Specific primer combination for distinguishing enteromorpha and sibling species, application of specific primer combination and molecular identification method

Abstract

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

Description

Technical Field

[0001] The present invention relates to the technical field of molecular detection, and in particular to a specific primer combination for distinguishing Enteromorpha from closely related species, and an application thereof and a molecular identification method. Background Art

[0002] Studies have shown that the species causing green tide outbreaks is Enteromorpha ( Ulva prolifera ) are the main species, and their unique physiological and ecological characteristics enable them to rapidly proliferate and form large-scale biomass accumulation under specific environmental conditions. However, during the occurrence of green tides, quantitative monitoring of the biomass of Enteromorpha in environmental samples still faces challenges. Traditional morphological identification methods are easily affected by sample fragmentation, morphological similarity between species (such as Ulva lentil The interference of closely related species (such as ect.) leads to insufficient species resolution and quantitative accuracy.

[0003] Molecular detection techniques (such as quantitative polymerase chain reaction, qPCR) have become important tools for 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 sequences of the two species share extremely high homology in the amplified region of the 5S rRNA gene, making conventional primers prone to nonspecific amplification, resulting in cross-reactions and biased quantitative results. This defect not only limits the accurate assessment of Enteromorpha biomass, but also affects the analysis of the early occurrence mechanism of green tides and interspecies competition relationships, restricting the improvement of disaster warning and prevention and control efficiency. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a specific detection primer combination for distinguishing the 5SrRNA gene of Enteromorpha and its closely related species Enteromorpha serrata and its application.

[0005] To achieve the above object, the technical solution adopted by the present invention is: A specific detection primer combination for distinguishing Enteromorpha from closely related species, the primer combination 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°C, 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°C, and a GC content of 55.6%.

[0006] An application of the specific detection primer combination for distinguishing Enteromorpha from closely related species, and an application of the primer combination in distinguishing Enteromorpha from closely related species.

[0007] An application of the specific detection primer combination for distinguishing Enteromorpha from closely related species, and an application of the primer combination in monitoring and quantitative detection of Enteromorpha.

[0008] A molecular identification method for distinguishing Enteromorpha from closely related species: S1: Extract the genomic DNA to be tested as a template; S2: Using the primer set SEQ1 and SEQ2 as primers, adopting a PCR amplification method to obtain a PCR amplification product, and performing gel electrophoresis detection using an electrophoresis detection method to distinguish Enteromorpha from closely related species.

[0009] If a DNA band with a molecular weight of 250 bp is present in the electrophoresis test, it is proved that the sample to be tested contains Enteromorpha.

[0010] The DNA bands from the gel electrophoresis were excised and recovered, and the DNA fragments were connected to the vector and transformed into competent cells. After the competent cells grew, the plasmids of the competent cells were extracted, and gradient dilutions were performed using the plasmids as standards. The abundance of Enteromorpha in the samples was quantified by qPCR.

[0011] The PCR reaction system in step (2) is: 2 μL of genomic DNA, 10 μL of Taq Mix, 1 μL of SEQ1 (10 μM), 1 μL of SEQ2 (10 μM), and 6 μL of ddH2O, for a total of 20 μL. The qPCR reaction system is: 2 μL of genomic DNA, 10 μL of SYBR Premix ExTaq Ⅱ, 0.4 μL of ROX Reference Dye Ⅱ, 0.4 μL of SEQ1 (10 μM), 0.4 μL of SEQ2 (10 μM), and 6.8 μL of ddH2O, for a total of 20 μL.

[0012] The PCR and qPCR reaction procedures were as follows: stage 1: pre-denaturation at 94°C for 5 min; stage 2: denaturation at 94°C for 30 s, annealing at 57°C for 45 s, and extension at 72°C for 30 s, for a total of 30 cycles; stage 3: extension at 72°C for 10 min; and stage 4: hold at 4°C.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention is based on Enteromorpha ( Ulva prolifera ) and screened its genomic characteristics with closely related species ( Ulva lentil ) and designed specific primers SEQ1 and SEQ2 accordingly. Using a qPCR detection system, a molecular detection method was established that can accurately distinguish and quantify Enteromorpha biomass in environmental samples, providing reliable technical support for early warning, source identification, and prevention and control strategy development of green tide outbreaks. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 In Example 2 of the present invention Ulva prolifera and Ulva lentil Specific amplification experiment results.

[0015] Figure 2 This is a graph showing the results of the environmental sample-specific amplification detection experiment in Example 3 of the present invention.

[0016] Figure 3 This is a photograph of the recombinant cell plate culture in Example 4 of the present invention.

[0017] Figure 4 This is the standard curve of the standard plasmid in Example 5 of the present invention. DETAILED DESCRIPTION

[0018] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0019] This method addresses the ambiguity and limitations of traditional morphological identification methods in species differentiation. Designed based on the genomic differences between the two algae species, this primer combination offers high specificity and sensitivity, effectively avoiding cross-reactions and ensuring accurate detection results. Furthermore, combined with real-time quantitative PCR (qPCR), this method can effectively quantify the abundance of Enteromorpha in environmental or laboratory samples, providing reliable technical support for early warning, source identification, and prevention and control strategies for green tide outbreaks.

[0020] In the following embodiments, the materials used are mainly as follows: LB medium was prepared as follows: 10 g tryptone, 5 g yeast powder, 10 g sodium chloride, and dilute to 1000 mL with deionized water, pH 7.0.

[0021] Blunt Zero gene cloning kit: purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0022] DH5α competent cells were purchased from Novozymes.

[0023] FastPure Gel DNA Extraction Mini Kit DNA gel recovery kit: purchased from Novozymes.

[0024] Dzup (Plant) Genomic DNA Isolation Reagent: purchased from Sangon Biotechnology Co., Ltd.

[0025] Example 1: Differentiation of Enteromorpha ( Ulva prolifera ) and Enteromorpha prolifera ( Ulva lentil ) Obtaining a specific primer combination for 5S rRNA gene detection Seven 5S rRNA gene sequences from Enteromorpha enteromorpha and four 5S rRNA gene sequences from Enteromorpha fringei were selected from NCBI for alignment analysis. A specific primer combination was designed based on unique fragments in the Enteromorpha 5S rRNA gene sequences: upstream primer SEQ1 and downstream primer SEQ2. The primer sequence for SEQ1 is TGCATCTGTGTATCGCGG (SEQ ID NO. 01), with an 18-bp sequence length, a melting temperature of 57.6°C, and a GC content of 55.6%. The primer sequence for SEQ2 is ATTGCGGGGGAATGGTCT (SEQ ID NO. 02), with an 18-bp sequence length, a melting temperature of 58.6°C, and a GC content of 55.6%.

[0026] The NCBI accession numbers of the seven 5S rRNA gene sequences of the Enteromorpha are HM031149.1, HM584780.1, HM584784.1, HM584783.1, HM031150.1, HM031152.1, and HM031139.1; the NCBI accession numbers of the four 5S rRNA gene sequences of Enteromorpha fringei are AB298685.1, AB299441.1, HM584771.1, and HM031140.1.

[0027] Example 2: Ulva prolifera and Ulva lentil Specific amplification experiment S1: Extract the genomic DNA to be tested as a template. Prepare fresh 3-5 cm long Ulva prolifera and Ulva lentil The genomic DNA of the two samples was extracted using Dzup (Plant) Genomic DNA Isolation Reagent. The extraction steps were referred to the reagent instructions.

[0028] S2: PCR amplification was performed using primers SEQ1 and SEQ2 to obtain a PCR product. The PCR reaction system consisted of 2 μL of genomic DNA, 10 μL of Taq Mix, 1 μL of SEQ1 (10 μM), 1 μL of SEQ2 (10 μM), and 6 μL of ddH2O, for a total of 20 μL. The PCR protocol was as follows: Stage 1: 94°C initial denaturation for 5 min; Stage 2: 30 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 45 s, and extension at 72°C for 30 s; Stage 3: 72°C extension for 10 min; Stage 4: 4°C hold.

[0029] S3: Detect PCR amplification results by electrophoresis. The specific method is as follows: prepare 1% agarose gel with 1×TAE, heat it in a microwave oven to completely dissolve it, cool it slightly to 45-55℃, add nucleic acid dye at a ratio of 1:10000 and shake it well, then pour it into the gel tank with the comb inserted, pull out the comb after the gel is completely solidified, take 4μL of PCR amplification product and add it to the sample well, run electrophoresis at 120V for 25 minutes, and take pictures with a gel imager after the electrophoresis is completed. Ulva prolifera After amplification of the genomic DNA, obvious bands can be seen, but no Ulva lentil The strips (see Figure 1 ).

[0030] Example 3: Environmental sample specific amplification detection experiment S1: Take a seawater sample collected in the field and filter 2 L of the seawater sample through a 0.2 μm pore size polycarbonate filter membrane. After filtration, the filter membrane is quickly frozen with liquid nitrogen.

[0031] S2: Grind the quick-frozen filter membrane into powder in a mortar and pestle. Then use Dzup (Plant) Genomic DNA Isolation Reagent to extract the genomic DNA on the filter membrane. Refer to the reagent manual for the extraction steps.

[0032] S3: Using SEQ1 and SEQ2 as primers, a PCR amplification method is used to obtain a PCR amplification product. The PCR reaction system and amplification procedure are described in Example 2.

[0033] S4: Detect the PCR amplification results by electrophoresis. The detection method is described in Example 2.

[0034] After the environmental DNA is amplified, obvious bands can be seen, proving that the specific detection primer combination of the present invention can effectively and specifically amplify the environmental samples. Ulva prolifera Gene (see Figure 2 ).

[0035] Example 4: Construction of qPCR standard strain experiment S1: PCR amplification of the target gene. Use SEQ1 and SEQ2 as primers. Ulva prolifera The genomic DNA was amplified by PCR. The PCR reaction system and amplification procedure are described in Example 2.

[0036] S2: Gel extraction: Use the FastPure Gel DNA Extraction Mini Kit to extract the target band according to the instructions to obtain the target DNA fragment.

[0037] S3: Construct the recombinant plasmid. Use the Blunt Zero Gene Cloning Kit to establish a cloning reaction system. The cloning system consists of the target DNA fragment (4 μL) and the pEASY®-Blunt Zero Cloning Vector (1 μL). Mix the mixture and incubate at 37°C for 10 minutes to generate the recombinant plasmid. After the reaction, place the centrifuge tube on ice.

[0038] S4: Obtain recombinant cells. Add 1 μL of recombinant plasmid to DH5α competent cells thawed on ice in advance. After reacting on ice for 20 minutes, heat shock the cells in a 42°C water bath for 45 seconds and then cool them on ice for 5 minutes. Transfer the cells to antibiotic-free LB liquid medium and resuspend them in a shaker at 37°C at 200 rpm for 1 hour. After resuscitation, spread the cells onto LB solid medium containing kanamycin (50 μg / mL) and incubate them at 37°C for 12 hours to obtain recombinant cells (see [1]). Figure 3 Finally, 8 recombinant cells were kept (i.e., H1, H2, H3, H4, H5, H6, S1, and S2).

[0039] Example 5 Absolute Quantification Experiment of Enteromorpha Abundance in Environmental Samples and Laboratory Culture Samples S1: 2 L of each of the field-collected seawater sample and the laboratory-grown seawater from Enteromorpha were filtered through a 0.2 μm pore size polycarbonate filter membrane. After filtration, the filter membrane was quickly frozen using liquid nitrogen.

[0040] S2: Grind the quick-frozen filter membrane into powder in a mortar and pestle. Then use Dzup (Plant) Genomic DNA Isolation Reagent to extract the genomic DNA on the filter membrane. Refer to the reagent manual for the extraction steps.

[0041] S3: The eight recombinant cells obtained in Example 4 were inoculated into 2 mL of LB liquid culture medium containing kanamycin (50 μg / mL) and revived in a shaker at 37°C and 200 rpm for 12 h. The plasmids in the bacterial solution were then extracted using the Omega Plasmid Mini Kit I (EZNA® Plasmid Mini Kit I D6943-02). A total of eight plasmid groups were obtained.

[0042] S4: qPCR was performed using the extracted DNA as a template, and the plasmids of the 8 recombinant cells were obtained for 10 -1 to 10 -8 The standard curve was drawn by quantitative serial dilution to verify the feasibility of qPCR.

[0043] The qPCR reaction system consisted of 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 SEQ1 (10 μM), 0.4 μL of SEQ2 (10 μM), and 6.8 μL of ddH2O, for a total of 20 μL. The qPCR reaction protocol was as follows: Stage 1: pre-denaturation at 94°C for 5 min; Stage 2: 30 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 45 s, and extension at 72°C for 30 s; Stage 3: extension at 72°C for 10 min; Stage 4: hold at 4°C.

[0044] S5: Calculate the absolute quantitative results of qPCR. Calculate the plasmid copy number using formula 1. Establish a standard curve between the lg value of the serially diluted plasmid copy number and the corresponding CT value determined by qPCR. A total of 8 standard curves were obtained (see Figure 4 ), the R2 values of the standard curve were all above 0.99. According to formula 2, the amplification efficiency of all eight plasmid groups was between 95% and 105%, which showed good amplification efficiency, proving that all eight plasmid groups could be effectively used in qPCR experiments. The CT values obtained by qPCR for environmental and laboratory samples were compared with the H1 standard curve, and the gene copy number of the two field samples was 7.43×10 6 copies / L and 8.45×10 6 The gene copy number of a laboratory culture sample is 1.95×10 8 copies / L.

[0045] Formula 1 C = (6.02 × C0 × 10 14 ) / M C: plasmid copy number (copies / μL) C0: plasmid concentration (ng / μL) M: Plasmid molecular weight, M = (plasmid base number + target fragment base number) × 660.

[0046] Formula 2 T = 10 -1 / k - 1 T: amplification efficiency k: slope of the standard curve.

[0047] It should be noted that the above examples are only intended to illustrate the technical solutions of the present invention and are not intended to limit the same. Although the present invention has been described in detail with reference to the examples given, those skilled in the art may modify or replace 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. A specific primer combination for distinguishing Enteromorpha from closely related species, characterized by: The primer combination includes 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.

2. A use of the specific primer combination for distinguishing Enteromorpha from closely related species according to claim 1, characterized in that: The primer combination is used to distinguish Enteromorpha from closely related species.

3. A use of the specific primer combination for distinguishing Enteromorpha from closely related species according to claim 1, characterized in that: Application of the primer combination in the monitoring and quantitative detection of Enteromorpha.

4. A molecular identification method for distinguishing Enteromorpha from closely related species, characterized by: S1: Extract the genomic DNA to be tested as a template; S2: Using the specific primer combination SEQ1 and SEQ2 for distinguishing Enteromorpha from closely related species as described in claim 1 as primers, a PCR amplification method is adopted to obtain a PCR amplification product, and gel electrophoresis detection is performed using an electrophoresis detection method to further distinguish Enteromorpha from closely related species.

5. The molecular identification method according to claim 4, characterized in that: If a DNA band with a molecular weight of 250 bp is present in the electrophoresis test, it is proved that the sample to be tested contains Enteromorpha.

6. The molecular identification method according to claim 4, characterized in that: The DNA bands from the gel electrophoresis were excised and recovered, and the DNA fragments were connected to the vector and transformed into competent cells. After the competent cells grew, the plasmids of the competent cells were extracted, and gradient dilutions were performed using the plasmids as standards. The abundance of Enteromorpha in the samples was quantified by qPCR.

7. The molecular identification method according to claim 4, characterized in that: The PCR reaction system in S2 is: 2 μL of genomic DNA, 10 μL of Taq Mix, 1 μL of SEQ1 (10 μM), 1 μL of SEQ2 (10 μM), and 6 μL of ddH2O, for 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 SEQ1 (10 μM), 0.4 μL of SEQ2 (10 μM), and 6.8 μL of ddH2O, for a total of 20 μL.

8. The molecular identification method according to claim 4 or 7, characterized in that: The PCR and qPCR reaction procedures were as follows: stage 1: pre-denaturation at 94°C for 5 min; stage 2: denaturation at 94°C for 30 s, annealing at 57°C for 45 s, and extension at 72°C for 30 s, for a total of 30 cycles; stage 3: extension at 72°C for 10 min; and stage 4: hold at 4°C.

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