Primer probe combination for detecting trachemys scripta elegans based on environmental DNA and application of primer probe combination
By designing a real-time quantitative PCR method combining specific primers and TaqMan probes, the problems of low sensitivity and poor specificity of red-eared slider detection methods in complex environments have been solved. This method achieves high-sensitivity, low-cost, non-invasive monitoring, which is suitable for large-scale dynamic tracking and quantitative evaluation at the grassroots level.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHENGDU INSTITUTE OF BIOLOGY CHINESE ACADEMY OF SCIENCES
- Filing Date
- 2026-03-25
- Publication Date
- 2026-05-08
AI Technical Summary
Existing methods for detecting red-eared sliders have low sensitivity and poor specificity in complex environments, making it difficult to achieve early and accurate monitoring and large-scale dynamic tracking. Furthermore, traditional equipment is costly and complex to operate.
A set of specific primers and TaqMan probes was designed for real-time quantitative PCR detection of environmental DNA in red-eared sliders. A triple recognition mechanism of "dual primers + specific probes" was constructed, and the specificity and sensitivity of the detection were improved by combining FAM fluorescent reporter group and MGB quencher group modification.
It enables highly sensitive, low-cost, and non-invasive monitoring of red-eared sliders in complex environments, making it suitable for large-scale dynamic tracking and quantitative assessment at the grassroots level, and reducing false positive and false negative results.
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Figure CN121992084A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biological detection, specifically relating to primer and probe combinations for detecting red-eared sliders based on environmental DNA and their applications. Background Technology
[0002] The red-eared slider (Trachemys scripta elegans) is listed by the International Union for Conservation of Nature (IUCN) as one of the world's 100 most threatening invasive species. Due to its extremely high adaptability, the red-eared slider's potential habitat range is vast and dynamically expanding. This not only intensifies its direct competition with native turtles for food resources and nesting sites but also leads to severe habitat loss for native species. Therefore, field monitoring of the red-eared slider is particularly important.
[0003] Traditional field monitoring of red-eared sliders relies heavily on visual observation and physical capture. However, this physical-based approach faces multiple challenges in practical application. Due to the target species' cryptic nature and environmental heterogeneity, traditional direct observation methods are easily affected by environmental noise in complex habitats such as those with abundant aquatic vegetation or turbid water, resulting in drastic fluctuations in detection success rates. Furthermore, red-eared slider populations are typically at low abundance in the early stages of an invasion, making it difficult for traditional methods to exceed detection thresholds and achieve early, accurate capture of invasion risks. Moreover, traditional survey methods suffer from inherent bottlenecks such as high labor costs, low monitoring frequency, and limited lateral spatial scaling, failing to meet the technical requirements of modern biosecurity systems for real-time, dynamic early warning over large areas. Physical contact surveys may also cause human disturbance to sensitive monitoring areas, contradicting the trend towards non-destructive monitoring technologies.
[0004] The rise of environmental DNA (eDNA) technology has provided a breakthrough for non-invasive monitoring of invasive species. This technology captures and analyzes trace genetic material, such as skin debris, mucus, excrement, and gametes, shed by target species into environmental media (e.g., water, sediment), enabling precise identification of biological distribution information. With its extremely high detection sensitivity, this technology can keenly capture very low concentrations of genetic residues during the initial low-density stage of red-eared slider invasion, effectively overcoming monitoring omissions caused by the strong concealment of species in traditional methods. Furthermore, through non-invasive sampling, eDNA technology is perfectly adaptable to complex habitats such as dense vegetation and turbid water, significantly improving detection efficiency in complex environments while avoiding ecosystem disturbance. Its highly standardized analytical process and broad spatial coverage provide scientific and technical support for the dynamic monitoring, distribution assessment, and early warning system construction of invasive species within large-scale watersheds.
[0005] In the field of molecular detection of environmental DNA (eDNA) in red-eared sliders, commonly used techniques include conventional PCR, droplet digital PCR (ddPCR), and quantitative real-time PCR (qPCR). While conventional PCR is simple to operate, it typically only provides a qualitative "detection or non-detection" result. Its detection limit is high, and its amplification efficiency is highly unstable when faced with complex chemical inhibitors in environmental samples, easily leading to false negatives. Although ddPCR offers high sensitivity and absolute quantification, its expensive equipment, complex procedures, and extremely low throughput make it difficult to implement at low cost in grassroots monitoring units or large-scale field surveys. Quantitative real-time PCR, due to its combination of high efficiency and cost-effectiveness, is considered a more promising approach for species identification.
[0006] However, most existing qPCR detection methods for red-eared sliders still rely solely on upstream and downstream primers combined with fluorescent dyes. When processing trace environmental samples with complex compositions, these methods are highly susceptible to interference from primer dimers or non-specific amplification signals from closely related species. This results in limited specificity of the detection system, leading to false positives and significant quantitative biases, making it difficult to meet the technical requirements for accurate monitoring of invasive species in their wild habitats.
[0007] In contrast, the TaqMan probe method constructs a triple recognition mechanism of "dual primers + specific probe" by introducing a specific fluorescent probe on top of the primers. An effective fluorescent signal is only triggered when all three components perfectly match the target sequence, which in principle greatly improves the specificity of the detection and effectively shields against interference from complex environmental backgrounds. However, when dealing with trace samples in complex habitats, red-eared slider eDNA primers and TaqMan probes that can simultaneously achieve ultra-high specificity recognition and extremely low detection limits are still lacking. Summary of the Invention
[0008] The purpose of this invention is to provide a primer-probe combination for detecting red-eared sliders based on environmental DNA and its application.
[0009] To achieve the above-mentioned objectives, the technical solution adopted by this invention is: a composition comprising an upstream primer, a downstream primer, and a TaqMan probe, wherein the sequence of the upstream primer is shown in SEQ ID NO:1, the sequence of the downstream primer is shown in SEQ ID NO:2, and the sequence of the TaqMan probe is shown in SEQ ID NO:3. Preferably, the 5' end of the TaqMan probe is modified with FAM, and the 3' end is modified with MGB.
[0010] Accordingly, kits, reagents, or test strips containing the said composition.
[0011] Accordingly, the composition or the kit, reagent or test strip is used in the detection or monitoring of red-eared sliders.
[0012] Accordingly, a method for detecting or monitoring red-eared sliders, the method comprising the following steps:
[0013] (1) Extract DNA from the sample to be tested;
[0014] (2) Using the DNA extracted in step (1) as a template, perform TaqMan real-time quantitative PCR using the aforementioned composition; the TaqMan real-time quantitative PCR reaction system is as follows: 10 μL of 2×T5 Fast qPCR Mix (probe), 0.7 μL each of the upstream and downstream primers at a concentration of 10 μM, 0.6 μL of the probe at a concentration of 10 μM, and 1 μL of DNA template, using dd... Add to 20 μL. The TaqMan real-time quantitative PCR reaction program is as follows: pre-denaturation at 95℃ for 2 min; followed by 40 cycles: denaturation at 95℃ for 15 s, annealing / extension at 60℃ for 30 s; fluorescence signal is acquired at the 60℃ stage.
[0015] (3) Result determination: If the test result shows a typical amplification curve and the Ct value is ≤40, it is determined to be positive, that is, it is determined that red-eared sliders exist in the tested environment; if the test result shows no amplification curve, no Ct value is obtained, or an amplification curve appears and the Ct value is >40, it is determined to be negative, that is, it is determined that red-eared sliders do not exist in the tested environment.
[0016] This invention offers the following advantages: Based on the red-eared slider turtle mitochondrial gene sequence, this invention designs a primer-TaqMan probe combination, constructing a triple recognition mechanism of "dual primers + specific probe." Compared to the expensive and complex droplet digital PCR (ddPCR) technology, this invention utilizes a real-time quantitative PCR (qPCR) platform, offering higher detection throughput and lower cost advantages. Through standardized reaction systems and program settings, it significantly improves detection robustness and is more suitable for large-scale, non-invasive dynamic tracking and quantitative assessment of red-eared slider turtle invasion dynamics at grassroots monitoring units. Attached Figure Description
[0017] Figure 1 This is an amplification curve used to verify the specificity of the primer-probe composition in this invention.
[0018] Figure 2 This is an amplification curve of the plasmid standard in this invention;
[0019] Figure 3 This is a schematic diagram of the standard curve of the plasmid standard in this invention;
[0020] Figure 4 Amplification curves of environmental water samples extracted for different experimental days;
[0021] Figure 5 A schematic diagram showing the changes in Ct values of environmental water samples extracted for different experimental days. Detailed Implementation
[0022] This invention provides a composition for detecting red-eared sliders, the composition comprising specific primers and a TaqMan probe. The specific primers comprise an upstream primer: 5'-TGGCTTTTACCCCCTTCATTAT-3' (SEQ ID NO:1) and a downstream primer: 5'-TATAGATGATGCCTGCAGCTAGG-3' (SEQ ID NO:2); the TaqMan probe sequence is: 5'-CGGAAATTTAGCCCACGCC-3' (SEQ ID NO:3).
[0023] A preferred approach is to modify the 5' end of the TaqMan probe with a fluorescent reporter group to enable signal detection during amplification. Preferably, the FAM fluorescent group is used for modification, as FAM has high luminescence intensity, good stability, and high compatibility with common qPCR instrument channels, providing clear and sensitive fluorescence signals. The 3' end of the TaqMan probe is modified with a quencher group to suppress background fluorescence signals, reduce the signal-to-noise ratio, and improve detection specificity and accuracy. Preferably, the MGB group is used for modification, as MGB, as a minor groove binding group, can significantly increase the probe's melting temperature (Tm) and binding stability, allowing even shorter probes to maintain good hybridization ability, thereby enhancing the ability to recognize single-base differences and reducing non-specific amplification signals. Appropriate modification of the TaqMan probe can improve the sensitivity and stability of the detection system, meeting the detection requirements for low-concentration environmental DNA.
[0024] The present invention also provides a method for detecting / determining the presence of red-eared sliders in a test environment based on the composition, the method comprising the following steps:
[0025] (1) Extracting environmental DNA from the sample to be tested;
[0026] (2) Using the DNA extracted in step (1) as a template, perform TaqMan real-time quantitative PCR reaction using the composition.
[0027] The preferred protocol is as follows: The TaqMan real-time quantitative PCR reaction system consists of: 10 μL of 2×T5 Fast qPCR Mix (probe), 0.7 μL each of 10 μM upstream and downstream primers, 0.6 μL of 10 μM probe, and 1 μL of DNA template, mixed with dd... Add to 20 μL;
[0028] The TaqMan real-time quantitative PCR reaction program is as follows: pre-denaturation at 95℃ for 2 min; followed by 40 cycles: denaturation at 95℃ for 15 s, annealing / extension at 60℃ for 30 s; fluorescence signal is acquired at the 60℃ stage.
[0029] (3) Result determination: If the test result shows a typical amplification curve and the Ct value is ≤40, it is determined to be positive, that is, it is determined that red-eared sliders exist in the tested environment; if there is no amplification curve, no Ct value is obtained, or an amplification curve appears and the Ct value is >40, it is determined to be negative, that is, it is determined that red-eared sliders do not exist in the tested environment.
[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the data obtained are all average values obtained after at least three repetitions, and each repetition yields valid data.
[0031] Example 1: Primer and Probe Design
[0032] To obtain a highly specific detection system for the environmental DNA of the red-eared slider turtle, mitochondrial gene sequences of the target species and its closely related species were compared and analyzed, and specific primers and TaqMan probes were designed. Specific steps included:
[0033] Mitochondrial gene sequences of the target species, the Red-eared Slider, and other sympatric turtles and tortoises were retrieved and downloaded from the NCBI (National Center for Biotechnology Information) database. The species involved include: Alligator Snapping Turtle (Macrochelys temminckii), Common Alligator Snapping Turtle (Chelydra serpentina), Annamite Box Turtle (Mauremys sannamensis), Yellow-margined Box Turtle (Mauremys mutica), Black-necked Turtle (Mauremys nigricans), Chinese Turtle (Mauremys reevesii), Painted Turtle (Mauremys sinensis), Florida Softshell Turtle (Apalone ferox), Chinese Softshell Turtle (Pelodiscus sinensis), Mountain Softshell Turtle (Palea steindachneri), and Sand Softshell Turtle (Pelodiscus axenaria).
[0034] The obtained mitochondrial gene sequences were subjected to multiple sequence alignment using MEGA 12 software. Through comparative analysis, highly conserved and significantly different regions of the red-eared slider mitochondrial COI gene from other species were selected as candidate targets. Specific mismatch sites were precisely designed at the 3' end of the primers and the middle of the probe to avoid shared bases, and the physical parameters of the primers and probes, such as length, GC content, and melting temperature, were made to meet the optimal thermodynamic standards for TaqMan quantitative amplification. After multiple rounds of design verification and thermodynamic stability evaluation, a set of red-eared slider eDNA-specific primers and probes with strong specific recognition ability and extremely high amplification efficiency was finally selected. The specific primers include the upstream primer: 5'-TGGCTTTTACCCCCTTCATTAT-3' (SEQ ID NO:1) and the downstream primer: 5'-TATATGTGATGCCTGCAGCTAGG-3' (SEQ ID NO:2); the TaqMan probe sequence is: 5'-CGGAAATTTAGCCCACGCC-3' (SEQ ID NO:3).
[0035] Example 2: Validation of Primer-Probe Specificity
[0036] Red-eared sliders and 11 closely related or sympatrically distributed control species as described in Example 1 were selected as validation subjects. Genomic DNA was extracted from tissues of each species using a commercially available animal genomic DNA extraction kit, and the extracted raw DNA solution was directly used as the detection template. Simultaneously, a template-free control (NTC) was set up, using ultrapure water instead of DNA template, to monitor for interference from environmental contamination, primer dimers, or non-specific probe amplification. Real-time quantitative PCR was performed using the primer-probe combination described in Example 1, with the generation of a typical S-shaped amplification curve and the corresponding Ct value as the core evaluation indicators to determine the specificity of the combination for different species templates. During the experiment, each sample underwent three independent replicate assays, thus constructing a complete specificity validation system including the target species, multiple control species, and a blank control.
[0037] Real-time quantitative PCR reaction system (20 μL): 10 μL 2×T5 Fast qPCR Mix (probe), 0.7 μL each of 10 μM upstream and downstream primers, 0.6 μL of 10 μM TaqMan specific probe, and 1 μL DNA template. Finally, dd... Add to the total volume.
[0038] Real-time quantitative PCR reaction procedure: 95℃ pre-denaturation for 2 min; followed by 40 cycles: 95℃ denaturation for 15 s, 60℃ annealing / extension for 30 s; fluorescence signal was acquired during the 60℃ annealing and extension phase.
[0039] The results are shown in Table 1 and... Figure 1 As shown.
[0040] Table 1 Comparison of PCR Detection Results
[0041]
[0042] The results showed that the primer-probe combination provided by this invention produced a typical S-shaped amplification curve only for the target species, the red-eared slider turtle, with an average Ct value of 17.518. No fluorescence signal was detected in the other 11 sympatric turtle species and NTC, and the Ct values were all undetermined. This demonstrates that the composition provided by this invention can accurately identify the red-eared slider turtle and completely shield against interference from complex background DNA in its habitat.
[0043] Example 3: Construction of Standard Curve and Sensitivity Analysis
[0044] To evaluate the amplification efficiency, linear range, and detection sensitivity of the composition provided in Example 1, this example constructs plasmid standards and establishes standard curves.
[0045] The red-eared slider product amplified in Example 2 was cloned into a vector, and the recombinant plasmid was sequenced for verification. Positive clones with completely correct sequences were selected, and plasmids were extracted using a plasmid extraction kit. These plasmids were then used as standard plasmids for subsequent amplification efficiency testing.
[0046] Calculate the copy number of plasmid concentration: C = A × B -1 ×6.02×10 14 A is the plasmid concentration, in ng / μL. B is the molecular weight of the plasmid DNA, in g / mol. C is the plasmid copy number concentration, in copies / μL. 6.02 × 10⁻⁶ 14 The constant is the sum of Avogadro's constant and the unit conversion factor. The calculated concentration of the red-eared slider standard plasmid stock solution was 6.05E+10 copies / μL, which was used as the experimental plasmid standard.
[0047] The red-eared slider standard plasmid stock solution was serially diluted with ultrapure water at concentrations of 10. A 10% concentration was selected. 3 Up to 10 times 7 A 1:1 dilution was used as the positive standard template. The real-time fluorescence PCR reaction system and amplification procedure of Example 2 were followed, with each dilution gradient repeated three times to construct a standard curve.
[0048] The probe gradient amplification curve of the COI-1 standard plasmid is shown below. Figure 2 As shown in the figure. The results show that the standards at each concentration gradient all produced typical S-shaped amplification curves, and the starting point shifted regularly as the concentration decreased, with good overlap in the three replicates.
[0049] The measured Ct values and copy number log values of each group were fitted to obtain the standard curve equation as follows: Figure 3 As shown: y = -3.4028x + 43.136.
[0050] Detecting R 2 The result showed that the red-eared slider gene reached 0.9997, indicating that the gene concentration was 6.05 × 10⁻⁶. 3 Up to 6.05×10 7 Within the range of copies / μL, the Ct value and the copy number log value showed a good linear relationship. Based on the established standard curve, the amplification efficiency was calculated to be 97%. The detection data are shown in Table 2.
[0051] Table 2 Comparison of test results at different dilution ratios
[0052]
[0053] The results showed that plasmid diluted 10 7 The good repeatability of the detection after doubling indicates that the sensitivity of the primers and probes provided by this invention meets the detection requirements, enabling accurate capture and quantitative monitoring of trace red-eared slider genetic residues in the environment.
[0054] Example 4: Monitoring of Environmental DNA Release and Dynamic Changes in Red-eared Slider
[0055] To verify the ability of the primer-probe combination provided by this invention to detect the dynamics of environmental DNA in red-eared sliders in an aquatic environment, this embodiment constructs a time-series experiment for continuous monitoring.
[0056] A closed indoor water system was set up, and 1.5 m³ of tap water was added to the tank. The tap water was allowed to stand to remove residual chlorine. After water collection was completed on the first day, three red-eared sliders were placed in the tank for rearing. After water collection was completed on the sixth day, the red-eared sliders were removed. Water samples were collected daily at fixed times after the red-eared sliders were placed in the tank: 1 liter of water sample was filtered through a 0.45 µm glass fiber membrane.
[0057] Environmental DNA was extracted from the filter membrane using the method described in Example 2. The extracted product was then used as a template for real-time quantitative PCR detection using the primer and probe combination from Example 1. The reaction system and procedure were consistent with those in Example 2. Three technical replicates were performed for each sample, and a template-free control (NTC) was included to monitor system contamination.
[0058] The results are as follows Figure 4 As shown ( Figure 4 The numbers 1, 2, 3...11 below the horizontal axis represent the experimental days. Except for day 1, samples at each time point produced typical S-shaped amplification curves. In the early stages of the experiment, the amplification curves of the samples started late, and the fluorescence signals were weak. As the red-eared sliders continued to exist in the water, the amplification curves of the samples entered the exponential amplification phase significantly earlier, and the fluorescence signals were significantly enhanced. After water collection and removal of the red-eared sliders on day 6, amplification signals could still be detected in samples at subsequent time points, but the cycle number of amplification signals gradually delayed, and the starting position of the amplification curves of some samples was significantly delayed, with relatively weaker amplification signals.
[0059] The results of Ct value changes for samples at each time point are shown in the figure. Figure 5 As the sampling time increased, the Ct value generally showed a trend of first decreasing and then increasing. From the initial stage of the experiment to day 6, the Ct value gradually decreased, indicating that the concentration of environmental DNA in the water was gradually increasing. After water collection and removal of the red-eared sliders on day 6, the Ct value significantly increased at subsequent time points and remained at a high level with a gradually increasing trend, indicating that the concentration of environmental DNA in the water gradually decreased after the release was stopped.
[0060] The above results indicate that the detection system constructed in this invention can effectively detect red-eared sliders both in the initial stage of their entry into the water and for a certain period of time after they leave, and can stably reflect the dynamic changes in the environmental DNA of red-eared sliders in the water.
[0061] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.
Claims
1. A group of compositions, characterized in that: The composition includes an upstream primer, a downstream primer, and a TaqMan probe, wherein the sequence of the upstream primer is shown in SEQ ID NO:1, the sequence of the downstream primer is shown in SEQ ID NO:2, and the sequence of the TaqMan probe is shown in SEQ ID NO:
3.
2. The composition according to claim 1, characterized in that: The 5' end of the TaqMan probe is modified with FAM, and the 3' end is modified with MGB.
3. A kit, reagent, or test strip containing the composition of claim 1 or 2.
4. The use of the composition of claim 1 or 2 or the kit, reagent or test strip of claim 3 in the detection or monitoring of red-eared sliders.
5. A method for detecting or monitoring red-eared sliders, characterized in that: The method includes the following steps: (1) Extract DNA from the sample to be tested; (2) Using the DNA extracted in step (1) as a template, a TaqMan real-time quantitative PCR reaction is performed using the composition; the composition includes an upstream primer, a downstream primer and a TaqMan probe, the sequence of the upstream primer is shown in SEQ ID NO:1, the sequence of the downstream primer is shown in SEQ ID NO:2 and the sequence of the TaqMan probe is shown in SEQ ID NO:
3. (3) Result determination: If the test results show a typical amplification curve and the Ct value is ≤40, it is determined that red-eared sliders exist in the tested environment; if the test results do not show an amplification curve, do not obtain a Ct value, or show an amplification curve and the Ct value is >40, it is determined that red-eared sliders do not exist in the tested environment.
6. The method according to claim 5, characterized in that: In step (2), the TaqMan real-time quantitative PCR reaction system is as follows: 10 μL of 2×T5 Fast qPCR Mix (probe), 0.7 μL each of the upstream and downstream primers with a concentration of 10 μM, 0.6 μL of the probe with a concentration of 10 μM, 1 μL of DNA template, and dd H2O to make up to 20 μL.
7. The method according to claim 5, characterized in that: In step (2), the TaqMan real-time quantitative PCR reaction program is as follows: pre-denaturation at 95℃ for 2 min; followed by 40 cycles: denaturation at 95℃ for 15 s, annealing / extension at 60℃ for 30 s; fluorescence signal is collected at the 60℃ stage.