Detection of harmful algae melting curve primer probe set and detection method and application

Abstract

This invention discloses a melting curve primer-probe set and detection method for detecting harmful dinoflagellates, as well as its application. The melting curve primer-probe set includes a first primer, a medium probe, and / or a second primer and a TaqMan probe. The detection method includes the following steps: S1, performing an amplification reaction; S2, if the nucleic acid to be tested contains one of AP, MP, KM, AAN, or GC, both the upstream and downstream primers of the first primer are extended; S3, forming a double-stranded product suitable for melting curve detection; S4, if the nucleic acid to be tested contains SA and an internal reference gene, both the upstream and downstream primers of the second primer are extended; S5, generating an amplification curve; S6, analyzing the melting curve and amplification curve of the double-stranded product to determine the detection result of harmful dinoflagellates. The melting curve primer-probe set can be used for the detection of harmful dinoflagellate content.

Description

Technical Field

[0001] This invention relates to the field of marine environmental monitoring, and in particular to a melting curve primer-probe set for detecting harmful dinoflagellates, as well as detection methods and applications. Background Technology

[0002] Harmful algal blooms are a common ecological phenomenon in nature, caused by the explosive proliferation or massive aggregation of specific algae under suitable environmental conditions. They mainly occur in marine, estuarine, and freshwater areas. Globally, more than 300 species of phytoplankton are known to form harmful algal blooms, about a quarter of which are toxic or harmful. Furthermore, phytoplankton that cause harmful algal blooms can be spread and dispersed through ship ballast water and its sediments.

[0003] Statistical data shows that approximately 75% of marine algal blooms are caused by dinoflagellates, which account for about 40% of harmful algal bloom species. Harmful algal blooms caused by dinoflagellates are driven not only by external environmental factors but also closely related to their unique survival strategies. Dormant cysts, a crucial stage in the dinoflagellate life cycle, not only provide the seed source for the interannual frequency of algal blooms but also expand their geographical range and even invade organisms through natural and anthropogenic pathways, such as cross-regional introduction through shellfish farming and the spread through ship ballast water and its sediments. Therefore, developing technologies for rapid detection of various toxic and harmful dinoflagellate vegetative cells and dormant cysts is particularly urgent. Such rapid detection methods have broad application prospects in monitoring and identifying the presence, distribution, and quantity of toxic and harmful dinoflagellates in my country's sea areas and in ballast water and sediments of inbound ships, and are expected to provide key technical support for predicting, warning, and even controlling and managing marine toxic and harmful dinoflagellate blooms.

[0004] Currently, the identification of dinoflagellates and their dormant cysts mainly relies on traditional morphological observation, molecular biological methods, and the combined application of multiple technologies.

[0005] (1) Morphological methods rely on optical or scanning electron microscopes to identify and classify cells based on their morphology, size, color, surface features, and internal structure. However, for cells or cysts that are tiny or have extremely simple structures, morphological methods often fail to achieve accurate identification, or may even be impossible to complete. Furthermore, this method is highly dependent on the experience of the identification personnel. In addition, even when identifying dormant cells through morphological analysis of post-germination vegetative cells, in most cases only the genus level can be determined, and only a few can be identified to the species level.

[0006] (2) Combining morphological observation with high-throughput sequencing or single-cell sequencing can improve the accuracy of identification, but the operation process is complicated and cumbersome, involving a variety of reagents and instruments. It is not only time-consuming and material-intensive, but may also introduce certain failure risks, and requires high skills from operators.

[0007] (3) Molecular biology techniques typically use rDNA sequences as molecular markers. Common methods include conventional PCR, real-time quantitative PCR, high-throughput sequencing, and fluorescence in situ hybridization. Real-time quantitative PCR is widely used in molecular biology. Its core principle is to introduce fluorescently labeled probes into the PCR reaction system and achieve quantitative analysis of nucleic acid targets by tracking changes in fluorescence intensity during amplification in real time. This technology has a standardized operating procedure and highly quantifiable results, and has been widely used for the rapid detection of various pathogens. However, this technology is limited by single-dimensional fluorescence signals—each fluorescence channel can only identify a single target, and multi-channel instruments are required for multiplex detection. This limits the number of targets that can be detected in a single reaction, and the high cost of equipment also hinders the widespread adoption of the technology. Summary of the Invention

[0008] The purpose of this invention is to address the above-mentioned problems by providing a melting curve primer and probe set for detecting harmful dinoflagellates, as well as a detection method and application, which improves detection throughput and reduces detection costs.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows: A primer-probe set for detecting the melting curve of harmful dinoflagellates. The melting curve primer-probe set includes: First primer and medium probe used to detect Alexandrium paciensis, Margherita polycyclic, Karenia mikimotoi, Alexandrium Anderson, and Gynostemma pentaphyllum; and / or The second primer and TaqMan probe used to detect Stellaria spidata; Both the upstream and downstream primers of the first primer contain a 5' universal amplification sequence that does not bind to the target nucleic acid and a 3' target nucleic acid-specific sequence that can specifically bind to the target nucleic acid; the medium probe contains a 5' tag sequence that does not bind to the target nucleic acid and a 3' target-specific sequence that can specifically bind to the target nucleic acid, and the 3' target-specific sequence of the medium probe is modified with a group that prevents elongation; The upstream primer, downstream primer, and TaqMan probe of the second primer can all specifically bind to the target nucleic acid. The 5' end of the TaqMan probe is modified with a fluorescent group, and the 3' end of the TaqMan probe is modified with a quenching group.

[0010] Furthermore, the melting curve primer-probe set also includes a detection probe, which is modified with RQP and has a complementary sequence to the 5' tag sequence in the medium probe.

[0011] A method for detecting a melting curve primer-probe set of harmful dinoflagellates, comprising the following steps: S1. Amplification reaction is carried out in a PCR amplification system containing the first primer, the second primer, the medium probe, the detection probe, the TaqMan probe, the nucleic acid to be tested, and Taq DNA polymerase; S2. If the nucleic acid to be tested contains one of the following: Alexandrium paclitaxum target nucleic acid, Margherita polycyclic anaerobic ... S3. When the extension reaches the probe site, Taq DNA polymerase cuts and releases the 5' tag sequence in the probe. The released 5' tag sequence is captured and extended by the detection probe. The extension product is complementary to the detection probe, forming a double-stranded product suitable for melting curve detection. S4. If the nucleic acid to be tested contains the target nucleic acid of Stellaria spicata and the internal reference gene, then both the upstream and downstream primers of the second primer will be extended. S5. When the extension reaches the TaqMan probe site, Taq DNA polymerase hydrolyzes and cuts the TaqMan probe, causing the modified fluorescent group and quenching group in the TaqMan probe to separate, the fluorescence signal is enhanced, and thus the corresponding amplification curve is generated. S6. Analyze the melting curve and amplification curve of the double-stranded product to determine the detection results of harmful dinoflagellates.

[0012] Furthermore, in the PCR amplification system of step S1, the amplification sequences of the amplification primers are consistent with the amplification sequences in the first primer and the second primer.

[0013] Furthermore, in step S6, if both the melting curve and amplification curve of the double-stranded product meet the reference range requirements, the detection result of the harmful dinoflagellate is determined to be positive; if the melting curve of the double-stranded product meets the reference range requirements and the amplification curve range is 38 < Ct ≤ 40, a retest is performed. If the retest result is consistent with the current result, the detection result of the harmful dinoflagellate is determined to be positive; otherwise, the detection result of the harmful dinoflagellate is determined to be negative.

[0014] An application of a melting curve primer-probe set for detecting harmful dinoflagellates, wherein the melting curve primer-probe set can be used for the detection of harmful dinoflagellate content.

[0015] Furthermore, the harmful dinoflagellates are one or more of the following: Alexandrium paciensis, Margherita polycyclic, Karenia mikimotoi, Alexandrium Anderson, Gynostemma pentaphyllum, and Stellaria spidata.

[0016] Compared with the prior art, the advantages and positive effects of this invention are: This invention proposes a primer-probe set for detecting harmful dinoflagellates based on melting curves, along with the detection method and its applications. It integrates melting curve analysis with real-time fluorescence PCR, achieving detection through a dual-dimensional approach of fluorescence signal and melting temperature (Tm value), thus enabling multiple target analysis within a single channel. This technology requires only two fluorescence channels to detect seven targets, significantly improving detection throughput. Meanwhile, the testing process does not require the addition of special equipment, which reduces testing costs. Furthermore, the testing operation does not require high operational skills from personnel, making it a technological evolution with practical application value and broad market prospects.

[0017] On the other hand, this invention employs asymmetric primers and universal primers: first, low-concentration specific primers bind to the template to reduce dimers, and the product carries a universal tag; then, high-concentration universal primers amplify the tag to eliminate bias and enable all targets to grow efficiently and uniformly; through innovative primer design, it solves the bias and dimer challenges in multiplex amplification and improves detection sensitivity. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention 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 the present invention. 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 of the single-target detection results of the primer-probe set using the melting curve method. Figure 2 This is a schematic diagram of the results of multiple single-target detection using the melting curve method for primer-probe sets. Figure 3a This is a schematic diagram showing the detection results of AP AAN, AP GC, AP MP, AP KM, AP SA, AAN GC, AAN MP, and AAN KM in the multiple dual-target detection of primer and probe sets using the melting curve method. Figure 3b This is a schematic diagram showing the detection results of AAN SA, GC MP, GC KM, GC SA, MP KM, MP SA, and KM SA in the multiple dual-target detection of primer and probe sets using the melting curve method. Figure 4a This is a schematic diagram showing the detection results of AP AAN GC, AP AAN MP, AP AANKM, AP AAN SA, AP GC MP, AP GC KM, AP GC SA, and AP MP KM in the multiplex triple-target detection of primer and probe sets using the melting curve method. Figure 4b This is a schematic diagram showing the detection results of AP MP SA, AP KM SA, AAN GCMP, AAN GC KM, AAN GC SA, AAN MP KM, AAN MP SA, and AAN KM SA in the multiplex triple-target detection of primer and probe sets using the melting curve method. Figure 4c This is a schematic diagram of the detection results of GC MP KM, GC MP SA, GC KMSA, and MP KM SA in the multiplex triple-target detection of primer and probe sets using the melting curve method. Figure 5 This is a schematic diagram of the results of multiple six-target detection using the melting curve method for primer-probe set; Figure 6a This is a schematic diagram of the detection results of PMIC, PMIN, KP, KB, and AAF in the specific detection of primer and probe sets using the melting curve method. Figure 6b This is a schematic diagram of the detection results of AM, AI, GM, and KD in the specific detection of primer-probe sets using the melting curve method. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art to all other embodiments obtained without creative effort should be included within the protection scope of the present invention.

[0021] This invention discloses a primer-probe set for detecting melting curves of harmful dinoflagellates, as well as a detection method and application. This primer-probe set can detect a variety of toxic and harmful dinoflagellates and has high sensitivity, good specificity, and uniform and stable performance.

[0022] Specific upstream primers, downstream primers, and vector probes were designed to target the nucleic acid sequences of various toxic and harmful dinoflagellates (Alexandrium pacificum (AP), Margalefidinium polykrikoides (MP), Karenia mikimotoi (KM), Alexandrium andersonii (AAN), and Gymnodinium catenatum (GC)). Both the upstream and downstream primers contain a universal amplification sequence at the 5' end and a target nucleic acid-specific sequence at the 3' end. The universal amplification sequence does not bind to the target nucleic acid, while the target nucleic acid-specific sequence can specifically bind to the target nucleic acid. The vector probe contains a tag sequence at the 5' end and a target-specific sequence at the 3' end. The tag sequence does not bind to the target nucleic acid, while the target-specific sequence can specifically bind to the target nucleic acid. Furthermore, the 3' end of the vector probe is modified with a group that prevents elongation.

[0023] A detection probe was designed for the 5' end tag sequence. This detection probe was modified with commercially available RQP, which contains a sequence complementary to the tag sequence, thereby enabling it to capture the tag sequence.

[0024] Upstream primers, downstream primers, and a TaqMan probe were designed to target the nucleic acid sequences of the dinoflagellate *Scrippsiella acuminata* (SA) and an internal reference. All three primers—upstream, downstream, and TaqMan—can specifically bind to the target nucleic acid. The TaqMan probe is modified with a fluorescent group at its 5' end and a quencher group at its 3' end.

[0025] Preferably, the primer-probe set includes: upstream primer AP-F1, downstream primer AP-R1, and medium probe M1-AP for detecting *Alexandrium pacificum*; upstream primer MP-F1, downstream primer MP-R1, and medium probe M2-MP for detecting *Margalefidinium polykrikoides*; upstream primer KM-F1, downstream primer KM-R1, and medium probe M3-KM for detecting *Karenia mikimotoi*; upstream primer AAN-F1, downstream primer AAN-R1, and medium probe M4-AAN for detecting *Alexandrium andersonii*; upstream primer GC-F1, downstream primer GC-R1, and medium probe M5-GC for detecting *Gymnodinium catenatum*; and a medium probe Scrippsiella for detecting *Scrippsiella*. The study included upstream primer SA-F1, downstream primer SA-R1, and TaqMan probe SA-P1-VIC for acuminata; and upstream primer 18s-F1, downstream primer 18s-R1, and TaqMan probe 18s-P1-FAM for internal control detection. Specifically, the 5' end of TaqMan probe SA-P1-VIC was modified with VIC, and the 3' end with BHQ1; the 5' end of TaqMan probe 18s-P1-FAM was modified with FAM, and the 3' end with BHQ1.

[0026] Based on the tag sequences of media probes M1-AP, M2-MP, and M3-KM, detection probe RQP-2 was designed; based on the tag sequences of media probes M4-AAN and M5-GC, detection probe RQP-1 was designed. Specifically, the 5' end of detection probe RQP-2 is modified with BF490, and the 3' end with RQP; the 5' end of detection probe RQP-1 is modified with BF533, and the 3' end with RQP.

[0027] The detection procedure for the aforementioned primer-probe set is as follows: Amplification is performed in a PCR amplification system containing the upstream primer, downstream primer, tag probe, detection probe, TaqMan probe, target nucleic acid, and Taq DNA polymerase. If the target nucleic acid contains the target nucleic acid (Alexandrium pacificum (AP), Margalefidinium polykrikoides (MP), Karenia mikimotoi (KM), Alexandrium andersonii (AAN), Gymnodinium catenatum (GC)), both the upstream and downstream primers will extend. When the extension reaches the mediator probe position, Taq DNA polymerase cleaves and releases the tag sequence. The released tag sequence is captured and extended by the detection probe. The tag sequence extension product is complementary to the detection probe, forming a double-stranded product. As the amplification reaction proceeds, the amount of mediator probe released by Taq DNA polymerase gradually increases, and the amount of tag sequence captured and extended by the detection probe increases accordingly, ultimately obtaining a double-stranded product suitable for melting curve detection. If the nucleic acid to be tested contains the target nucleic acid *Scrippsiella acuminata* (SA) and the internal reference gene, both the upstream and downstream primers will extend. When the extension reaches the TaqMan probe site, Taq DNA polymerase hydrolyzes and cleaves the TaqMan probe, causing the fluorescent group and quencher group to separate, resulting in enhanced fluorescence signal and the generation of a corresponding amplification curve. The melting curve of the double-stranded product and the amplification curve are analyzed to determine the detection result of harmful dinoflagellates.

[0028] Design a universal amplification primer (BP primer) whose sequence is consistent with the universal amplification sequences in the upstream and downstream primers of the detection target.

[0029] The sequences of each upstream primer, downstream primer, medium probe, universal amplification primer, and TaqMan probe are shown in Table 1, where the underlined sequences are universal amplification sequences: Table 1 The reaction system of the above-mentioned melting curve primer-probe set is shown in Table 2: Table 2 The amplification and detection procedure for the above-mentioned melting curve primer-probe set is shown in Table 3: Table 3 The judgment criteria are shown in Table 4: Table 4 Based on Table 4, the criteria for judging the test results are as follows: Positive: A result is considered positive if both the amplification curve and melting curve analysis results meet the reference range requirements. Suspicious: If the melting curve analysis results meet the reference range requirements, and the amplification curve shows 38 < Ct ≤ 40, a retest is required. If the retest results are consistent, the result is considered positive; otherwise, it is considered negative. Negative: Except for the above situations, all other results are judged as negative; The effectiveness of this technical solution will be verified through the following examples; Example 1: Single-target detection results of primer-probe set using melting curve method like Figure 1 As shown, according to the designed primer and probe ratio scheme, the internal control, Alexandrium pacificum (AP), Margalefidinium polykrikoides (MP), Karenia mikimotoi (KM), Alexandrium andersonii (AAN), Gymnodinium catenatum (GC), and Scrippsiella acuminata (SA) were all detected normally.

[0030] Example 2: Results of Multiplex Single-Target Detection Using Primer-Probe Sets Based on Melting Curve Method like Figure 2 As shown, according to the designed primer-probe ratio scheme, Alexandrium pacificum (AP), Margalefidinium polykrikoides (MP), Karenia mikimotoi (KM), Alexandrium andersonii (AAN), Gymnodinium catenatum (GC), and Scrippsiella acuminata (SA) were all detected normally, and the internal control was detected in all samples.

[0031] Example 3: Detection Results of Dual-Target Primer-Probe Sets Using Melting Curve Method like Figure 3a , Figure 3bAs shown, according to the designed primer-probe ratio scheme, any two targets of Alexandrium pacificum (AP), Margalefidinium polykrikoides (MP), Karenia mikimotoi (KM), Alexandrium andersonii (AAN), Gymnodinium catenatum (GC), and Scrippsiella acuminata (SA) can be detected normally, and internal controls are detected in all samples.

[0032] Example 4: Detection Results of Three Targets Using Primer-Probe Sets Based on Melting Curve Method like Figure 4a , Figure 4b , Figure 4c As shown, following the designed primer-probe ratio scheme, any three targets from Alexandrium pacificum (AP), Margalefidinium polykrikoides (MP), Karenia mikimotoi (KM), Alexandrium andersonii (AAN), Gymnodinium catenatum (GC), and Scrippsiella acuminata (SA) could be detected normally, and all internal controls were also detected.

[0033] Example 5: Detection Results of Six Targets Using the Melting Curve Method Primer-Probe Set like Figure 5 As shown, according to the designed primer-probe ratio scheme, all six targets—Alexandrium pacificum (AP), Margalefidinium polykrikoides (MP), Karenia mikimotoi (KM), Alexandrium andersonii (AAN), Gymnodinium catenatum (GC), and Scrippsiella acuminata (SA)—were detected normally, and all internal controls were also detected.

[0034] Example 6: Specificity Detection Results of Primer-Probe Sets Using Melting Curve Method like Figure 6a , Figure 6bAs shown, according to the designed primer-probe ratio scheme, the negative controls—Prorocentrum micans (PMIC), Prorocentrum minimum (PMIN), Karenia papilionacea (KP), Karenia brevis (KB), Alexandrium affine (AAF), Alexandrium minutum (AM), Alexandrium insuetum (AI), Gymnodinium microreticulatum (GM), and Karlodinium decipens (KD)—were all undetectable.

[0035] The present invention has the following beneficial effects: 1. This invention utilizes bioinformatics analysis technology to perform high-throughput differential alignment of target sequences (including Alexandrium pacificum (AP), Margalefidinium polykrikoides (MP), Karenia mikimotoi (KM), Alexandrium andersonii (AAN), Gymnodinium catenatum (GC), and Scrippsiella acuminata (SA)), and also compares them with the gene sequences of closely related species. This ensures the high specificity of the primer and probe set, thereby guaranteeing the specificity of the detection results.

[0036] 2. The melting curve primer-probe set for detecting various toxic and harmful dinoflagellates provided by this invention effectively solves the problem of insufficient detection throughput compared to traditional molecular biology techniques. This invention only requires two fluorescence channels to detect seven target sites, thereby significantly improving detection throughput while reducing reagent and time costs.

[0037] 3. In the technical solution of this invention, the tag sequence and the detection probe complementarily combine to form a double-stranded product. By obtaining its melting curve, the melting curve corresponding to the target nucleic acid (Alexandrium pacificum (AP), Margalefidinium polykrikoides (MP), Karenia mikimotoi (KM), Alexandrium andersonii (AAN), Gymnodinium catenatum (GC)) can be obtained, thus achieving detection. This method is target-independent and belongs to the target-independent melting curve method; both the tag sequence and the detection probe sequence are known designs, and the melting point (Tm value) of each double strand can be calculated in advance. In melting curve analysis, by identifying the melting peak with a specific Tm value, it can be determined whether the corresponding target nucleic acid sequence exists in the sample. This avoids peak shift and misjudgment caused by target nucleic acid mutation, improving accuracy; at the same time, since the sequence is known and the melting point can be pre-calculated, this method can also improve detection throughput.

[0038] 4. This invention describes a melting curve primer-probe set and its technical solution, innovatively combining melting curve analysis with the TaqMan probe method, effectively improving the limitations of traditional melting curve analysis in terms of the sufficiency of amplification curves. Traditional melting curve technology requires simultaneous targeting of multiple targets within the same detection channel, making it difficult to distinguish specific targets in the amplification curve, thus reducing its application value. The melting curve primer-probe set involved in this invention, through technical means and specific modification of the detection probe RQP, enables targets for melting curve detection (such as Alexandrium pacificum (AP), Margalefidinium polykrikoides (MP), Karenia mikimotoi (KM), Alexandrium andersonii (AAN), and Gymnodinium catenatum (GC)) to not generate amplification curves during the amplification stage, while targets for TaqMan probe detection (such as Scrippsiella acuminata (SA) and internal controls) generate amplification curves during the amplification stage but do not form melting curves. This achieves the technical effect of non-interference between the two methods and alleviates the high dependence of detection throughput on the number of device channels.

[0039] 5. This technology addresses the bias and dimer challenges in multiplex amplification through innovative primer design, improving detection sensitivity. In multiplex PCR, primer dimers increase exponentially with the number of primer pairs, consuming components and interfering with background noise. Amplification bias stems from target differences, leading to competitive inhibition and false negatives. This invention employs asymmetric and universal primers: first, low-concentration specific primers bind to the template to reduce dimers, resulting in a product with a universal tag; then, high-concentration universal primers amplify the tag, eliminating bias and ensuring efficient and consistent amplification of all target sites.

Claims

1. A primer-probe set for detecting the melting curve of harmful dinoflagellates, characterized in that: The melting curve primer-probe set includes: First primer and medium probe used to detect Alexandrium paciensis, Margherita polycyclic, Karenia mikimotoi, Alexandrium Anderson, and Gynostemma pentaphyllum; and / or The second primer and TaqMan probe used to detect Stellaria spidata; Both the upstream and downstream primers of the first primer contain a 5' universal amplification sequence that does not bind to the target nucleic acid and a 3' target nucleic acid-specific sequence that can specifically bind to the target nucleic acid; the medium probe contains a 5' tag sequence that does not bind to the target nucleic acid and a 3' target-specific sequence that can specifically bind to the target nucleic acid, and the 3' target-specific sequence of the medium probe is modified with a group that prevents elongation; The upstream primer, downstream primer, and TaqMan probe of the second primer can all specifically bind to the target nucleic acid. The 5' end of the TaqMan probe is modified with a fluorescent group, and the 3' end of the TaqMan probe is modified with a quenching group.

2. The primer and probe set for detecting harmful dinoflagellates as described in claim 1, characterized in that: The melting curve primer-probe set also includes a detection probe, which is modified with RQP and has a complementary sequence to the 5' tag sequence in the medium probe.

3. A detection method for a melting curve primer-probe set for detecting harmful dinoflagellates as described in claim 1 or 2, characterized in that: Includes the following steps: S1. Amplification reaction is carried out in a PCR amplification system containing the first primer, the second primer, the medium probe, the detection probe, the TaqMan probe, the nucleic acid to be tested, and Taq DNA polymerase; S2. If the nucleic acid to be tested contains one of the following: Alexandrium paclitaxum target nucleic acid, Margherita polycyclic anaerobic ... S3. When the extension reaches the probe site, Taq DNA polymerase cuts and releases the 5' tag sequence in the probe. The released 5' tag sequence is captured and extended by the detection probe. The extension product is complementary to the detection probe, forming a double-stranded product suitable for melting curve detection. S4. If the nucleic acid to be tested contains the target nucleic acid of Stellaria spicata and the internal reference gene, then both the upstream and downstream primers of the second primer will be extended. S5. When the extension reaches the TaqMan probe site, Taq DNA polymerase hydrolyzes and cuts the TaqMan probe, causing the modified fluorescent group and quenching group in the TaqMan probe to separate, the fluorescence signal is enhanced, and thus the corresponding amplification curve is generated. S6. Analyze the melting curve and amplification curve of the double-stranded product to determine the detection results of harmful dinoflagellates.

4. The detection method for the melting curve primer-probe set of harmful dinoflagellates as described in claim 3, characterized in that: In the PCR amplification system of step S1, the amplification sequences of the amplification primers are consistent with the amplification sequences of the first primer and the second primer.

5. The detection method for the melting curve primer-probe set of harmful dinoflagellates as described in claim 4, characterized in that: In step S6, if both the melting curve and amplification curve of the double-stranded product meet the reference range requirements, the detection result of the harmful dinoflagellate is judged to be positive; if the melting curve of the double-stranded product meets the reference range requirements and the amplification curve range is 38 < Ct ≤ 40, a retest is performed. If the retest result is consistent with the current result, the detection result of the harmful dinoflagellate is judged to be positive; otherwise, the detection result of the harmful dinoflagellate is judged to be negative.

6. An application of the melting curve primer-probe set for detecting harmful dinoflagellates as described in claim 1 or 2, characterized in that: The melting curve primer-probe set can be used for the detection of harmful dinoflagellates.

7. The application of the primer-probe set for detecting harmful dinoflagellates as described in claim 6, characterized in that: The harmful dinoflagellates are one or more of the following: Alexandrium paciensis, Margherita polycyclic, Karenia mikimotoi, Alexandrium Anderson, Gynostemma pentaphyllum, and Stellaria spidata.

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