Environmental DNA (Deoxyribose Nucleic Acid) sequencing primer aiming at Phyla amyloliquefaciens

Abstract

The invention discloses an environmental DNA (deoxyribonucleic acid) sequencing primer and a fluorescent quantitative PCR (polymerase chain reaction) detection kit aiming at phylum paragliflora, the primer comprises an upstream primer 28SF and a downstream primer 28SR, the sequence of the 28SF is 5 '-GCGAGYGAASDGRGADNAGCCCA-3', the sequence of the 28SR is 5 '-CTCTYTTCARAGTHCTTKC-3', and the size of a target fragment is 305bp. According to the invention, a pair of broad-spectrum PCR primers which are suitable for a plurality of types of phylum paragliflora and can synchronously detect a plurality of species is designed for the first time, the problems of lack of universal phylum paragliflora sequencing primers and high detection cost in the prior art are solved, the detection operation is simple, the specificity is strong, and the application range is wide. The method is suitable for the fields of field water monitoring, aquaculture, biodiversity protection and the like, can realize rapid screening of the species of the phylum amyloliquefaciens, such as epidemiological investigation and prevention and control effect evaluation of parasitic diseases represented by flukes, tapeworms and the like, and has important practical application value.

Description

Technical Field

[0001] This invention relates to the field of aquatic environmental biological monitoring, specifically to a universal sequencing primer for environmental DNA of flatworms and a matching PCR detection kit. Background Technology

[0002] Flatworms encompass multiple groups, including planarians, trematodes, and tapeworms. Some of these groups are parasitic, with a wide host range, including fish, birds, and mammals. These parasitic groups can cause severe economic losses to aquaculture and livestock farming, and some species can also infect humans, affecting public health and safety.

[0003] Currently, relevant detection mainly relies on morphological identification and group-specific molecular markers. Morphological methods require professional operation and are easily affected by subjective human factors; existing molecular detection methods mostly design primers based on specific or limited species, lacking universal environmental DNA detection primers that can cover multiple groups of flatworms. This leads to the need to use multiple sets of primers for multiple tests during large-scale screening, resulting in high costs and low efficiency, which restricts the development of related monitoring and prevention work.

[0004] Therefore, developing a broad-spectrum, efficient, and low-cost PCR primer and kit suitable for environmental DNA detection in flatworms is of great technical significance for the rapid screening and monitoring of flatworms, and can also provide basic tool support for the early warning of parasitic species. Summary of the Invention

[0005] To address the aforementioned problems in the existing technology, this invention provides environmental DNA sequencing primers and PCR detection kits for flatworms.

[0006] To achieve the above-mentioned technical objectives and effects, the present invention is implemented through the following technical solution:

[0007] A sequencing primer for flatworms, comprising an upstream primer 28SF and a downstream primer 28SR;

[0008] The sequence of the upstream primer 28SF is as follows:

[0009] 5'-GCGAGYGAASDGRGADNAGCCCA-3';

[0010] The sequence of the downstream primer 28SR is as follows:

[0011] 5'-CTCTYTTCARAGTHCTTTKC-3'.

[0012] Preferably, the molar ratio of the upstream primer 28SF to the downstream primer 28SR is 1:1.

[0013] A real-time PCR detection kit for flatworms includes the following components:

[0014] Components Working fluid concentration / specification Sample loading per 20 μL reaction system Upstream primer 28SF 5μM 0.8μL Downstream primer 28SR 5μM 0.8μL reaction solution 2×Pro Taq 10μL Ultrapure water <![CDATA[ddH2O]]> Make up to 20 μL Positive control <![CDATA[1×10 6 Copy / μL plasmid]]> 1μL (add as needed) negative control <![CDATA[Sterile ddH2O]]> 1μL (add as needed)

[0015] The positive control was a recombinant plasmid containing a 305bp target gene fragment, used to verify the effectiveness of the PCR reaction system; the negative control was sterile ddH2O, used to eliminate contamination interference.

[0016] A method for detecting environmental DNA in flatworms includes the following steps:

[0017] DNA template preparation: Take the sample to be tested, extract genomic DNA using the conventional phenol-chloroform method or a DNA extraction kit, determine the concentration using a nucleic acid quantification instrument, adjust to 10 ng / μL, and store at -20℃ for later use;

[0018] PCR amplification: Perform programmed temperature cycling on the PCR instrument according to the reaction conditions parameters in the table above to ensure specific amplification of the DNA template.

[0019] Reaction system preparation: Add each component in sequence according to the proportions in the table above, vortex mix well, and then briefly centrifuge to avoid the generation of bubbles;

[0020] Reaction conditions were set using ABI GeneAmp. ® The reaction program for the 9700 PCR instrument was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, 35 cycles for routine samples, 40 cycles for low concentration samples; 72℃ final extension for 10 min, and incubation at 10℃.

[0021] Results identification: Agarose gel electrophoresis was used to detect the quality of the amplified products, verifying whether the fragment size was consistent with the target region and whether there were any non-specific bands.

[0022] Electrophoresis detection: Prepare a 2% agarose gel, mix 3 μL of PCR product with the loading buffer, load the sample, electrophoresis at 120V for 20-30 min, and observe the results using a gel imaging system.

[0023] Result interpretation: If a specific band of 305 bp appears, and the positive control has a band while the negative control has no band, the result is considered positive; if there is no specific band, the result is considered negative; if the positive control has no band or the negative control has a band, it indicates that the reaction system is abnormal and retesting is required.

[0024] Preferably, the samples to be tested include, but are not limited to, aquatic DNA samples, parasite samples, fish or mammalian host tissues and fecal samples.

[0025] The beneficial effects of this invention are:

[0026] A primer for environmental DNA sequencing and a real-time PCR detection kit for flatworms;

[0027] The primers of this invention are designed for the conserved 28S rRNA gene of flatworms and can cover multiple groups such as planarians, monogenean trematodes, digenean trematodes, and tapeworms. This solves the problem of narrow detection range of existing primers, eliminates the need for multiple primer changes, and reduces detection costs.

[0028] The PCR reaction system is simple to configure, the reaction conditions are easy to control, and the electrophoresis detection results are intuitive, making it suitable for rapid and efficient detection of environmental samples such as water samples and fish tissue samples collected in the field.

[0029] The primers are highly specific and sensitive, the kit is stable, and the detection results are accurate. It can effectively distinguish between flatworm species and non-target organisms, avoiding false positive and false negative results.

[0030] This method is applicable to fields such as field water monitoring, aquaculture, and biodiversity conservation. It can enable rapid screening of species in the phylum Platyhelminthes, such as epidemiological investigations and evaluation of control effects for parasitic diseases represented by trematodes and tapeworms, and has important practical application value.

[0031] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time. Attached Figure Description

[0032] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 PCR products were detected by 2% agarose gel electrophoresis; 3 μl of sample was loaded and the electrophoresis image was detected.

[0034] Figure 2 DL2000 stripe distribution diagram;

[0035] Figure 3 Electrophoresis results of a small-batch sample validation experiment;

[0036] Figure 4 Electrophoresis results of a single-batch, dual-sample comparison experiment;

[0037] Figure 5 Electrophoresis results of a large-scale sample verification experiment. Detailed Implementation

[0038] The technical solutions of the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit this disclosure or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this disclosure without creative effort are within the scope of protection of this disclosure.

[0039] Example 1: Primer Design and Screening

[0040] Primer design: Gene sequences of different groups of flatworms, including monogenean trematodes, digenean trematodes, and tapeworms, were downloaded from GenBank. Homologous sequences of non-flatworm organisms, including bacteria, fungi, and vertebrates, were also downloaded as references. Clustal X was used for sequence alignment to screen for conserved regions specific to flatworms.

[0041] Primer screening: Three sets of candidate primers were designed using primer design software, and the optimal primers were selected based on the following criteria:

[0042] The amplification product length is between 200-400 bp, which facilitates electrophoresis detection;

[0043] The GC content is between 40% and 60%, and the Tm value is close to 55℃;

[0044] It lacks secondary structures such as self-complementation and primer dimers;

[0045] The sequence similarity with non-target organisms is less than 70%.

[0046] Screening results: The 28SF / 28SR primer combination was finally determined to be the optimal primer combination. Its amplification products are single and without impurities. The amplification efficiency for different groups of flatworms is between 90% and 110%, and the specificity is the best.

[0047] Example 2: Optimization of PCR reaction conditions

[0048] Annealing temperature optimization: Five annealing temperature gradients were set at 50℃, 52℃, 55℃, 58℃, and 60℃, and PCR amplification was performed using flatworm DNA as a template. The results showed that the amplified band was brightest and there were no impurities at 55℃, thus 55℃ was determined to be the optimal annealing temperature.

[0049] Cycle number optimization: Four cycle number gradients of 30, 35, 40, and 45 were set for amplification of different template concentrations (10 ng / μL, 1 ng / μL, and 0.1 ng / μL). The results showed that when the template concentration was ≥1 ng / μL, a clear band was obtained after 35 cycles; when the template concentration was 0.1 ng / μL, a clear band was obtained after 40 cycles. Therefore, 35 cycles were used for routine detection, and 40 cycles were used for low-concentration samples.

[0050] Example 3: Reagent Kit Performance Validation

[0051] Specificity validation: The kit was used to detect DNA samples from both flatworm and non-flatworm species. Results showed that all flatworm samples amplified a specific 305 bp band, while no band was observed in non-flatworm samples, indicating high specificity of the kit.

[0052] Stability verification: After storing the kit at -20℃ for 3 months and 4 months, the same batch of positive samples were tested. The results showed that clear specific bands were amplified in both cases, with no significant difference, indicating that the kit has good stability.

[0053] Actual sample testing: Eighteen water samples were collected from different environments and tested using both the kit and traditional morphological methods. Results showed that both the kit and the morphological methods detected 18 positive samples. The kit demonstrated a high detection rate, while also reducing testing time and labor costs, resulting in a significant improvement in testing efficiency.

[0054] Example 4: Validation Experiment of Small-Batch Sample Amplification Effectiveness

[0055] Experimental Objective

[0056] The study aimed to verify the amplification effectiveness of 28SF / 28SR primers on small batches of samples from different sources under 35 cycles, clarify the influencing factors of sample amplification under low cycle numbers, and provide a basis for subsequent optimization of detection conditions.

[0057] Experimental samples

[0058] Three relevant samples were selected and numbered A20, A21, and A22, corresponding to gel image numbers 10, 11, and 12. The samples were obtained from the filter membranes obtained after filtration of water samples from river environments in the wild. After routine processing, genomic DNA was extracted and the concentration was adjusted to 10 ng / μL.

[0059] PCR reaction system and conditions

[0060] Reaction system: A 20-μL system was used, containing 10 μL of 2×Pro Taq, 0.8 μL of upstream primer 28SF (5 μM), 0.8 μL of downstream primer 28SR (5 μM), 10 ng / μl of template DNA, and ddH2O was added to make up to 20 μL.

[0061] Reaction conditions: An ABI GeneAmp ® Model 9700 PCR instrument was used. Pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s, for a total of 35 cycles; final extension at 72°C for 10 min, and incubation at 10°C until detection.

[0062] Result detection

[0063] The PCR products were detected by 2% agarose gel electrophoresis. 3 μL of the products were loaded, and with DL2000 Marker as a reference, the band situation was observed through a gel imaging system.

[0064] Experimental results

[0065] Sample A20: A clear specific band of 305 bp appeared in the electrophoresis pattern, without any non-specific bands, meeting the size requirement of the target product, and was judged as qualified.

[0066] Sample A21: No obvious band was detected in the electrophoresis pattern, and there was no amplification of the target product, so it was judged as unqualified.

[0067] Sample A22: No obvious band was detected in the electrophoresis pattern, and there was no amplification of the target product, so it was judged as unqualified.

[0068] Samples A21 and A22 were subjected to secondary extraction and detection, and the number of cycles was changed to 40. A clear specific band of 305 bp appeared in the electrophoresis pattern, without any non-specific bands, meeting the size requirement of the target product, and was judged as qualified.

[0069] Result analysis

[0070] The verification results of small-batch samples showed that under the condition of 35 cycles, only 1 sample (A20) was amplified successfully, and 2 samples failed to amplify. After increasing the number of cycles and performing secondary extraction and detection, it was successful. It was speculated that the reason might be that the template concentration of the samples was too low or the DNA purity was insufficient, resulting in ineffective amplification at low cycle numbers. This result indicates that in routine detection, the number of cycles needs to be adjusted according to the actual situation of the samples. For suspected low-concentration samples, the number of cycles can be increased to 40 to improve the amplification success rate.

[0071] Example 5: Single-batch double-sample comparative verification experiment

[0072] Experimental purpose

[0073] By comparing the amplification results of two fish intestinal contents samples, the influence of sample characteristics (such as parasite infectivity and template quality) on the PCR amplification results was analyzed, and the amplification specificity and detection stability of the primers were further verified.

[0074] Experimental Materials and Methods

[0075] Experimental samples

[0076] Two samples of fish gastrointestinal contents, numbered A9 and A10 and corresponding to gel images 9 and 10, were collected from the same aquatic environment sampling point and from the same sampling batch. Genomic DNA was extracted from both samples using the same method. Sample A9 was a high-concentration sample, while sample A10 was a low-concentration sample.

[0077] PCR reaction system and conditions

[0078] Reaction system: Same as in Example 4, 20 μL system contains 10 μL of 2×Pro Taq, 0.8 μL each of forward and reverse primers (5 μM), 1 μL of template DNA, and ddH2O added to 20 μL.

[0079] Reaction conditions: ABI GeneAmp ® 9700 PCR instrument, 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, 35 cycles; 72℃ final extension for 10 min, 10℃ incubation.

[0080] Result detection

[0081] The presence and clarity of the target bands were observed using 2% agarose gel electrophoresis with a DL2000 marker as a molecular weight reference.

[0082] Experimental results

[0083] A9 sample: A clear, single 305bp specific band appears in the electrophoresis image, without any extraneous bands, and is judged to be qualified.

[0084] Sample A10: No bands were detected in the electrophoresis image, and no target product was amplified, so it was judged to be unqualified.

[0085] Results Analysis

[0086] Comparison of two samples from the same batch showed that A9 amplification was successful, while A10 amplification was unsuccessful, with both having a cycle number of 35. This result validates the hypothesis in Example 4 that low concentrations might lead to amplification failure.

[0087] Example 6: Stability Verification Experiment for Large-Scale Sample Amplification

[0088] Experimental Objective

[0089] To verify the stability and versatility of the 28SF / 28SR primers and optimized PCR conditions (55℃ annealing, 35 cycles) in large-scale sample detection, and to evaluate the feasibility of the kit for large-scale field screening.

[0090] Experimental Materials and Methods

[0091] Experimental samples

[0092] Fifteen bioaccumulation samples from different aquatic environments were selected, numbered A90-A104, corresponding to gel image numbers 61-75. Genomic DNA was extracted using a standardized method and the concentration was adjusted to 10 ng / μL.

[0093] PCR reaction system and conditions

[0094] Reaction system: 20 μL system containing 10 μL of 2×Pro Taq, 0.8 μL of upstream primer 28SF (5 μM), 0.8 μL of downstream primer 28SR (5 μM), 1 μL of template DNA, and ddH2O added to 20 μL.

[0095] Reaction conditions: ABI GeneAmp ® 9700 PCR instrument, 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, 35 cycles; 72℃ final extension for 10 min, 10℃ incubation.

[0096] Result detection

[0097] The bands of each sample were detected by 2% agarose gel electrophoresis with DL2000 Marker as a reference. The bands of each sample were observed and recorded using a gel imaging system. The criterion for judgment was whether a specific band appeared.

[0098] Experimental results

[0099] After PCR amplification, all 15 samples (A90-A104) showed clear, single 305bp specific bands in the electrophoresis images, with no extraneous bands or false positive amplifications. All samples were deemed qualified.

[0100] Results Analysis

[0101] Large-scale sample validation results showed that the 28SF / 28SR primers could effectively amplify 15 samples from different sources under 35 cycles and annealing at 55℃, with a success rate of 100%. This result demonstrates the primers' strong versatility and stability, enabling them to adapt to the detection needs of different aquatic environments and meet the application scenarios of large-scale on-site screening, providing a solid experimental basis for the industrialization and promotion of the kit.

[0102] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0103] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A primer for environmental DNA sequencing targeting flatworms, characterized in that: Including upstream primer 28SF and downstream primer 28SR; The sequence of the upstream primer 28SF is as follows: 5'-GCGAGYGAASDGRGADNAGCCCA-3'; The sequence of the downstream primer 28SR is as follows: 5'-CTCTYTTCARAGTHCTTTKC-3'.

2. The environmental DNA sequencing primers for flatworms as described in claim 1, characterized in that: The molar ratio of the upstream primer 28SF to the downstream primer 28SR is 1:1, and the working solution concentration of both the upstream primer 28SF and the downstream primer 28SR is 5 μM.

3. A real-time PCR detection kit for environmental DNA sequencing primers targeting flatworms, characterized in that: Includes the following components: Primer solution: Contains upstream primer 28SF and downstream primer 28SR as described in claim 1, with a working solution concentration of 5 μM for both. Reaction solution: 2×Pro Taq; Ultrapure water: ddH2O; Positive control: Plasmid containing a target gene fragment from a species of flatworm, at a concentration of 1 × 10⁻⁶. 6 Copy / μL; Negative control: sterile ddH2O.

4. The real-time PCR detection kit as described in claim 3, characterized in that: The target gene fragment sequence of the positive control corresponds to the amplification product of primers 28SF / 28SR, which is 305 bp in length.

5. A method for detecting species of flatworms, characterized in that, The detection using the kit described in claim 3 includes the following steps: DNA template preparation: Take the sample to be tested, extract genomic DNA using conventional DNA extraction methods, and adjust the concentration to 10 ng / μL for later use; PCR amplification: Prepare a 20 μL PCR reaction system, including 10 μL of 2×Pro Taq, 0.8 μL of upstream primer 28SF, 0.8 μL of downstream primer 28SR, 10 ng / μL of template DNA, and add ddH2O to a final volume of 20 μL; PCR reaction conditions are as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for 35-40 cycles; 72℃ final extension for 10 min, and incubation at 10℃ until detection; Results identification: PCR products were detected by 2% agarose gel electrophoresis. If a specific band appeared, the sample was determined to contain a species of flatworm; if no specific band appeared, the sample was determined to be negative.

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