Digital PCR kit for detecting red-eared slider and bullfrog and application thereof

Abstract

The application provides a digital PCR kit for detecting highland invasive species red-eared slider and bullfrog, a set of red-eared slider specific primer probes, which are composed of a primer pair for a specific segment of a red-eared slider mitochondrial CYTB gene and a red-eared slider specific TaqMan probe matched therewith; a set of bullfrog specific primer probes, which are composed of a primer pair for a specific segment of a bullfrog mitochondrial 12S rRNA gene and a bullfrog specific TaqMan probe matched therewith, and a set of internal standard (IPC) primer pairs and internal standard TaqMan probes for monitoring the nucleic acid extraction and amplification reaction process monitoring. The application is provided with the primer pair for the specific segment of the red-eared slider mitochondrial CYTB gene and the red-eared slider specific TaqMan probe, the primer pair for the specific segment of the bullfrog mitochondrial 12S rRNA gene and the bullfrog specific TaqMan probe, and a common internal standard to construct a triple detection reaction system, thereby avoiding the quantitative uncertainty caused by the single species detection in the prior art which needs to be carried out in multiple reactions or relies on a standard curve for relative quantification.

Description

Technical Field

[0001] This invention relates to the field of biosafety monitoring and molecular detection technology, and in particular to a digital PCR kit and its application for detecting the invasive species red-eared slider turtle and bullfrog from the plateau region. Background Technology

[0002] Biological invasion is one of the major factors leading to global biodiversity loss and ecosystem service degradation. Red-eared sliders ( Trachemys scripta elegans (Commonly known as the Brazilian turtle) and bullfrog ( Rana catesbeiana As one of the most harmful invasive species worldwide, these two species have established wild populations in many parts of my country. In recent years, with the increase in tourism, trade, and artificial release activities, they have appeared in some waters of the Qinghai-Tibet Plateau. Their strong adaptability, reproductive capacity, and predatory nature pose direct competition and predation pressure on the fragile aquatic organisms native to the plateau (such as endemic fish, amphibians, and aquatic insects), seriously threatening regional ecological security. Therefore, establishing a technology that can achieve early, rapid, and accurate monitoring of their distribution and abundance is an urgent need for implementing effective interception and eradication and protecting the unique ecosystem of the plateau. Traditional invasive species monitoring mainly relies on field surveys (such as transect methods, trap methods, and call counting) and community reports. However, these methods have significant drawbacks when applied to the vast, remote, and habitat-complex areas of the Qinghai-Tibet Plateau: 1) Lag: Invasive species are often only detected after they have established themselves and caused significant ecological damage; 2) Low efficiency and high cost: Large-scale surveys consume enormous human and material resources; 3) Insufficient sensitivity: Small numbers of hidden or juvenile individuals are easily missed; 4) High level of expertise required: Identification of juveniles or traces is difficult. Environmental DNA (eDNA) technology infers the presence of species by detecting DNA contained in shed skin cells, secretions, and other substances in the environment, providing a revolutionary solution to overcome the aforementioned bottlenecks. This technology has the advantages of being non-invasive, sensitive, and capable of large-scale simultaneous screening, making it particularly suitable for the early monitoring of invasive species. Currently, eDNA analysis mostly employs real-time quantitative PCR (qPCR). However, in practical applications of invasive species monitoring, especially in complex environmental sample contexts, qPCR technology has the following key limitations: 1) Relative quantitative uncertainty: Its quantitative results depend on the standard curve. PCR inhibitors commonly found in environmental samples can severely interfere with amplification efficiency, leading to distorted quantitative results and making it impossible to accurately assess the size of invasive populations; 2) Insufficient early warning sensitivity: In the early stages of invasion, the concentration of target DNA in the environment is extremely low, and the detection limit and quantitative accuracy of qPCR are limited, making it easy to miss the optimal control window; 3) False negative risk: Inhibitors may cause the reaction to fail completely, and conventional internal controls cannot completely identify this situation. Digital PCR (dPCR), as the third generation of PCR technology, divides the reaction system into tens of thousands of independent nanoliter reaction units, performs endpoint fluorescence detection, and directly calculates the absolute copy number of target DNA based on the Poisson distribution. Compared with qPCR, dPCR has significant advantages such as absolute quantification, stronger resistance to inhibitors, and more accurate and reliable detection at ultra-low concentrations. Theoretically, it is the most ideal platform for eDNA quantification monitoring. Despite the significant advantages of dPCR, developing ready-to-use detection solutions for specific invasive species, especially suitable for the stringent monitoring needs of high-altitude regions, still faces challenges: First, genetically highly specific primers and probes must be designed for red-eared sliders and bullfrogs to ensure accurate and unique identification of invasive species signals from complex environmental sample backgrounds (which may contain native relatives or large amounts of unknown DNA); second, to ensure the reliability of monitoring results, an effective internal standard (IPC) system must be integrated to eliminate false negatives; finally, a stable multiplex detection system that has undergone rigorous and comprehensive experimental validation needs to be constructed.

[0003] Currently, there is a lack of commercially available dPCR kits capable of simultaneously and absolutely quantifying the eDNA of red-eared sliders and bullfrogs, with built-in quality controls. Existing research mainly focuses on qPCR methods for single species, whose early detection capabilities and quantitative accuracy in complex environmental samples are insufficient to meet the precise management needs of dynamic monitoring of invasive species on the Tibetan Plateau. Therefore, developing an integrated, standardized, and fully validated dPCR dual detection kit is of crucial technical value and urgent practical significance for improving biosecurity control capabilities on the Qinghai-Tibet Plateau. Therefore, a digital PCR kit for detecting the invasive species *Red-eared Slider* and *Bullfrog* in the high-altitude region and its application are proposed. Summary of the Invention

[0004] In view of this, the present invention provides a digital PCR kit and its application for detecting the invasive species red-eared slider turtle and bullfrog in high-altitude areas, in order to solve or alleviate the technical problems existing in the prior art, and at least provide a beneficial alternative.

[0005] The technical solution of this invention is achieved as follows: a digital PCR kit for detecting the invasive species *Red-eared Slider* and *Bullfrog*, the plateau invasive species, the kit comprising the following components, which synergistically constitute a triple digital PCR detection reaction system: A set of red-eared slider turtle-specific primer-probe pairs consists of primer pairs targeting a specific region of the red-eared slider turtle mitochondrial CYTB gene and matching red-eared slider turtle-specific TaqMan probes. A set of bullfrog-specific primer-probe pairs consists of primer pairs targeting a specific region of the bullfrog mitochondrial 12S rRNA gene and matching bullfrog-specific TaqMan probes. A set of internal standard primers and probes, designed for a specific region shared by the 16S rRNA genes of red-eared sliders and bullfrogs, are used to monitor the nucleic acid extraction and amplification reaction process; Digital PCR premix contains hot-start DNA polymerase, dNTPs, MgCl2, and a buffer system; And positive control samples and negative control samples; In this process, the nucleic acid of the sample to be tested is mixed with the digital PCR premix, red-eared slider turtle-specific primers and probes, bullfrog-specific primers and probes, and internal standard primers and probes, and then digital PCR is performed for partitioned amplification. The amplification status of the red-eared slider turtle target DNA, bullfrog target DNA, and internal standard sequence is determined by the endpoint fluorescence signals of the red-eared slider turtle-specific TaqMan probe, bullfrog-specific TaqMan probe, and internal standard probe, respectively.

[0006] More preferably, the red-eared slider-specific primer probe targets a specific segment of the red-eared slider mitochondrial cytochrome b (CYTB) gene.

[0007] More preferably, the bullfrog-specific primer probe targets a specific segment of the bullfrog mitochondrial 12S rRNA gene.

[0008] More preferably, the red-eared slider turtle-specific TaqMan probe, the bullfrog-specific TaqMan probe, and the internal standard probe are each labeled with different fluorescent reporter groups.

[0009] More preferably, the fluorescent reporter group of the red-eared slider-specific TaqMan probe is FAM, the fluorescent reporter group of the bullfrog-specific TaqMan probe is ROX, and the fluorescent reporter group of the internal standard probe is HEX.

[0010] More preferably, the internal standard sequence corresponding to the internal standard primer probe is distinct from the target region of the red-eared slider and the target region of the bullfrog in terms of sequence origin, and the internal standard primer probe does not amplify in a matching manner with the target region of the red-eared slider and the target region of the bullfrog.

[0011] More preferably, the positive control is recombinant plasmid DNA containing red-eared slider target sequences and bullfrog target sequences and / or artificially synthesized nucleic acid fragments, and the negative control is nuclease-free water.

[0012] More preferably, the nucleic acid of the sample to be tested is derived from biological tissue samples of red-eared sliders or bullfrogs, and / or from environmental DNA extracted from environmental samples.

[0013] More preferably, the environmental samples include water samples, soil samples, and / or sediment samples. Water samples may be enriched first to improve the recovery rate of environmental DNA before DNA extraction; the enrichment method may be filtration enrichment, centrifugation enrichment, or other equivalent methods, followed by the DNA extraction process to obtain a DNA extract solution suitable for PCR amplification.

[0014] This invention also provides the application of a digital PCR kit in the simultaneous identification and absolute quantitative detection of red-eared sliders and bullfrogs.

[0015] The embodiments of the present invention have the following advantages due to the adoption of the above technical solutions: I. This invention simultaneously configures primer pairs and TaqMan probes specific to the CYTB gene segment of the red-eared slider turtle mitochondria and specific to the bullfrog mitochondrial 12S rRNA gene segment within the same digital PCR reaction system. Combined with digital PCR partitioning amplification and endpoint fluorescence interpretation mechanisms, the target DNA of the red-eared slider turtle and the target DNA of the bullfrog can be distinguished and identified in a single detection, and the absolute copy number result can be output. This avoids the quantitative uncertainty caused by the need for multiple reactions or reliance on standard curves for relative quantification in single-species detection, which is common in existing studies. This makes it more suitable for the dynamic monitoring needs of invasive species in environmental samples such as plateau water bodies.

[0016] Second, this invention introduces an internal standard sequence and its corresponding internal standard primer probe that are distinct from the target regions of red-eared sliders and bullfrogs into the kit, and enables parallel detection of the internal standard probe channel and the two target probe channels. This allows for monitoring and interpretation of the reaction process status in complex environmental sample backgrounds. When the target signal is missing, the detection status of the internal standard channel can still be combined to confirm whether the reaction is in an interpretable state, avoiding interpretation bias caused by relying solely on the target amplification results. This enhances the reliability of results in trace eDNA monitoring scenarios and meets the requirements for detection consistency in early warning and precise management of invasive species.

[0017] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the invention will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram illustrating the interpretation of the triple digital PCR detection method of the present invention; Figure 2 This is a schematic diagram of the multi-channel endpoint fluorescence interpretation logic of the present invention.

[0020] Figure 3 The images show the droplet 1D plots and determination results for linearity testing of single-target digital PCR systems provided in this embodiment of the invention. A represents the droplet 1D plot for linearity testing of the red-eared slider target, B represents the determination result for the red-eared slider target, C represents the droplet 1D plot for linearity testing of the bullfrog target, and D represents the determination result for the bullfrog target.

[0021] Figure 4 The above is a statistical chart of the linear results of the single-target digital PCR system provided in the embodiments of the present invention.

[0022] Figure 5 The images shown are 1D droplet diagrams for each channel of the multiplex digital PCR system provided in this embodiment of the invention. A is a 1D droplet diagram of the red-eared slider target in the FAM channel, B is a 1D droplet diagram of the internal standard in the HEX channel, and C is a 1D droplet diagram of the bullfrog target in the ROX channel.

[0023] Figure 6 The images shown are 2D droplet diagrams for each channel of the multiplex digital PCR system provided in this embodiment of the invention. A represents the 2D droplet diagrams for the FAM and HEX channels, B represents the 2D droplet diagrams for the FAM and ROX channels, and C represents the 2D droplet diagrams for the HEX and ROX channels.

[0024] Figure 7 The images show the droplet 1D plots and determination results for linear assays of a multiplex digital PCR system provided in this embodiment of the invention. A represents the droplet 1D plot for linear assays of the red-eared slider target, B represents the determination results for the red-eared slider target, C represents the droplet 1D plot for linear assays of the internal standard, D represents the determination results for the internal standard, E represents the droplet 1D plot for linear assays of the bullfrog target, and F represents the determination results for the bullfrog target.

[0025] Figure 8 A statistical graph showing the linear results of the multiplex digital PCR system provided in this embodiment of the invention.

[0026] Figure 9The images show 1D droplet plots and value determination results for repeatability testing of a multiplex digital PCR system provided in this embodiment of the invention. A represents the 1D droplet plot for repeatability testing of the red-eared slider target, B represents the value determination result for the red-eared slider target, C represents the 1D droplet plot for repeatability testing of the internal standard, D represents the value determination result for the internal standard, E represents the 1D droplet plot for repeatability testing of the bullfrog target, and F represents the value determination result for the bullfrog target.

[0027] Figure 10 This document presents a comparison of 1D droplet plots for multiplex digital PCR and singlex digital PCR for each target, provided in this embodiment of the invention. A represents the 1D droplet plot of the red-eared slider target in both the multiplex and singlex systems; B represents the target value determination results in both systems; C represents the droplet plot of the bullfrog target in both systems; and D represents the target value determination results in both systems. Detailed Implementation

[0028] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0029] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0030] like Figure 1-2 As shown, this embodiment of the invention provides a digital PCR kit for detecting the invasive plateau species, the red-eared slider and the bullfrog, comprising the following components and constituting a triple digital PCR detection reaction system: Red-eared slider turtle-specific primers and probes: consisting of primer pairs targeting a specific region of the red-eared slider turtle mitochondrial CYTB gene and a matching red-eared slider turtle-specific TaqMan probe; Bullfrog-specific primers and probes: consisting of primer pairs targeting specific regions of the bullfrog mitochondrial 12S rRNA gene and matching bullfrog-specific TaqMan probes; Internal control (IPC) primers and probes: designed from a specific region shared by the 16S rRNA genes of red-eared sliders and bullfrogs, used to monitor the nucleic acid extraction and amplification process, and the internal control primers and probes do not match and amplify the target regions of red-eared sliders and bullfrogs; Digital PCR premix: contains hot-start DNA polymerase, dNTPs, MgCl2, and a buffer system; Positive control: Recombinant plasmid DNA and / or artificially synthesized nucleic acid fragments containing red-eared slider target sequences and bullfrog target sequences; Negative control: A control system without nucleic acid template, preferably nuclease-free water.

[0031] In this embodiment, the above components can be prepared into independent tubes, or the red-eared slider specific primer probe, bullfrog specific primer probe and internal standard primer probe can be pre-mixed into a "specific primer probe mixture" according to a predetermined ratio and then packaged separately; the digital PCR premix, positive control and negative control are packaged separately; the above different assembly forms do not affect the composition and use of the triple detection reaction system of the present invention.

[0032] To achieve differentiated detection within the same reaction system, the red-eared slider turtle-specific TaqMan probe, the bullfrog-specific TaqMan probe, and the internal standard TaqMan probe were each labeled with different fluorescent reporter groups. In a preferred embodiment, the fluorescent reporter group of the red-eared slider turtle-specific TaqMan probe is FAM, the fluorescent reporter group of the bullfrog-specific TaqMan probe is ROX, and the fluorescent reporter group of the internal standard probe is HEX. By outputting the endpoint fluorescence signal of the three different fluorescent reporter groups, the amplification status of the red-eared slider turtle target DNA, the bullfrog target DNA, and the internal standard sequence can be distinguished and interpreted.

[0033] The nucleic acids in the test samples of this invention can be derived from the following two types: Biological tissue samples: including tissue samples derived from red-eared sliders or bullfrogs; Environmental samples: These include water samples, soil samples, and / or sediment samples. Environmental DNA (eDNA) is extracted from these samples and used as the nucleic acid to be tested. For water samples, enrichment treatment can be performed first to improve the recovery rate of environmental DNA before DNA extraction. Enrichment methods can include filtration enrichment, centrifugation enrichment, or other equivalent methods. Subsequently, a DNA extract solution suitable for PCR amplification is obtained according to the DNA extraction procedure.

[0034] In this embodiment, DNA can be obtained from tissue samples and environmental samples using conventional nucleic acid extraction methods. Environmental samples may contain various background DNAs and potential inhibitors. To ensure that the detection process can be monitored, this invention introduces an internal standard (IPC) primer probe into the triple reaction system as a monitoring channel for the reaction process, which is amplified and interpreted in parallel with the target channel. Based on the above-mentioned reagent kit components, the detection process of this embodiment of the invention includes the following steps: S1, Reaction System Configuration Take the nucleic acid of the sample to be tested, add digital PCR premix, and add red-eared slider turtle-specific primers and probes, bullfrog-specific primers and probes and internal standard primers and probes. Mix well to obtain digital PCR reaction mixture. Simultaneously, positive and negative quality control reactions were prepared separately: Positive control reaction: Replace the nucleic acid of the test sample with a positive control, and keep the other components the same as the test reaction; Negative control reaction: The negative control is used to replace the nucleic acid in the test sample, and the remaining components are the same as those in the test reaction.

[0035] S2, Partitioning and Amplification The above reaction mixture is loaded into the partitioning system of the digital PCR platform to form multiple independent reaction units; then PCR amplification is performed to obtain the endpoint amplification product and output the endpoint fluorescence signal.

[0036] S3. Acquisition and interpretation of endpoint fluorescence signal The endpoint fluorescence detection of the partitioned reaction unit was performed, and the fluorescence signals corresponding to the red-eared slider turtle-specific TaqMan probe channel, the bullfrog-specific TaqMan probe channel, and the internal standard probe channel were collected respectively; in the preferred embodiment using the FAM / ROX / HEX configuration, the FAM, ROX, and HEX channel signals were collected respectively.

[0037] S4, Absolute Quantitative Output Interpretation is based on the endpoint fluorescence signal: when a positive signal appears in the red-eared slider-specific channel, it is determined that red-eared slider target DNA is present in the nucleic acid of the sample to be tested; when a positive signal appears in the bullfrog-specific channel, it is determined that bullfrog target DNA is present in the nucleic acid of the sample to be tested; the internal standard probe channel is used to monitor the reaction process status to confirm that the reaction system is in an interpretable state; The digital PCR platform calculates the absolute copy number of red-eared slider target DNA and bullfrog target DNA based on the ratio of positive / negative reaction units in the partition and combined with the Poisson distribution model, and outputs the corresponding quantitative detection data; the quantitative detection data can be used for subsequent statistical analysis and monitoring records.

[0038] Application of reagent kits The kit of this invention is mainly used for the detection reaction system of the target DNA of red-eared sliders and bullfrogs in samples; based on the digital PCR partitioning amplification and endpoint fluorescence interpretation method of this invention, the kit of this invention can be used in the following scenarios: (1) Early diagnosis and confirmation of invasive species: DNA from tissue samples or environmental swabs of suspected individuals is tested, and the results of the presence status determination of red-eared slider target DNA and / or bullfrog target DNA are output for species identification and confirmation. (2) Monitoring of invasion dynamics and distribution range: Batch testing of environmental DNA (eDNA) samples from rivers, lakes, wetlands, ponds and other water bodies in plateau areas, outputting absolute quantitative data of red-eared sliders and bullfrogs. The quantitative data can be used as input data for spatial distribution statistics, abundance distribution analysis and monitoring database construction, and is used for monitoring the invasion distribution range and dynamic changes. (3) Evaluation of prevention and control effectiveness and risk assessment: Comparative detection of environmental DNA samples in the corresponding areas before and after the implementation of prevention and control measures such as artificial removal and ecological restoration, output absolute quantitative results of target DNA, which are used to evaluate the trend of changes in detection results after the implementation of measures, and are used for diffusion monitoring and risk assessment in the frontier area of ​​invasion, supporting the data needs of biosafety management decision-making.

[0039] Based on the feasibility and detection performance of the reagent kit of the present invention, the following performance verification was carried out on the reagent kit of the present invention: Specificity (qPCR initial screening and dPCR verification): Initial screening was performed using qPCR and validation was performed using digital PCR; The genomic DNA of red-eared sliders and bullfrogs was used as a positive control template; the genomic DNA of various non-target species coexisting in Tibet was used as a negative template for testing. The non-target species included black-spotted loach, Tibetan croaker, Tibetan blackhead fish, plateau naked carp, Medog new-lipped fish, light brown loach, flat-lipped loach, yellow-spotted croaker, flat-finned naked-snout fish, naked-bellied leaf-whiskered fish, pointed naked carp, Asian salmon, Lhasa naked-splitter, Lhasa naked-splitter (Himalayan subspecies), curved-lipped schizothorax, whole-lipped schizothorax, Lancang schizothorax, Medog schizothorax, Qi-mouthed schizothorax, Lhasa schizothorax, and thin-tailed plateau loach, etc. The results showed that specific fluorescent signals were detected only in the target species responses (FAM channel or its corresponding red-eared slider channel for red-eared sliders, and ROX channel or its corresponding bullfrog channel for bullfrogs). No signals were detected in the target channels for any non-target species responses. Simultaneously, the internal control (HEX channel or its corresponding internal control channel) amplified normally. This indicates that the primer-probe combination of this invention is species-specific, and no cross-reactions were observed in the aforementioned non-target species background.

[0040] Linearity and Quantitative Range: Digital PCR analysis was performed using the kit of this invention, and the measured copy numbers of the two targets were compared with the theoretical input copy numbers. The results showed that the logarithmic values ​​of the measured copy numbers of the two targets had a good linear relationship with the theoretical input copy numbers, and the linear correlation coefficients R² were all greater than 0.98, indicating that the kit of this invention has a quantitative response relationship for absolute quantitative analysis.

[0041] Limit of detection: Repeated testing was performed using low-concentration real samples. Digital PCR results showed that the kit of this invention can achieve detection output at a concentration level of 10 copies / μL for both red-eared slider and bullfrog targets, making it suitable for the detection of trace target DNA.

[0042] Repeatability: Eight replicate tests were performed on a single concentration sample using the digital PCR kit of this invention, and the coefficient of variation (CV) within each group was calculated. The results showed that the coefficient of variation (CV) values ​​within each of the eight replicates were all less than 2%, indicating that the kit of the present invention has stable detection consistency under the repeated test conditions.

[0043] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in the present invention, and these should all be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

[0044] Example 1: Primer and probe design The mitochondrial genomes of the red-eared slider turtle (Genebank: KM216748.1) and the bullfrog (Genebank: NC_022696.1) were downloaded from Genbank. At the same time, the mitochondrial genomes of non-target species coexisting in Tibet were also downloaded for sequence alignment. The species names, Latin names and Genebank accession numbers of the non-target species are shown in Table 1.

[0045] IPC internal standards are designed based on conserved regions of the 16S rRNA gene in each species to fully cover more non-target species. After mitochondrial gene sequence alignment of each species using SnapGene software, relatively conserved gene regions of red-eared sliders and bullfrogs were selected. Based on primer and TaqMan probe design principles and relevant national standards, relevant primer and probe sequences were designed using PrimerPremier 5 and Oligo Analyzer software. The designed primer and probe sequences are summarized in Table 2. The designed primers and probes were then synthesized by Sangon Biotech Co., Ltd.

[0046] Table 1. Statistics on the names, Latin names, and Genebank accession numbers of coexisting non-target species in Tibet.

[0047] Table 2 Summary of specific primer and probe sequences for red-eared sliders and bullfrogs

[0048] Example 2: Screening and optimization of target and internal standard primers and probes After designing detection primers and probes for red-eared sliders, bullfrogs, and their shared internal standard, further screening of primer-probe combinations is necessary. Primer sets with similar amplification efficiencies should be selected to avoid mutual interference between primers. Following primer and probe screening and optimization, the final target and internal standard primer-probe sequences were determined. The TaqMan probe for the red-eared slider target has the fluorophore FAM, the TaqMan probe for the bullfrog target has the fluorophore ROX, and the TaqMan probe for the shared internal standard has the fluorophore HEX (see Table 3).

[0049] Table 3 Primer and probe sequences determined by multiplex digital PCR detection method for red-eared sliders and bullfrogs.

[0050] Example 3: Sample processing and specificity testing (1) Sample processing: Genomic DNA was extracted from the target species, red-eared slider and bullfrog, using a commercially available kit (blood / cell / tissue genomic DNA extraction kit), and simultaneously extracted from other species coexisting in Tibet. Genomic DNA from various non-target species, including Black-spotted Loach, Tibetan Gudgeon, Tibetan Blackhead Fish, Plateau Naked Carp, Medog New-lipped Fish, Light Brown Loach, Flat-lipped Loach, Yellow-spotted Folded Loach, Flat-finned Naked-snout Fish, Naked-belly Leaf-whiskered Fish, Pointed Naked Carp, Asian Salmon, Lhasa Naked Schizothorax, Lhasa Naked Schizothorax (Himalayan subspecies), Arc-lipped Schizothorax, Whole-lipped Schizothorax, Lancang Schizothorax, Medog Schizothorax, Qi-mouthed Schizothorax, Lhasa Schizothorax, and Fine-tailed Plateau Loach.

[0051] (2) Specificity test of the test samples: The optimized target and internal standard primers and probes were used for initial screening by qPCR. The genomic DNA of red-eared sliders and bullfrogs was used as positive templates, and the genomic DNA of non-target species was used as negative templates. Nuclease-free water was used as a negative control. The reaction system configuration for each well is shown in Table 4. The fluorescent PCR reaction was carried out according to the procedure shown in Table 5. The equipment used was the JLM QX600 from Jeremy Laboratories.

[0052] Table 4. Preparation of singleton qPCR reaction system for target and internal standard

[0053] Table 5 qPCR reaction parameter settings

[0054] The results showed that specific fluorescent signals (FAM or ROX channels) were detected only in the wells of the target species, while no signals were detected in all wells of non-target species. The internal standard (HEX channel) amplified normally, indicating that the primer-probe combination had high species specificity and no cross-reactivity. The results are shown in Table 6.

[0055] Table 6. Specificity test results of red-eared sliders, bullfrogs, and internal standard single-weight systems.

[0056] Example 4: Testing of a singlet digital PCR system Using the optimized target primers and probes, linearity testing of the targets was performed using singlet digital PCR. Genomic DNA from red-eared sliders and bullfrogs was used as the sample stock solution, and each sample was diluted 4-fold to seven concentration gradients before digital PCR reactions. Nuclease-free water was used as a negative control. The reaction system is shown in Table 7. Singlet digital PCR reactions were performed according to the reaction conditions in Table 8 to evaluate whether the target primers and probes could be used for the construction of subsequent multiplex digital PCR systems. The equipment used was the JLM Digital Matrix-5000 digital PCR system from Jeremy Laboratories. Sample concentration (Copies / μL) = (Copies / μL) × 20 (total reaction volume) ÷ 5 (sample loading amount).

[0057] The linear assay results of singlet digital PCR for the target showed that the number of droplets was greater than 10,000, which met the experimental analysis requirements. The droplet plot results are shown below. Figure 3 As shown. A linear standard curve is constructed with the dilution concentration gradient on the x-axis and the logarithm of the constant concentration on the y-axis. Figure 4 The results show that the correlation coefficient (R) of the standard curve 2 The linearity of the singlet digital PCR method is greater than 0.98, indicating that the linearity of the singlet digital PCR method meets the performance requirements and can be used for the construction of subsequent multiplet digital PCR systems.

[0058] Table 7 Preparation of Singleton Digital PCR System

[0059] Table 8. Digital PCR reaction parameter settings for this kit

[0060] Example 5: Construction and testing of a multiplex digital PCR system Multiplex digital PCR system was constructed by mixing the optimized target and internal standard primers and probes. Genomic DNA of red-eared sliders and bullfrogs was mixed, with the mixed sample as a positive control and nuclease-free water as a negative control. The reaction system is shown in Table 9. Multiplex digital PCR reaction was carried out according to the reaction conditions in Table 8.

[0061] The 1D plot of the droplet test results is as follows: Figure 5 As shown, the 2D diagram of the droplets is as follows: Figure 6As shown in the 1D image, the positive and negative bands in each channel are clearly layered and do not interfere with each other in detection. As shown in the 2D image, the targets are clearly distinguished and the bands are clearly divided. The 1D and 2D images show good results, proving that the multiplex digital PCR reaction system has been successfully established.

[0062] Table 9. Preparation of the Multiplex Digital PCR System for This Kit

[0063] Example 6: Establishment of standard curve and determination of limit of detection for multiplex digital PCR system Multiplex digital PCR systems were constructed by mixing the optimized target and internal standard primers and probes. Linearity testing of the multiplex digital PCR system was performed by mixing genomic DNA from red-eared sliders and bullfrogs, using the mixed sample as the stock solution, and then performing digital PCR reactions after 4-fold dilutions at 7 concentration gradients. Nuclease-free water was used as a negative control. The reaction system is shown in Table 9. Multiplex digital PCR reactions were performed according to the reaction conditions in Table 8 to evaluate the standard curve and limit of detection of the established multiplex digital PCR system. The equipment used was the JLM Digital Matrix-5000 digital PCR system from Jeremy Laboratories. Sample concentration (Copies / μL) = (Copies / μL) × 20 (total reaction volume) ÷ 5 (sample loading amount).

[0064] The results of the multiplex digital PCR linear assay showed that the number of droplets was greater than 10,000, which met the experimental analysis requirements. (See attached results). Figure 7 As shown. A linear standard curve is constructed with the dilution concentration gradient on the x-axis and the logarithm of the constant concentration on the y-axis. Figure 8 The results show that the correlation coefficient (R) of the standard curve 2 The linearity values ​​were all greater than 0.98, indicating that the linearity of the multiplex digital PCR system met the performance requirements. In the multiplex digital PCR system, the limits of detection for red-eared sliders, bullfrogs, and internal standards were 1.11 copies / μL, 1.79 copies / μL, and 1.08 copies / μL, respectively. All limits of detection were below 10 copies / μL, meeting the performance requirements. The results are shown in Table 10.

[0065] Table 10. Statistics on the lowest detection limits of each target in the multiplex digital PCR system of this kit.

[0066] Example 7 Repeatability test of multiplex digital PCR system Using a mixed sample of red-eared sliders and bullfrogs as a template, a multiplex digital PCR system was prepared for eight replicate tests at a single concentration. The experimental results were statistically analyzed, the coefficient of variation was calculated, and the repeatability of the method was evaluated.

[0067] The repeatability test results of multiplex digital PCR showed that the number of droplets was greater than 10,000, which met the experimental analysis requirements. (See attached results). Figure 9 As shown in Table 11, the coefficient of variation (CV) within the group for red-eared sliders was 1.737%, for bullfrogs it was 0.358%, and for the internal standard it was 0.175%, all less than 2%, indicating that the kit has good reproducibility.

[0068] Table 11 Statistical analysis of repeatability test results for each target in the multiplex digital PCR system of this kit.

[0069] Example 8 Comparison of singleton and multiplex digital PCR reaction systems Using a mixed sample of red-eared sliders and bullfrogs as templates, we conducted comparative tests on singlet and multiplet digital PCR systems for both red-eared sliders and bullfrogs. We compared the setpoint concentrations corresponding to the same template concentration, calculated the coefficient of variation of the setpoint results for the singlet and multiplet systems, and compared the consistency between singlet and multiplet digital PCR.

[0070] The results showed that the number of droplets was greater than 10,000, which met the requirements for experimental analysis. (See attached figures.) Figure 10 The coefficients of variation (CV) of the logarithmic values ​​of single- and multiplex PCR concentrations were both less than 2%, indicating that the single- and multiplex digital PCR reaction systems showed little difference in the results for the same sample, meeting the experimental requirements. The results are shown in Table 12.

[0071] Table 12 Statistical analysis of the comparison test results of single-particle and multiplex digital PCR systems of this kit

[0072] After designing and optimizing primers and probes for the two target species, red-eared sliders and bullfrogs, and a common internal standard, a triple digital PCR system including the internal standard was established through singlet fluorescent PCR and digital PCR tests on each target. After testing for specificity, linearity, limit of detection, repeatability, and comparison of singlet and multiplet digital PCR, the developed digital PCR kit for detecting the invasive species red-eared sliders and bullfrogs in the plateau region fully meets the development requirements.

Claims

1. A digital PCR kit for detecting the invasive species *Red-eared Slider* and *Bullfrog* from the plateau region, characterized in that... The kit comprises the following components, which work synergistically to form a triple digital PCR detection reaction system: A set of red-eared slider turtle-specific primer-probe pairs consists of primer pairs targeting a specific region of the red-eared slider turtle mitochondrial CYTB gene and matching red-eared slider turtle-specific TaqMan probes. A set of bullfrog-specific primer-probe pairs consists of primer pairs targeting a specific region of the bullfrog mitochondrial 12S rRNA gene and matching bullfrog-specific TaqMan probes. A set of internal standard primers and probes, designed for a specific region shared by the 16S rRNA genes of red-eared sliders and bullfrogs, are used to monitor the nucleic acid extraction and amplification reaction process; Digital PCR premix contains hot-start DNA polymerase, dNTPs, MgCl2, and a buffer system; And positive control samples and negative control samples; In this process, the nucleic acid of the sample to be tested is mixed with the digital PCR premix, red-eared slider turtle-specific primers and probes, bullfrog-specific primers and probes, and internal standard primers and probes, and then digital PCR is performed for partitioned amplification. The amplification status of the red-eared slider turtle target DNA, bullfrog target DNA, and internal standard sequence is determined by the endpoint fluorescence signals of the red-eared slider turtle-specific TaqMan probe, bullfrog-specific TaqMan probe, and internal standard probe, respectively.

2. The reagent kit according to claim 1, characterized in that: The red-eared slider-specific primer probe targets a specific segment of the red-eared slider mitochondrial cytochrome b (CYTB) gene.

3. The reagent kit according to claim 1, characterized in that: The bullfrog-specific primer probe targets a specific segment of the bullfrog mitochondrial 12S rRNA gene.

4. The kit according to claim 1, characterized in that: The red-eared slider turtle-specific TaqMan probe, the bullfrog-specific TaqMan probe, and the internal standard probe are each labeled with different fluorescent reporter groups.

5. The reagent kit according to claim 4, characterized in that: The fluorescent reporter group of the red-eared slider-specific TaqMan probe is FAM, the fluorescent reporter group of the bullfrog-specific TaqMan probe is ROX, and the fluorescent reporter group of the internal standard probe is HEX.

6. The reagent kit according to claim 1, characterized in that: The internal standard sequence corresponding to the internal standard primer probe is distinct from the target regions of the red-eared slider turtle and the bullfrog in terms of sequence origin, and the internal standard primer probe does not amplify the target regions of the red-eared slider turtle and the bullfrog.

7. The kit according to claim 1, characterized in that: The positive control is recombinant plasmid DNA containing red-eared slider target sequences and bullfrog target sequences and / or artificially synthesized nucleic acid fragments, and the negative control is nuclease-free water.

8. The reagent kit according to claim 1, characterized in that: The nucleic acid in the sample to be tested is derived from biological tissue samples of red-eared sliders or bullfrogs, and / or from environmental DNA extracted from environmental samples.

9. The digital PCR kit according to claim 8, characterized in that: The environmental samples include water samples, soil samples, and / or sediment samples. Water samples may be enriched first to improve the recovery rate of environmental DNA before DNA extraction; enrichment methods may include filtration enrichment, centrifugation enrichment, or other equivalent methods. Subsequently, a DNA extract solution suitable for PCR amplification is obtained according to the DNA extraction procedure.

10. The application of the digital PCR kit according to any one of claims 1 to 9 in the simultaneous identification and absolute quantitative detection of red-eared sliders and bullfrogs.

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