furthermore, the amplification domain is only 102 bp, making it suitable for the detection of short environmental DNA fragments.

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the design concept of the present invention should be included within the protection scope of the present invention.

Claims

1. A highly sensitive environmental DNA-specific detection kit for Chinese sturgeon, characterized in that, This includes a primer set and probes for detecting Chinese sturgeon. The primer set includes a matched upstream primer and a downstream primer. The sequences of the upstream primer, the downstream primer, and the probe are as follows: Upstream primer: 5'-AATCACGGCCGTGCTTCTC-3'; Downstream primer: 5'-GCTGGGTCAAAGAAGGTGGT-3'; Probe: 5'-FAM-CCAGTGCTGGCTGCGGGGAT-TAMRA-3'.

2. A detection method based on a highly sensitive environmental DNA-specific detection kit for Chinese sturgeon, characterized in that, This includes using the highly sensitive Chinese sturgeon environmental DNA-specific detection kit described in claim 1 to detect Chinese sturgeon environmental DNA.

3. The detection method based on a high-sensitivity environmental DNA-specific detection kit for Chinese sturgeon according to claim 2, characterized in that, Includes the following steps: Step 1: Extract environmental DNA from water samples; Step 2: Perform PCR amplification on the extracted environmental DNA; Step 3: Perform a comprehensive analysis of the PCR amplification results; If the amplification curve after the PCR amplification reaction is S-shaped, the test result of the water sample is positive, indicating the presence of Chinese sturgeon environmental DNA in the water sample; conversely, if the test result of the water sample is negative, the water sample does not contain Chinese sturgeon environmental DNA.

4. The detection method based on a high-sensitivity environmental DNA-specific detection kit for Chinese sturgeon according to claim 3, characterized in that, The extraction of environmental DNA from water samples includes the following steps: Step 1: Collect water samples from the target river area; Step 2: Seal and preserve the collected water samples; Step 3: Vacuum filter the membrane and retain the filter membrane containing the target DNA vector after vacuum filtration. The filter membrane needs to be stored below -20℃. Step 4: Use a DNA extraction kit to extract environmental DNA from the filter membrane containing the target DNA vector. The environmental DNA needs to be stored below -20°C.

5. The detection method based on a highly sensitive environmental DNA-specific detection kit for Chinese sturgeon according to claim 4, characterized in that, The water sample was stored at room temperature and immediately vacuum filtered using a filter membrane.

6. The detection method based on a high-sensitivity environmental DNA-specific detection kit for Chinese sturgeon according to claim 4, characterized in that, The water samples are stored at low temperatures. A reagent to prevent DNA degradation needs to be added to the water samples, and vacuum filtration is completed using a filter membrane within 24 hours. At the same time, the storage temperature at low temperatures does not exceed 4°C.

7. The detection method based on a high-sensitivity environmental DNA-specific detection kit for Chinese sturgeon according to claim 3, characterized in that, The PCR amplification reaction system includes 10 µL of PCR reaction premix, 1.8 µL of upstream primer, 1.8 µL of downstream primer, 0.25 µL of probe, 2 µL of DNA template, and 4.15 µL of ddH2O, with the concentrations of upstream primer, downstream primer, and probe all set to 10 µM.

8. The detection method based on a high-sensitivity environmental DNA-specific detection kit for Chinese sturgeon according to claim 3, characterized in that, The PCR amplification reaction conditions were: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 15 s, 60℃ annealing extension for 1 min, and 55 cycles.

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