Specific primer combination for distinguishing enteromorpha from related species, application and molecular identification method thereof

Abstract

The present application relates to the technical field of molecular detection, and particularly relates to a specific primer combination for distinguishing Enteromorpha prolifera and related species, application and molecular identification method thereof. The primer combination comprises an upstream primer SEQ1 and a downstream primer SEQ2. The primer sequence of SEQ1 is shown as SEQ ID NO. 01, and the primer sequence of SEQ2 is shown as SEQ ID NO. 02. The primer combination of the present application is designed based on the genomic difference of two kinds of algae, has high specificity and sensitivity, can effectively avoid cross reaction, and ensures the accuracy of the detection result. Meanwhile, combined with the real-time fluorescent quantitative PCR (qPCR) technology, the method can effectively quantify the abundance of Enteromorpha prolifera in environmental samples or laboratory samples, and provides reliable technical support for early warning, tracing analysis and prevention and control strategy of green tide outbreak.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology, specifically to a specific primer combination for distinguishing Ulva prolifera from closely related species, its application, and a molecular identification method. Background Technology

[0002] Studies have shown that green tide outbreaks are mainly caused by *Ulva prolifera* (a type of seaweed). Ulva prolifera The unique physiological and ecological characteristics of *Ulva prolifera* enable it to proliferate rapidly and form large-scale biomass accumulations under specific environmental conditions. However, quantitative monitoring of *Ulva prolifera* biomass in environmental samples during green tides remains challenging. Traditional morphological identification methods are susceptible to sample fragmentation and interspecific morphological similarities (such as...). Ulva lentil Interference from closely related species (such as stigmata) leads to insufficient species resolution and quantitative accuracy.

[0003] Molecular detection techniques (such as quantitative polymerase chain reaction, qPCR) have become important tools for the quantitative monitoring of green tide species due to their high sensitivity. However, the 5S rRNA gene primers used in existing methods are difficult to effectively distinguish because they target conserved sequence regions. Ulva prolifera Closely related species with highly similar morphological and genetic characteristics Ulva lentil Studies have shown that the two species share extremely high sequence homology in the 5S rRNA gene amplification region, making it easy for conventional primers to induce non-specific amplification, resulting in cross-reactions and deviations in quantitative results. This deficiency not only limits the accurate assessment of *Ulva prolifera* biomass but also affects the understanding of the early occurrence mechanism of green tides and interspecific competition, thus hindering the improvement of disaster early warning and control efficiency. Summary of the Invention

[0004] To address the shortcomings of the existing technology, this invention provides a specific primer combination for distinguishing the 5S rRNA gene of *Ulva prolifera* from its closely related species *Ulva tragus* and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A specific primer combination for distinguishing Ulva prolifera from closely related species, comprising an upstream primer SEQ1 and a downstream primer SEQ2; the primer sequence of SEQ1 is shown in SEQ ID NO.01, with a sequence length of 18 bp, a melting temperature of 57.6℃, and a GC content of 55.6%; the primer sequence of SEQ2 is shown in SEQ ID NO.02, with a sequence length of 18 bp, a melting temperature of 58.6℃, and a GC content of 55.6%.

[0007] An application of a specific detection primer combination for distinguishing between *Ulva prolifera* and closely related species, wherein the primer combination is used to distinguish between *Ulva prolifera* and closely related species.

[0008] An application of the specific detection primer combination for distinguishing Ulva prolifera from closely related species, and the application of the primer combination in the monitoring and quantitative detection of Ulva prolifera.

[0009] A molecular identification method to distinguish Ulva prolifera from closely related species:

[0010] S1: Extract the genomic DNA to be tested as a template;

[0011] S2: Using the primer set SEQ1 and SEQ2 as primers, PCR amplification was performed to obtain PCR amplification products, and gel electrophoresis was performed to detect the products, thereby distinguishing Ulva prolifera from closely related species.

[0012] If the electrophoretic detection shows a DNA band with a molecular weight of 250 bp, it proves that the sample contains Ulva prolifera.

[0013] The DNA bands obtained from the above gel electrophoresis were cut and recovered, and the DNA fragments were ligated to the vector and transformed into competent cells. After the competent cells grew, the plasmids of the competent cells were extracted, and the plasmids were serially diluted as standards. The abundance of Ulva prolifera in the samples was quantified by qPCR.

[0014] The PCR reaction system for step (2) is as follows: 2 μL genomic DNA, 10 μL Taq Mix, 1 μL SEQ1 (10 μM), 1 μL SEQ2 (10 μM), 6 μL ddH2O, totaling 20 μL; the qPCR reaction system is as follows: 2 μL genomic DNA, 10 μL SYBR Premix ExTaq II, 0.4 μL ROX Reference Dye II, 0.4 μL SEQ1 (10 μM), 0.4 μL SEQ2 (10 μM), 6.8 μL ddH2O, totaling 20 μL.

[0015] The PCR and qPCR reaction procedures are as follows: Stage 1: 94℃ pre-denaturation for 5 min; Stage 2: 94℃ denaturation for 30 s, 57℃ annealing for 45 s, 72℃ extension for 30 s, for a total of 30 cycles; Stage 3: 72℃ extension for 10 min; Stage 4: 4℃ hold.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] This invention is based on seaweed (Ulva prolifera) Ulva prolifera The genomic characteristics of ) were used to screen for its relationship with closely related species ( Ulva lentil Specific genetic marker sites between the two species were identified, and specific primers SEQ1 and SEQ2 were designed accordingly. A molecular detection method for accurately distinguishing and quantifying the biomass of *Ulva prolifera* in environmental samples was established using a qPCR detection system, providing reliable technical support for early warning, source tracing, and prevention and control strategies for green tide outbreaks. Attached Figure Description

[0018] Figure 1 In Embodiment 2 of the present invention Ulva prolifera and Ulva lentil Figure showing the results of a specific amplification experiment.

[0019] Figure 2 This is a diagram showing the experimental results of environmental sample-specific amplification detection in Example 3 of the present invention.

[0020] Figure 3 This is a photograph of the recombinant cell plate culture in Example 4 of the present invention.

[0021] Figure 4 This is the standard curve of the standard plasmid in Example 5 of the present invention. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] This invention addresses the ambiguity and limitations of traditional morphological identification methods in species differentiation. The primer combination, designed based on genomic differences between two algae, exhibits high specificity and sensitivity, effectively avoiding cross-reactivity and ensuring accurate detection results. Furthermore, combined with real-time quantitative PCR (qPCR) technology, this method can effectively quantify the abundance of *Ulva prolifera* in environmental or laboratory samples, providing reliable technical support for early warning, source tracing, and control strategy development for green tide outbreaks.

[0024] The materials used in the following embodiments are mainly as follows:

[0025] LB medium consisted of 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and deionized water to a final volume of 1000 mL, pH 7.0.

[0026] Blunt Zero Gene Cloning Kit: Purchased from Beijing TransGen Biotech Co., Ltd.

[0027] DH5α competent cells: purchased from Novizan.

[0028] FastPure Gel DNA Extraction Mini Kit: Purchased from Novizan.

[0029] Dzup (Plant) Genomic DNA Isolation Reagent: Purchased from Sangon Biotech.

[0030] Example 1: Differentiating Ulva prolifera ( Ulva prolifera ) and Enteromorpha marginata ( Ulva lentil Obtaining primer combinations for specific detection of the 5S rRNA gene

[0031] Seven 5S rRNA gene sequences from *Ulva prolifera* and four 5S rRNA gene sequences from *Ulva tubiflora* were selected from NCBI for comparative analysis. Specific primer combinations were designed based on unique fragments in the *Ulva prolifera* 5S rRNA gene sequences; these primer combinations are the upstream primer SEQ1 and the downstream primer SEQ2. Specifically, the primer sequence for SEQ1 is TGCATCTGTGTATCGCGG (SEQ ID NO. 01), with a sequence length of 18 bp, a melting temperature of 57.6℃, and a GC content of 55.6%; the primer sequence for SEQ2 is ATTGCGGGGGAATGGTCT (SEQ ID NO. 02), with a sequence length of 18 bp, a melting temperature of 58.6℃, and a GC content of 55.6%.

[0032] The NCBI accession numbers for the seven 5S rRNA gene sequences of *Ulva prolifera* are HM031149.1, HM584780.1, HM584784.1, HM584783.1, HM031150.1, HM031152.1, and HM031139.1, respectively; and the NCBI accession numbers for the four 5S rRNA gene sequences of *Ulva prolifera* are AB298685.1, AB299441.1, HM584771.1, and HM031140.1, respectively.

[0033] Example 2: Ulva prolifera and Ulva lentil Specific amplification experiment

[0034] S1: Extract the genomic DNA to be tested as a template. Prepare fresh DNA 3-5 cm long. Ulva prolifera and Ulva lentil Genomic DNA was extracted from both samples using Dzup (Plant) Genomic DNA Isolation Reagent, following the instructions in the reagent manual.

[0035] S2: Using SEQ1 and SEQ2 as primers, PCR amplification was performed to obtain the PCR amplification products. The PCR reaction system was as follows: 2 μL genomic DNA, 10 μL Taq Mix, 1 μL SEQ1 (10 μM), 1 μL SEQ2 (10 μM), 6 μL ddH2O, totaling 20 μL. The PCR reaction program was as follows: Stage 1: 94℃ pre-denaturation for 5 min; Stage 2: 94℃ denaturation for 30 s, 57℃ annealing for 45 s, 72℃ extension for 30 s, for a total of 30 cycles; Stage 3: 72℃ extension for 10 min; Stage 4: 4℃ hold.

[0036] S3: Electrophoresis detection of PCR amplification results. The specific method is as follows: Prepare 1% agarose gel with 1×TAE, heat it completely in a microwave oven, let it cool slightly to 45-55℃, add nucleic acid dye at a ratio of 1:10000 and shake well, then pour it into the gel tank with comb teeth inserted. After the gel has completely solidified, remove the comb teeth, take 4μL of PCR amplification product and add it to the sample well, electrophoresis at 120V for 25min, and take pictures with a gel imaging device after electrophoresis. Ulva prolifera After amplification, the genomic DNA shows obvious bands, but not visible bands. Ulva lentil The bands (see) Figure 1 ).

[0037] Example 3: Environmental Sample Specific Amplification Detection Experiment

[0038] S1: Take a seawater sample collected in the field and filter 2L of the seawater sample through a polycarbonate filter membrane with a pore size of 0.2μm. After filtration, quickly freeze the filter membrane with liquid nitrogen.

[0039] S2: Grind the quick-frozen filter membrane into powder in a mortar, and then extract the genomic DNA on the filter membrane using Dzup (Plant) Genomic DNA Isolation Reagent. Refer to the reagent instructions for the extraction steps.

[0040] S3: Using SEQ1 and SEQ2 as primers, PCR amplification was performed to obtain the PCR amplification products. The PCR reaction system and amplification procedure are described in Example 2.

[0041] S4: Electrophoresis is used to detect the PCR amplification results. The detection method is described in Example 2.

[0042] After amplification, clear bands were observed in the environmental DNA, demonstrating that the specific detection primer combination described in this invention can effectively and specifically amplify the DNA in environmental samples. Ulva prolifera Genes (see) Figure 2 ).

[0043] Example 4: Construction of qPCR standard strain experiment

[0044] S1: PCR amplification of the target gene. Using SEQ1 and SEQ2 as primer pairs. Ulva prolifera The genomic DNA was amplified by PCR. The PCR reaction system and amplification procedure are described in Example 2.

[0045] S2: Gel recovery. The target band was recovered using the FastPure Gel DNA Extraction Mini Kit according to the instructions to obtain the target DNA fragment.

[0046] S3: Constructing the recombinant plasmid. The cloning reaction system was constructed using the Blunt Zero Gene Cloning Kit. The cloning system consisted of the target DNA fragment (4 μL) and pEASY®-Blunt Zero Cloning Vector (1 μL), mixed and incubated at 37°C for 10 min to obtain the recombinant plasmid. After the reaction, the centrifuge tubes were placed on ice.

[0047] S4: Obtaining Recombinant Cells. Add 1 μL of recombinant plasmid to DH5α competent cells that have been thawed on ice beforehand. Incubate on ice for 20 min, then heat-shock in a 42°C water bath for 45 s, followed by cooling on ice for 5 min. Transfer to antibiotic-free LB liquid medium and incubate at 37°C and 200 rpm for 1 h. After incubation, plate onto LB solid medium containing kanamycin (50 μg / mL) and incubate at 37°C for 12 h to obtain recombinant cells (see [link to article]). Figure 3 Finally, eight recombinant cells were preserved (namely H1, H2, H3, H4, H5, H6, S1, and S2).

[0048] Example 5: Absolute Quantification Experiment of Ulva Abundance in Environmental Samples and Laboratory Cultured Samples

[0049] S1: Take 2L of seawater samples collected in the field and seawater from seaweed cultured in the laboratory, and filter them through a polycarbonate membrane with a pore size of 0.2μm. After filtration, flash freeze the membrane with liquid nitrogen.

[0050] S2: Grind the quick-frozen filter membrane into powder in a mortar, and then extract the genomic DNA on the filter membrane using Dzup (Plant) Genomic DNA Isolation Reagent. Refer to the reagent instructions for the extraction steps.

[0051] S3: The eight recombinant cells obtained in Example 4 were seeded into 2 mL of LB liquid medium containing kanamycin (50 μg / mL) and incubated at 37°C and 200 rpm for 12 h. The plasmids in the bacterial culture were then extracted using the Omega Plasmid Mini Kit I (EZNA® Plasmid Mini Kit I D6943-02), resulting in a total of eight plasmids.

[0052] S4: qPCR was performed using the extracted DNA as a template, and the plasmids from the 8 recombinant cells were subjected to 10... -1 Up to 10 -8 Standard curves were plotted by serial dilution and quantification to verify the feasibility of qPCR.

[0053] The qPCR reaction system consisted of: 2 μL genomic DNA, 10 μL SYBR Premix Ex Taq II, 0.4 μL ROX Reference Dye II, 0.4 μL SEQ1 (10 μM), 0.4 μL SEQ2 (10 μM), and 6.8 μL ddH2O, totaling 20 μL. The qPCR reaction program was as follows: Stage 1: 94℃ pre-denaturation for 5 min; Stage 2: 94℃ denaturation for 30 s, 57℃ annealing for 45 s, and 72℃ extension for 30 s, for a total of 30 cycles; Stage 3: 72℃ extension for 10 min; Stage 4: hold at 4℃.

[0054] S5: Calculate the absolute quantification results of qPCR. Calculate the plasmid copy number using Formula 1. Establish a standard curve by comparing the lg value of the plasmid copy number from each serial dilution with the corresponding CT value determined by qPCR. A total of 8 standard curves were obtained (see [link to standard curve]). Figure 4 The R² values ​​of the standard curves were all above 0.99. Amplification efficiency was calculated using Formula 2, and the amplification efficiencies of all eight plasmids were between 95% and 105%, indicating good amplification efficiency and demonstrating that all eight plasmids could be effectively used in qPCR experiments. The CT values ​​obtained from qPCR of environmental and laboratory samples were compared with the H1 standard curve, revealing that the copy number of the *Ulva prolifera* gene in the two field environmental samples was 7.43 × 10⁻⁶. 6 copies / L and 8.45×10 6 The copy number of the *Ulva prolifera* gene in a laboratory culture sample was 1.95 × 10⁻⁶ copies / L. 8 copies / L.

[0055] Formula 1 C = (6.02 × C0 × 10) 14 ) / M

[0056] C: Plasmid copy number (copies / μL)

[0057] C0: Plasmid concentration (ng / μL)

[0058] M: Molecular weight of plasmid, M = (number of plasmid bases + number of target fragment bases) × 660.

[0059] Formula 2 T = 10 -1 / k - 1

[0060] T: Amplification efficiency

[0061] k: Slope of the standard curve.

[0062] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can make modifications or equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. The use of a specific primer combination for distinguishing Enteromorpha from related species, characterized in that: The primer combination is applied to the monitoring and quantitative detection of Enteromorpha in seawater. The primer combination comprises an upstream primer SEQ1 and a downstream primer SEQ2; the primer sequence of SEQ1 is shown in SEQ ID NO. 01, and the primer sequence of SEQ2 is shown in SEQ ID NO.

02.

2. A molecular identification method for distinguishing Enteromorpha from related species, characterized in that: S1: extracting the genomic DNA to be tested as a template; S2: using the specific primer combination SEQ1 and SEQ2 for distinguishing Enteromorpha from related species according to claim 1 as primers, adopting a PCR amplification method to obtain a PCR amplification product, and performing gel electrophoresis detection by an electrophoresis detection method, so as to distinguish Enteromorpha from related species; If the electrophoresis detection exists a DNA band with a molecular weight of 250 bp, it is proved that the sample to be detected contains Enteromorpha.

3. The method of molecular identification of claim 2, wherein: The DNA band of the gel electrophoresis is cut and recovered, the DNA fragment is connected with a vector, and is transformed into a competent cell; after the growth of the competent cell, the plasmid of the competent cell is extracted, the plasmid is used as a standard product to perform gradient dilution, and the abundance of Enteromorpha in the environmental sample is quantified by qPCR.

4. The method of molecular identification of claim 3, wherein: The PCR reaction system in S2 is 2 μL of genomic DNA, 10 μL of Taq Mix, 1 μL of 10 μM SEQ1, 1 μL of 10 μM SEQ2, 6 μL of ddH2O, and a total of 20 μL; the qPCR reaction system is 2 μL of genomic DNA, 10 μL of SYBR Premix Ex Taq II, 0.4 μL of ROX Reference Dye II, 0.4 μL of 10 μM SEQ1, 0.4 μL of 10 μM SEQ2, 6.8 μL of ddH2O, and a total of 20 μL.

5. The method of molecular identification according to claim 3 or 4, characterized in that: The PCR and qPCR reaction programs are as follows: stage 1: 94℃ pre-denaturation for 5 min; stage 2: 94℃ denaturation for 30 s, 57℃ annealing for 45 s, 72℃ extension for 30 s, a total of 30 cycles; stage 3: 72℃ extension for 10 min; and stage 4: 4℃ keeping.

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