An environmental DNA-based primer probe combination for oncorhynchus masonii and application thereof

Abstract

The application discloses a primer probe combination for Salmo platycephalus based on environmental DNA and application thereof, and belongs to the technical field of molecular biology and endangered aquatic organism monitoring. The application provides the primer probe combination for Salmo platycephalus, which comprises upstream and downstream primers and a probe P, and the amplified fragment has a length of 179 bp; the primer probe combination for Salmo platycephalus can specifically detect Salmo platycephalus in environmental DNA, can efficiently monitor and protect Salmo platycephalus, and can effectively avoid the interference of related species including Salmo, Oncorhynchus and Leucichthys bampsii; the provided primer probe combination for Salmo platycephalus can be applied to the monitoring of Salmo platycephalus in different water bodies to be detected such as streams, rivers and reservoirs, is easy to popularize, and has important significance for the protection and monitoring of Salmo platycephalus.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology and endangered aquatic organism monitoring technology, specifically relating to a primer-probe combination for Sichuan and Shaanxi taimen based on environmental DNA and its application. Background Technology

[0002] The Sichuan-Shaanxi taimen is a rare and endangered cold-water fish species endemic to my country and is listed as a Class I protected animal in China. It is mainly distributed in the Minjiang River, Dadu River, and the upper reaches of the Hanjiang River. The wild population is extremely small and its habitat is severely fragmented. Traditional resource surveys rely on fishing, electrofishing, or underwater observation, which cause great harm to endangered individuals, have low detection rates, and make long-term monitoring difficult.

[0003] Environmental DNA (eDNA) technology enables rapid, non-invasive species detection by collecting cell-free DNA shed from organisms in water and then specifically amplifying it via PCR. It has been widely used for monitoring the distribution and populations of rare fish species. Combining qPCR with specific probes can significantly improve sensitivity and specificity, making it suitable for detecting low concentrations of eDNA.

[0004] Currently reported detection methods for Sichuan-Shaanxi taimen eDNA mostly use ordinary PCR primers, lacking a TaqMan probe system. These methods suffer from insufficient sensitivity, specificity, and quantification capabilities, are susceptible to interference from closely related species (such as taimen and Qinling lenok), and have a high risk of false negatives / false positives in complex water samples from the field. Therefore, developing a highly specific, highly sensitive, and quantifiable primer-probe combination for Sichuan-Shaanxi taimen eDNA is of significant practical importance for its conservation and monitoring. Summary of the Invention

[0005] Based on the above-mentioned technological needs, the purpose of this invention is to provide a primer-probe combination for Sichuan-Shaanxi taimen based on environmental DNA and its application. This primer-probe combination overcomes the shortcomings of existing Sichuan-Shaanxi taimen eDNA detection technologies, providing a highly specific, sensitive, and adaptable primer-probe combination for Sichuan-Shaanxi taimen environmental DNA. It also provides applications, detection kits, and detection methods for this primer-probe combination, enabling non-invasive, accurate, and rapid monitoring of Sichuan-Shaanxi taimen, reducing the risk of false positives and false negatives, and meeting monitoring needs in different scenarios.

[0006] To achieve the above objectives, the present invention is implemented through the following solution:

[0007] This invention provides a primer-probe combination for Sichuan-Shaanxi taimen based on environmental DNA, the primer-probe combination for Sichuan-Shaanxi taimen comprising:

[0008] (1) Upstream primer F: The nucleotide sequence of the upstream primer F is shown in SEQ ID NO.11;

[0009] (2) Downstream primer R: The nucleotide sequence of the downstream primer R is shown in SEQ ID NO.12;

[0010] (3) Probe P: The nucleotide sequence of the probe P is shown in SEQ ID NO.13.

[0011] Furthermore, the amplified fragment length of the Sichuan-Shaanxi taimen primer-probe combination is 179 bp.

[0012] The present invention also provides a kit for the aforementioned primer and probe combination of Sichuan and Shaanxi taimen.

[0013] The present invention also provides the application of the aforementioned primer-probe combination or kit for the specific detection of Sichuan-Shaanxi taimen in environmental DNA.

[0014] This invention also provides the application of the aforementioned primer-probe combination or kit for Sichuan-Shaanxi taimen in the monitoring of Sichuan-Shaanxi taimen populations.

[0015] This invention also provides the application of the aforementioned Sichuan-Shaanxi taimen primer-probe combination or kit in protecting Sichuan-Shaanxi taimen populations in variable water bodies.

[0016] Furthermore, the method of application specifically includes:

[0017] (1) Sample collection: Collect environmental samples to be tested, filter them through a 0.45μm cellulose acetate membrane to enrich eDNA, and extract environmental DNA to obtain DNA templates to be tested;

[0018] (2) qPCR detection: The Sichuan-Shaanxi taimen primer and probe combination was used to perform qPCR detection on the DNA template to be tested;

[0019] (3) Testing and Judgment Criteria

[0020] S1: If a typical S-type amplification curve appears in the fluorescence channel and the Ct value is ≤38, then the presence of Sichuan-Shaanxi taimen eDNA in the environmental sample to be tested is determined.

[0021] S2: If there is no amplification curve in the fluorescence channel, it is determined that there is no eDNA of Sichuan-Shaanxi taimen in the sample to be tested;

[0022] S3: If there is an amplification curve but no S-type characteristic or Ct value > 38, it is determined that no Sichuan-Shaanxi taimen eDNA was detected in the environmental sample to be tested.

[0023] Furthermore, the qPCR amplification system in step (2) includes: a system of 10 μL: DNA template to be tested: 2 μL; 2×qPCR mix: 5.0 μL; upstream primer F / downstream primer R: 10 pmol / μL × 0.2 μL; P probe: 10 pmol / μL × 0.1 μL; ddH2O: to make up to 10 μL.

[0024] Furthermore, the qPCR amplification conditions are as follows: initial denaturation at 95°C for 30 seconds; 45 cycles of denaturation at 95°C for 10 seconds and annealing at 60°C for 30 seconds.

[0025] Furthermore, the Sichuan-Shaanxi taimen primer-probe combination can effectively avoid interference from closely related species, including at least one of taimen, rainbow trout, and Qinling lenok.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] 1. High specificity: The Cytb2 primer-probe combination provided by this invention can target the unique mutation sites of Sichuan and Shaanxi taimen, and has no cross-amplification with closely related species such as taimen, Qinling lenok, and rainbow trout, effectively avoiding false positives.

[0028] 2. High sensitivity: The TaqMan probe system (Cytb2 primer-probe combination) provided by this invention can detect up to 10-100 copies / μL eDNA, which is significantly better than ordinary PCR and is suitable for low-concentration field water samples.

[0029] 3. Rapid quantification: The entire qPCR process takes about 1.5 hours and can simultaneously perform qualitative and quantitative analysis, supporting large-scale field monitoring and population dynamic assessment, and achieving efficient detection.

[0030] 4. Non-invasive protection: No need to catch or disturb wild individuals, in line with the principles of endangered species protection.

[0031] 5. Wide applicability: The method for specific detection of Sichuan-Shaanxi taimen in environmental DNA provided by this invention can be used in different water bodies such as streams, rivers, and reservoirs. The method has strong resistance to inhibitors such as humus and silt. The TaqMan probe system (Cytb2 primer-probe combination) provided has promotional value and is of great significance for the protection and monitoring of Sichuan-Shaanxi taimen. Attached Figure Description

[0032] Figure 1 This is a phylogenetic tree of NJ constructed based on the Cytb gene sequence.

[0033] Figure 2 This is a gel image for nucleic acid electrophoresis detection.

[0034] Figure 3The image shows the amplification curve of the Dloop primers.

[0035] Figure 4 The amplification curve is for Cytb primer 1.

[0036] Figure 5 The amplification curve is for Cytb primer 2.

[0037] Figure 6 This is the sensitivity standard curve. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments can be obtained commercially.

[0039] Example 1: Design and Synthesis of Primers and Probes

[0040] Based on the phylogenetic relationships of salmonid fish (see...) Figure 1 The two species most closely related to the Sichuan-Shaanxi taimen (Hucho bleekeri) and the Qinling lenok (Brachymytax lenok tsinlingensis) were used as differential samples, with 5 samples for each species, for a total of 15 samples (Table 1).

[0041] 1. DNA extraction

[0042] First, DNA was extracted from tissue samples of the corresponding species using an adsorption-based animal tissue genomic DNA extraction kit (Table 1). The extracted DNA concentration was then determined using electrophoresis gel chromatography. Figure 2 ).

[0043] Table 1 Nucleic acid concentration detection results

[0044]

[0045] 2. PCR amplification and sequencing of mitochondrial D-loop and Cytb

[0046] The mitochondrial D-loop and Cytb genes were selected as target fragments, and the D-loop and Cytb gene sequences of 15 individuals were amplified using universal primers (Table 2). The PCR products were bidirectionally sequenced and assembled to obtain the D-loop and Cytb sequences of 15 samples, which were used to further design specific primers and probes.

[0047] Table 2 Universal primer sequence information

[0048]

[0049] 3. Primer and TaqMan probe design

[0050] Based on the D-loop and Cytb sequences obtained above, 15 sample sequences were compared. Regions with conserved intraspecific and interspecific variant sites were selected, and three sets of amplification primers and probes for Sichuan and Shaanxi taimen were designed using Primer 5.

[0051] Table 3 Probe Primer Sequence Information

[0052]

[0053] Example 2: Specificity Verification Experiment

[0054] This embodiment focuses on specific detection using the Dloop primer-probe combination, Cytb1 primer-probe combination, and Cytb2 primer-probe combination provided in Example 1. qPCR tests were performed using DNA from Sichuan-Shaanxi taimen, Qinling lenok, and taimen.

[0055] The qPCR amplification system is 10 μL:

[0056] DNA template: 2μL;

[0057] 2×qPCR mix: 5.0 μL;

[0058] F / R primers: 10 pmol / μL × 0.2 μL;

[0059] P probe: 10 pmol / μL × 0.1 μL;

[0060] ddH2O: Add to a final volume of 10 μL.

[0061] qPCR thermal cycling conditions: initial denaturation at 95℃ for 30 s; 45 cycles of denaturation at 95℃ for 10 s and annealing at 60℃ for 30 s.

[0062] The results showed that the Cytb2 primer-probe combination provided in this embodiment only showed a typical S-shaped amplification curve in Sichuan and Shaanxi taimen, and no amplification curve was observed in the other two species. Figure 5 The other two primer sets both showed positive amplification for another species, making it impossible to specifically distinguish between Sichuan and Shaanxi taimen (Hucho chuanxiong). Figure 3 ,4).

[0063] Example 3: Sensitivity Verification Experiment

[0064] This embodiment demonstrates the sensitivity of the Cytb2 primer-probe combination for gene detection in Sichuan and Shaanxi taimen, specifically including the following:

[0065] 1. Experimental Materials: Recombinant plasmids of the Cytb region of mitochondrial salmon from Sichuan and Shaanxi were used as positive standards for primers and probes, with a concentration of 10. 9 copies / μL, serially diluted 10-fold with enzyme-free water to obtain 10 8 -10 1 A series of dilutions per copy / μL; (the sensitivity standard curve is shown in the figure) Figure 6 (as shown)

[0066] 2. qPCR detection was performed using a primer-probe combination (Cytb2 primer-probe combination);

[0067] 3. Experimental Results: The target primer-probe combination (Cytb2 primer-probe combination) can detect a minimum concentration of 10. 2 Copy / μL, Ct value 37.24±0.25; suitable for the detection of trace amounts of eDNA in water.

[0068] Example 4: Application of Cytb2 primer-probe combination in actual water sample detection

[0069] This embodiment provides the results of Cytb2 primer-probe combination DNA detection in the Sichuan-Shaanxi taimen environment, specifically including the following:

[0070] 1. Sample collection: Water from the aquaculture of Sichuan and Shaanxi taimen and other fish was selected. The water was filtered through a 0.45μm cellulose acetate membrane to enrich eDNA. Environmental DNA was extracted using a DNA extraction kit to obtain the DNA template to be tested. Three replicate samples were set up for each sample.

[0071] 2. The above samples, positive controls, and negative controls were tested by qPCR using the (Cytb2 primer-probe combination).

[0072] 3. Experimental Judgment Criteria

[0073] (1) Determination of experimental validity: If the positive control shows a typical S-type amplification curve and the Ct value is ≤38; and the negative control does not show an amplification curve, then the experimental results are valid;

[0074] (2) Target species determination: If the experiment is effective, the fluorescence channel shows a typical S-type amplification curve and the Ct value is ≤38, then it is determined that the Sichuan-Shaanxi taimen eDNA exists in the water sample to be tested.

[0075] (3) Negative determination: If the experiment is effective, there is no amplification curve in the fluorescence channel, or the amplification curve has no S-shaped characteristics and the Ct value is >38, then it is determined that no Sichuan-Shaanxi taimen eDNA was detected in the water sample to be tested.

[0076] 4. Experimental Results

[0077] Three samples from the aquaculture water of taimen from Sichuan and Shaanxi showed S-shaped amplification curves, with two samples having Ct values ​​of 33.8-34.53 and one sample having a Ct value of 39.85, which were judged as positive; the three samples from non-aquaculture water did not show any amplification curves and were judged as negative.

[0078] The Cytb2 primer-probe combination provided by this invention is a set of environmental DNA primer-probe combinations for Sichuan-Shaanxi taimen with high specificity, high sensitivity, and good adaptability. The Cytb2 primer-probe combination can effectively avoid interference from closely related species (including taimen, rainbow trout, and Qinling lenok), and can be used in different water bodies such as streams, rivers, and reservoirs, which is of great significance for the protection and monitoring of Sichuan-Shaanxi taimen.

[0079] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A primer-probe combination for Sichuan and Shaanxi taimen based on environmental DNA, characterized in that, The primer and probe combination for Sichuan and Shaanxi taimen includes: (1) Upstream primer F: The nucleotide sequence of the upstream primer F is shown in SEQ ID NO.11; (2) Downstream primer R: The nucleotide sequence of the downstream primer R is shown in SEQ ID NO.12; (3) Probe P: The nucleotide sequence of the probe P is shown in SEQ ID NO.

13.

2. A kit containing the primer and probe combination of Sichuan and Shaanxi taimen as described in claim 1.

3. The application of the primer-probe combination of Sichuan and Shaanxi taimen as described in claim 1 in the specific detection of Sichuan and Shaanxi taimen using environmental DNA.

4. The application of the primer-probe combination of Sichuan and Shaanxi taimen as described in claim 1 in the population monitoring of Sichuan and Shaanxi taimen.

5. The application of the Sichuan-Shaanxi taimen primer-probe combination as described in claim 1 in protecting Sichuan-Shaanxi taimen populations in variable water bodies.

6. The application according to any one of claims 3-5, characterized in that, The application method includes: (1) Sample collection: Collect environmental samples to be tested, filter them through a 0.45μm cellulose acetate membrane to enrich eDNA, and extract environmental DNA to obtain DNA templates to be tested; (2) qPCR detection: The DNA template to be tested was amplified by qPCR using the primer and probe combination of the Sichuan and Shaanxi taimen. (3) Testing and Judgment Criteria S1: If a typical S-type amplification curve appears in the fluorescence channel and the Ct value is ≤38, then the presence of Sichuan-Shaanxi taimen eDNA in the environmental sample to be tested is determined. S2: If there is no amplification curve in the fluorescence channel, it is determined that there is no eDNA of Sichuan-Shaanxi taimen in the sample to be tested; S3: If there is an amplification curve but no S-type characteristic or Ct value > 38, it is determined that no Sichuan-Shaanxi taimen eDNA was detected in the environmental sample to be tested.

7. The application according to claim 6, characterized in that, The qPCR amplification system in step (2) includes: 10 μL of DNA template to be tested: 2 μL; 2×qPCR mix: 5.0 μL; upstream primer F / downstream primer R: 10 pmol / μL × 0.2 μL; P probe: 10 pmol / μL × 0.1 μL; ddH2O: to bring the total to 10 μL.

8. The application according to claim 7, characterized in that, The qPCR amplification conditions were as follows: initial denaturation at 95°C for 30 seconds; 45 cycles of denaturation at 95°C for 10 seconds and annealing at 60°C for 30 seconds.

9. The application according to claim 6, characterized in that, The amplified fragment was 179 bp in length.

10. The application according to claim 6, characterized in that, The Sichuan-Shaanxi taimen primer-probe combination can effectively avoid interference from closely related species, including at least one of taimen, rainbow trout, and Qinling lenok.

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