A probe primer composition, method, product for quantitative detection of karenia longicauda and karenia brevis

Abstract

The present application provides a kind of probe primer composition, method, product for the quantitative detection of harmful red tide causing species-Karen long groove and Karen butterfly that has been newly discovered in China offshore in recent years, belongs to the field of microbial detection technology.The present application provides a set of probe primer composition for the quantitative detection of Karen long groove and Karen butterfly shown in SEQ ID NO.1-6, the composition can be quantitatively detected for Karen long groove and Karen butterfly, and has strong specificity and high sensitivity.The method overcomes the limitations of traditional morphological identification, saves time and effort, and the method is simple, can realize the rapid and accurate qualitative identification and quantitative detection of a large number of samples.The present application can be used for the accurate identification of Karen long groove and Karen butterfly, provides more accurate and effective technical support for the detection of harmful red tide in China offshore, and can also provide a new technical means for Karen algal red tide early warning and large-area monitoring.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, and in particular to a probe primer composition, method, and product for the quantitative detection of Karenia longiformis and Karenia butterflyi. Background Technology

[0002] Currently, red tides have become a serious ecological disaster in many sea areas around the world, especially in nearshore regions. Harmful dinoflagellates are the main group causing marine disasters. Among them, red tides caused by Karenia algae have attracted widespread attention from all sectors of society due to their wide distribution, high frequency of occurrence, and large-scale damage.

[0003] For a long time, *Karenella mikimotoi* has been considered the main causative species of algal blooms in my country's coastal waters and the most harmful marine dinoflagellate causing the most severe economic losses to my country's coastal aquaculture fisheries. However, in recent years, with the applicant's systematic and in-depth research on the diversity of *Karenella* in my country's coastal waters, we have found that the causative species of *Karenella* in my country's coastal waters is not just *Karenella mikimotoi*, which is widely recognized in the academic community. In addition to *Karenella mikimotoi*, other *Karenella* species such as *Karenella longiformis* and *Karenella davidii* often appear simultaneously with *Karenella mikimotoi*, forming complex *Karenella* red tides that cause enormous damage to the marine ecological environment and aquaculture fisheries.

[0004] Karenia longiformis produces polyunsaturated fatty acids, thus exhibiting hemolytic activity, while Karenia davidii produces the short toxin PbTx-2 intracellularly, suggesting potential toxicity. Both types of red tides could threaten marine ecosystems and consequently endanger human health. Furthermore, the applicant's mixed culture experiments with several Karenia species revealed interspecific interactions: Karenia longiformis promotes the growth of Karenia davidii, while Karenia saddle-shaped and Karenia mikimotoi inhibit its growth. Moreover, the toxicity of each Karenia species significantly increased when mixed with other Karenia species.

[0005] In 2024, the applicant further discovered that the globally reported species of *Karenella longiformis* and *Karenella pachymorpha* can be divided into three groups, and the *Karenella longiformis* found in my country's coastal waters may belong to one of these separate groups (Five Karenia species along the Chinese coast: with the description of a newpecies, Karenia Hui sp. nov. (Kareniaceae, Dinophyta), Jingyi Cen et al., 2024, Harmful Algae 137, 102645, Fig. 7).

[0006] Therefore, to better prevent and control harmful Karenia red tides in my country's coastal waters and reduce disaster losses, it is first necessary to develop species-specific detection methods or technologies for several Karenia species in my country. Secondly, in order to understand the occurrence mechanisms of red tides caused by different Karenia species, such as *Karenella longiformis* and *Karenella pachycarpa*, it is also necessary to establish a technology capable of quantitatively detecting the cell counts of *Karenella longiformis* and *Karenella pachycarpa*.

[0007] Currently, the detection technologies for these two algae mainly include morphological detection technology, cytochrome-based detection technology, and high-throughput detection technology based on molecular biology. In my country, the most common method for identifying red tide algae is to use optical microscopy to identify and count algal species.

[0008] While optical microscopy is the most intuitive and commonly used method for red tide species detection, morphological classification requires a high level of expertise from the identification personnel and is time-consuming, making rapid, large-scale detection of algal species difficult. Furthermore, the detection capability of microscopes is limited when the target algae in environmental samples are at low densities. Karenia, a type of dinoflagellate algae with individuals only about 20 μm in size, is difficult to accurately identify specific species under an optical microscope. However, existing studies have found that different species of Karenia exhibit variations in toxin composition and toxicity. Therefore, it is necessary to develop simpler and more sensitive detection methods to supplement traditional microscopic detection methods, in order to achieve accurate species-level identification of toxic and harmful Karenia.

[0009] PCR detection technology is one of the fastest-growing and most widely used techniques in molecular biology. However, conventional PCR technology can only identify target species and cannot quantify their abundance. Quantitative real-time PCR (qPCR) is a molecular biology technique developed based on PCR. Quantitative PCR methods can be divided into SYBR Green I dye method and TaqMan probe method according to the different binding mechanisms of fluorescent groups. The SYBR Green I dye method is highly versatile, but because this dye is non-specific, it may bind to amplification products or non-target sequences during the reaction, resulting in false positives. Therefore, there is an urgent need to find a faster, more sensitive, and more specific quantitative detection method, such as molecular detection based on the TaqMan probe method. Summary of the Invention

[0010] The purpose of this invention is to provide a probe primer composition, method, and product for the quantitative detection of Karenia longiformis and Karenia davidii, which can realize the quantitative detection of Karenia longiformis and Karenia davidii with good specificity and high sensitivity, providing accurate and effective technical support for the detection of red tides.

[0011] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a probe-primer composition for TaqMan quantitative PCR detection of *Karenella longiformis* and *Karenella pachymorpha*, characterized in that the composition of the probe-primer composition is as follows: Karenia longifolia: SEQ ID NO.1-L2F:GCTTCAGTGTCAATATGC; SEQ ID NO.2-L2R: CAGACAAAGCAACAGATG; SEQ ID NO.3-L2P: VIC-TATGCCACCGTCAACCTGTCA-MGB; Butterfly-shaped Karenia: SEQ ID NO.4-P-F1: GGTGTTGTCTAATGTGATG; SEQ ID NO.5-P-R1: CCTCAATTATGAACTGACG; SEQ ID NO. 6-P-P1: FAM-CAAGCAACCAGTATCGCATCCAG-BHQ1.

[0012] Preferably, the target genes for TaqMan quantitative PCR detection of Karenia longiformis and Karenia davidii are the ITS region of Karenia longiformis ribosomal DNA and the LSU D1-D3 region of Karenia davidii ribosomal DNA, respectively.

[0013] This invention also provides a TaqMan fluorescence quantitative detection method for Karenia longiformis and Karenia butterflyi, comprising the following steps: (1) Extract DNA from the sample to be tested, and use the probe and primer combination shown in SEQ ID NO.1~SEQ ID NO.6 to amplify the extracted DNA by TaqMan qPCR. After the amplification is completed, the Ct values ​​of Karenia longiformis and Karenia butterfly are detected respectively. (2) Based on the Ct value obtained in step (1), the cell number of *Karenella longiformis* and *Karenella davidii* is calculated using the plasmid standard curve, cell standard curve, and cell-plasmid standard curve, respectively. The method is to substitute the Ct values ​​obtained from the amplification of *Karenella longiformis* and *Karenella davidii* into the regression equation of the plasmid standard curve of *Karenella longiformis* and *Karenella davidii*, respectively, to calculate the copy number of recombinant plasmid DNA of *Karenella longiformis* and *Karenella davidii* in the sample; then, through the regression relationship between the cell number and the plasmid copy number established in the cell-plasmid standard curve obtained by combining the cell standard curve and the plasmid standard curve, the cell number of *Karenella longiformis* and *Karenella davidii* is calculated, respectively.

[0014] A plasmid standard curve was established because recombinant plasmids are stable even after long-term storage, and their quantity can be easily calculated by measuring optical density. Furthermore, they can be used as positive templates to calculate the stability of qPCR assays. We used two standard curves instead of a cell-based standard curve to improve the accuracy of cell counting caused by changes in DNA copy number within cells.

[0015] Preferably, the amplification system for TaqMan qPCR amplification in step (1) is: 10 μL Probe qPCRSuper PreMix, 0.4 μL forward primer, 0.4 μL reverse primer, 0.2 μL TaqMan probe, 8 μL ddH2O, and 1 μL DNA template; the amplification program is: 37℃ for 2 min, 95℃ for 10 min, 95℃ for 10 sec, 60℃ for 34 sec, for 40 cycles.

[0016] Preferably, the plasmid standard curve in step (2) is: Karenia longifolia: y = -3.457x + 38.727; Papilionella Karenia: y = -3.27x + 41.015.

[0017] Where x represents the logarithm of the recombinant plasmid copy number (base 10), and y represents the Ct value.

[0018] Preferably, the cell standard curve in step (2) is: Karenia longifolia: y = -3.729x + 29.751; Papilionella Karenia: y = -3.545x + 26.672.

[0019] Where x represents the logarithm of the recombinant plasmid copy number (base 10), and y represents the Ct value.

[0020] Preferably, the cell-plasmid standard curve in step (2) is: Karenia longifolia: y = 0.927x - 2.407; Papilionella Karenia: y = 0.922x - 4.046.

[0021] Where x represents the logarithm of the recombinant plasmid copy number (base 10), and y represents the logarithm of the algal cell number (base 10).

[0022] The present invention also provides a product for TaqMan quantitative fluorescence detection of Karenia longiformis and Karenia davidii, the product comprising the probe and primer composition for TaqMan quantitative fluorescence detection of Karenia longiformis and Karenia davidii as described in claim 1.

[0023] Preferably, the product includes any one of the following: a detection reagent, a detection kit, and a detection chip.

[0024] This invention also provides the application of a TaqMan fluorescence quantitative detection product for the detection of Karenia longiformis and Karenia davidii.

[0025] The beneficial effects of this invention compared to the prior art are as follows: This invention provides a set of probe primer compositions, as shown in SEQ ID NO.1~6, for the quantitative detection of Karenia longiformis and Karenia davidii. These compositions can quantitatively detect both Karenia longiformis and Karenia davidii with high specificity, high sensitivity, and low detection limits for algal cells. They overcome the limitations of traditional morphological identification, saving time and effort, and are simple to implement. They enable rapid and accurate qualitative and quantitative identification of large batches of samples, providing accurate identification of Karenia longiformis and Karenia davidii, offering relatively accurate and effective technical support for red tide detection, and providing a new technical means for early warning and large-scale monitoring of Karenia red tides. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in 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.

[0027] Figure 1 The results of specific amplification of Karenia longiformis in Example 1 of the present invention are shown in the figure. 1 represents Karenia longiformis DNA, 2 represents Karenia saddle-shaped DNA, 3 represents Karenia mikimotoi DNA, 4 represents Karenia butterfly-shaped DNA, 5 represents mixed DNA of control algae, and 6 represents negative control. Figure 2 The results of specific amplification of *Karenella davidii* in Example 1 of this invention are shown in the figure. 1 represents *Karenella davidii* DNA, 2 represents *Karenella saddle-shaped* DNA, 3 represents *Karenella mikimotoi* DNA, 4 represents *Karenella longiformis* DNA, 5 represents mixed DNA from control algae, and 6 represents negative control. Figure 3 This is a standard curve diagram of the plasmid of Karenia longifolia in Example 2 of the present invention; Figure 4 This is a standard curve diagram of the *Karenella pulvinata* plasmid in Example 2 of the present invention; Figure 5 This is a standard curve diagram of *Karenella longspinipes* cells in Example 2 of the present invention; Figure 6 This is a standard curve diagram of *Karenella pulcherrima* cells in Example 2 of the present invention; Figure 7 This is a standard curve diagram of *Karenella longifolia* cells and plasmids in Example 2 of the present invention; Figure 8 This is a standard curve diagram of *Karenella pulvinata* cells and plasmids in Example 2 of the present invention. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0033] Example 1 Example 1 of this invention prepared a probe-primer composition for the quantitative detection of Karenia longiformis and Karenia butterflyi, and tested the detection effect of the probe-primer composition. The specific steps are as follows: The ribosomal RNA (rRNA) of the genus *Karenella* is encoded by ribosomal DNA (rDNA) located in the nucleolar organizer region. This rDNA can be divided into untranscribed regions (NTS), external transcribed spacers (ETS), 18S rDNA (SSU rDNA), internal transcribed spacers (ITS, including ITS1, 5.8S rDNA, and ITS2), and 28S rDNA (LSU rDNA). Among these, the 18S rDNA sequence exhibits high sequence conservation in biological evolution and is commonly used for species analysis at the phylum, class, and order levels. The LSU and ITS sequences, compared to 18S rDNA, evolve relatively faster and exhibit significant sequence polymorphism across different species, making them suitable for species identification at different genera and species levels. Therefore, primer design targeting these two regions can enhance the specificity of species identification. This invention targets Karenia longiformis and Karenia davidii, and identifies specific sites based on the ITS region of Karenia longiformis and the LSU region of Karenia davidii. Corresponding specific primers and TaqMan probes are designed to construct a TaqMan qPCR detection technology for algal species, aiming to accurately identify them and provide targeted technical support for the monitoring of harmful algal blooms.

[0034] (1) Take 1 mL of algal cells of Karenia longiformis and Karenia butterflyi that have grown to the exponential stage, add Lugo reagent to fix and mix well, take 100 μL of sample and observe under a microscope in a plankton counting frame, then filter 300 mL of algal solution with a magnetic filter and a 0.22 μm mixed cellulose filter membrane, discard the filtrate, transfer the filter membrane to a 1.5 mL centrifuge tube and store it in a -80℃ refrigerator.

[0035] The filtered membrane was cut into small pieces with sterile scissors, and algal DNA was extracted according to the instructions of the TaKaRa MiniBEST Universal GenomicDNA Extraction Kit Ver.5.0 (Takara Bio Inc. (Dalian)). The DNA was dissolved in 40 μL of elution buffer and stored at -20℃ for later use. (2) Use universal primers for the ITS region of eukaryotic algae: SEQ ID NO.7-TW28: 5'-GGGATCCGTTTCCGTAGGTGAACCTGC; SEQ ID NO.8-AB81: 5'-GGGATCCATATGCTTAAGTTCAGCGGGT; DNA from Karenia longiformis was amplified by PCR.

[0036] Use universal primers for LSU D1-D3 regions. SEQ ID NO.9-D1R: 5'-ACCCGCTGAATTTAAGCATA; SEQ ID NO.10-D3Ca: 5'-ACGAACGATTTGCACGTCAG; PCR amplification was performed on *Karenella paisleyi*.

[0037] The PCR reaction system was as follows: 10 μL Super Mix (TransGen Biotech Co., Ltd. (Beijing)), 7 μL ddH2O, 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), and 1 μL DNA sample. The amplification program for the ITS region primers was: 94℃ for 3 min, 94℃ for 30 sec, 57℃ for 30 sec, 72℃ for 1 min, 38 cycles, 72℃ for 6 min; the amplification program for the LSU region primers was: 94℃ for 3 min, 94℃ for 1 min, 55℃ for 1.5 min, 72℃ for 1 min, 35 cycles, 72℃ for 10 min. After preliminary observation by 1% agarose gel electrophoresis confirming that the PCR products met the target fragment length, they were sent to BGI Genomics (Shenzhen) for sequencing.

[0038] The Clustal software was used to analyze the sequence differences between the target algae's ITS or LSU region and the control algae to identify the specific regions of the target algae. Specific primers and TaqMan probes were designed in Beacon Designer 7, and the specificity of the primers and probes was initially verified using NCBI-Primer Plast. The successfully verified primers and probes were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. Fluorescent reporter groups and fluorescent quencher groups were labeled at the 5' and 3' ends of the TaqMan probes, respectively.

[0039] (3) Primer and TaqMan probe specificity test Using *Karenella longifolia* and *Karenella butterflyi* as target algae, Karenia hui, Coscinodiscus radiatus, Gymnodinium impudicum, Karlodinium australe, Karlodinium decipien, Karlodinium digitatum,Karlodinium elegans,Karlodinium veneficum,Karlodinium zhouanum、Takayama acrotrocha As a control algae, L1 medium (salinity approximately 33±2, pH approximately 8.0±2) was used in a light incubator (temperature 20±1℃, light intensity 80-100 μmol·s⁻¹). -1 ·m -2 Algal culture was carried out in a light-dark ratio of 12h:12h.

[0040] Take 1 mL of algal cells that have grown to the exponential stage, fix them with Luger's reagent and mix well. Take 100 μL of the sample and observe it under a microscope in a plankton counting frame. Filter 300 mL of algal solution using a magnetic filter and a 0.22 μm mixed cellulose membrane. Discard the filtrate, transfer the membrane to a 1.5 mL centrifuge tube, and store it in a -80℃ freezer.

[0041] Cut the filtered membrane into small pieces with sterile scissors and extract algal DNA according to the instructions of TaKaRa MiniBEST Universal GenomicDNA Extraction Kit Ver.5.0. Dissolve the DNA in 40 μL of elution buffer and store at -20℃ for later use.

[0042] Equal volumes of 5 μL of various control algal DNA solutions were mixed to prepare control algal DNA solutions. The DNA of *Karenella longiformis*, *Karenella butterflyi*, control algal DNA, and deionized water (negative control) were amplified using the TaqMan qPCR reaction system to verify the specificity of the primers and TaqMan probes for the two algae species. The amplified products of the target algae were sent to BGI Genomics (Shenzhen) for sequencing, and the sequences were compared for homology on NCBI-Blast to further determine the specificity of the primers and probes.

[0043] (4) Specificity test results of primers and TaqMan probes Primers L2F and L2R and probe L2P only amplified DNA from *Karenella longifolia*, while no amplification signal was observed in the control algae and negative control (results as shown in Figure 1). Figure 1 (As shown); sequencing results of the qPCR products, compared with the NCBI-Blast genome, revealed that the specific amplification product was *Karenella longiformis*, further confirming that the qPCR had species-level specific amplification for *Karenella longiformis*. P-F1, P-R1, and P-P1 generated positive amplification signals for *Karenella pachycarpa* DNA, while the control algae and negative control showed no amplification curves (results are shown in Figure 1). Figure 2 (As shown in the figure); the sequencing results of the qPCR products were compared with the species source using NCBI-Blast, and the results showed that the amplified products all came from *Karenella papilioni*, further confirming that the qPCR has species-specific amplification of *Karenella papilioni*.

[0044] Depend on Figure 1 , 2It can be seen that the specific primers and probes for *Karenella longiformis* and *Karenella davidii* designed in this invention only amplify the DNA of *Karenella longiformis* and *Karenella davidii*, further confirming that this qPCR has species-level specificity for *Karenella longiformis* and *Karenella davidii*. Therefore, the primer sets and probes designed in this invention for *Karenella longiformis* and *Karenella davidii* are highly specific and can be used for the accurate identification of these two algae, providing relatively accurate and effective technical support for the detection of red tides.

[0045] Example 2 In Example 2 of this invention, plasmid standard curves, cell standard curves, and cell-plasmid standard curves were established for *Karenella longiformis* and *Karenella pachymorpha*, respectively. The specific steps are as follows: (1) Establishment of plasmid standard curve: A. Preparation of recombinant plasmids: DNA from *Karenella longifolia* was amplified by PCR using universal primers TW28 (SEQ ID NO.7: 5'-GGGATCCGTTTCCGTAGGTGAACCTGC) and AB81 (SEQ ID NO.8: 5'-GGGATCCATATGCTTAAGTTCAGCGGGT) for the ITS region of eukaryotic algae.

[0046] PCR amplification of *Karenella papilosa* was performed using LSU universal primers D1R (SEQ ID NO.9: 5'-ACCCGCTGAATTTAAGCATA) and D3Ca (SEQ ID NO.10: 5'-ACGAACGATTTGCACGTCAG) for the D1-D3 regions.

[0047] The PCR reaction system was as follows: 10 μL SuperMix, 7 μL ddH2O, 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), and 1 μL DNA sample. The amplification program for the ITS region primers was: 94℃ for 3 min, 94℃ for 30 sec, 57℃ for 30 sec, 72℃ for 1 min, 38 cycles, 72℃ for 6 min; the amplification program for the LSU region primers was: 94℃ for 3 min, 94℃ for 1 min, 55℃ for 1.5 min, 72℃ for 1 min, 35 cycles, 72℃ for 10 min. After preliminary observation by 1% agarose gel electrophoresis confirming that the PCR products met the target fragment length, they were sent to BGI Genomics (Shenzhen) for sequencing. After confirming the PCR amplified sequence as the desired algal species by comparison on the NCBI website, BGI Genomics synthesized the recombinant plasmid. The mass concentration of the recombinant plasmid was measured using a Picodrop ultra-micro UV spectrophotometer. The formula was: Copy number concentration (copies / μL) = [Mass concentration (ng / μL) × 10] -9 ×6.02×10 23 ] / (base pairs × 660), converting the mass concentration of the recombinant plasmid to the copy number concentration.

[0048] The recombinant plasmid was serially diluted 10-fold, and at 10 2 ~10 8 Six consecutive concentrations of recombinant plasmids (copies / μL) were used as templates for TaqMan qPCR amplification of *Karenella longiformis* and *Karenella davidii*. The amplification system for both species was: 10 μL Probe qPCR Super PreMix, 0.4 μL forward primer (10 μM), 0.4 μL reverse primer (10 μM), 0.2 μL TaqMan probe (10 μM), 8 μL ddH2O, and 1 μL DNA template. The amplification program was: 37℃ for 2 min, 95℃ for 10 min, 95℃ for 10 sec, and 60℃ for 34 sec, for 40 cycles. Amplification curves for different algal species were obtained by plotting the number of reaction cycles on the x-axis and the real-time fluorescence signal intensity on the y-axis. Using the logarithm of the recombinant plasmid copy number (lgX) as the x-axis and the cycle threshold (Ct) as the y-axis, standard curves for plasmids of *Karenella longiformis* and *Karenella butterflyi* were obtained, and their amplification efficiency (E) and correlation coefficient r were calculated. The results are as follows: Karenia longifolia: Its plasmid standard curve regression equation is: y = -3.457x + 38.727 (where: x represents the logarithm of the recombinant plasmid copy number (base 10), y represents the Ct value), the correlation coefficient r = -0.997, and the amplification efficiency is 94.76%. Figure 3 As shown.

[0049] *Karenella paisleyi*: Its plasmid standard curve regression equation is: y = -3.27x + 41.015 (where: x represents the logarithm of the recombinant plasmid copy number (base 10), and y represents the Ct value), with a correlation coefficient r = -0.998 and an amplification efficiency of 102.20%. Figure 4 As shown.

[0050] (2) Establishment of cell standard curve: Algal cells of *Karenella longiformis* and *Karenella papilioni* that had reached the exponential growth stage were counted using an optical microscope. 30 mL of the algal solution was filtered, and DNA was extracted. The number of algal cells per microliter of DNA solution was calculated. The DNA solution was serially diluted 10-fold to obtain a target algal density of 10-1. 0 -10 4 DNA standard samples were prepared at a concentration of cells / μL. TaqMan qPCR amplification was performed using the DNA solution as a template. A standard curve was obtained by plotting the logarithm of cell number (lgX) on the x-axis and Ct value on the y-axis. The amplification efficiency and correlation coefficient r were calculated, and the results are as follows: *Karenella longifolia*: Its cell standard curve regression equation is: y = -3.729x + 29.751 (where: x represents the logarithm of the algal cell number (base 10), y represents the Ct value), correlation coefficient r = -0.999, amplification efficiency of 85.43%, such as... Figure 5 As shown.

[0051] *Carya chapensis*: Its cell standard curve regression equation is: y = -3.545x + 26.672 (where: x represents the logarithm of the algal cell number (base 10), y represents the Ct value), correlation coefficient r = -0.997, amplification efficiency of 91.45%, such as... Figure 6 As shown.

[0052] (3) Establishment of cell-plasmid standard curve Combining the plasmid standard curve and cell standard curve mentioned above, with the horizontal axis representing the logarithm of the recombinant plasmid copy number (base 10) and the vertical axis representing the logarithm of the number of cells per microliter of DNA solution (base 10), the linear relationship between plasmid copy number and algal cell number was obtained, as shown below: Karenia longifolia: The regression equation for the cell-plasmid standard curve of Karenia longifolia is y = 0.927x - 2.407, as shown below. Figure 7As shown in the figure. Where x represents the logarithm of the recombinant plasmid copy number base 10, and y represents the logarithm of the cell number base 10.

[0053] *Karenella papilosa*: The regression equation for the cell-plasmid standard curve of *Karenella papilosa* is y = 0.922x - 4.046, as shown below. Figure 8 As shown in the figure. Where x represents the logarithm of the recombinant plasmid copy number base 10, and y represents the logarithm of the cell number base 10.

[0054] (4) Sensitivity test: 10 respectively 0 ~10 8 copies / μL recombinant plasmid, 10 -2 ~10 4 DNA extracted from algal cells per μL was used as a template for TaqMan qPCR amplification. The plasmid copy number of four Karenia species and the minimum detectable concentration in cells were calculated.

[0055] Karenia longiformis: The limit of detection (LOD) for the recombinant plasmid of Karenia longiformis is 2.67 copies / μL, indicating high detection sensitivity. The minimum detectable cell count per μL of DNA solution is 0.039 cells. Based on the formula: Algal cells in water sample (cells / L) = [Number of cells corresponding to DNA solution (cells / μL) × Total DNA (μL)] / Water sample volume (L), the minimum algal density of Karenia longiformis in the water is calculated to be 5 cells / L.

[0056] *Karenella papilosa*: The limit of detection (LOD) for the recombinant plasmid of *Karenella papilosa* is 1.73 copies / μL, indicating high detection sensitivity. The minimum detectable cell count per μL of DNA solution is 0.044 cells. Based on the formula: Algal cells in water sample (cells / L) = [Number of cells corresponding to DNA solution (cells / μL) × Total DNA (μL)] / Water sample volume (L), the minimum algal density corresponding to *Karenella papilosa* in the water is calculated to be 5 cells / L.

[0057] (5) Repeatability test With 10 4 ~10 6 Recombinant plasmids of copies / μL, 10 2 ~10 4DNA extracted from cells at a concentration of cells / μL was used as a template for intra-group and inter-group repeatability tests. Intra-group repeatability: Three copies of each of the three concentrations of recombinant plasmid and DNA solution were used as templates. The Ct values ​​were obtained and substituted into the following formula to calculate the intra-group coefficient of variation. Inter-group repeatability: Three independent experiments were conducted using the three concentrations of recombinant plasmid and DNA solution as one group, and the coefficient of variation was calculated. Coefficient of variation (CV%) = Standard deviation (SD) / Mean × 100%. If the coefficient of variation is less than 2%, the repeatability is considered good.

[0058] Karenia longifolia: The concentration range selected was 2.67 × 10⁻⁶. 4 -2.67×10 6 Using recombinant plasmids of *Karenella longifolia* (copies / μL) as templates, intra- and inter-group reproducibility were tested. The intra- and inter-group coefficients of variation of the recombinant plasmids of *Karenella longifolia* at different concentration gradients were both less than 2%, indicating good experimental reproducibility.

[0059] Papilionella kallenii: The selected concentration range was 1.73 × 10⁻⁶. 4 -1.73×10 6 Using recombinant plasmids of *Karenella pulvinata* (copies / μL) as templates, intra- and inter-group reproducibility were tested. The intra- and inter-group coefficients of variation of the recombinant plasmids of *Karenella pulvinata* at different concentration gradients were both less than 2%, indicating good experimental reproducibility.

[0060] Example 3 Example 3 of this invention detected the number of *Karenella longiformis* and *Karenella butterflyi* in simulated field samples. The specific steps are as follows: (1) Preparation of field simulation samples: Take 30 mL of each algae, with a density of 2.4 × 10⁻⁶. 4 cells / mL, 4.5×10 4 cells / mL, 5.2×10 4 cells / mL, 5.8 × 10 4 A solution of *Karenella mikimotoi*, *Karenella saddle-shaped*, *Karenella longiformis*, and *Karenella butterfly-shaped* was prepared by mixing these solutions with 1000 mL of unsterilized seawater from the Shazikou sea area of ​​Qingdao to prepare laboratory-scale field simulation samples. DNA was extracted from the simulation samples and diluted to three different concentrations to ensure that each DNA sample contained a different number of cells.

[0061] (2) Using the DNA from the above-obtained field-simulated samples as templates, the probe and primer combinations shown in SEQ ID NO.1~SEQ ID NO.6 were used. The amplification system and amplification program of Karenia longiformis and Karenia davidii in step (1) of Example 2 were adopted to perform TaqMan qPCR fluorescence quantitative amplification to obtain Ct values. The Ct values ​​obtained from the amplification of Karenia longiformis and Karenia davidii were substituted into the regression equation of the standard curve of the plasmid of Karenia longiformis and Karenia davidii in Example 2 to calculate the copy number of recombinant plasmid DNA of Karenia longiformis and Karenia davidii in the sample. Then, the cell number and plasmid copy number were calculated by using the regression relationship between the cell number and plasmid copy number established in the cell-plasmid standard curve obtained by combining the cell standard curve and the plasmid standard curve (Table 2). The differences between the cell density of the four algae under microscopic examination and the cell density obtained by fluorescence quantitative PCR were compared by SPSS t-test.

[0062] The results of the single-sample t-test showed that there was no significant difference between the TaqMan qPCR detection results and the number of algal cells observed under a microscope (P>0.05), with a detection rate of 100%. This indicates that the method has a good detection rate for the target algal species, and the TaqMan qPCR detection method established in this invention can be further used for on-site sample detection.

[0063] Table 2 compares the results of TaqMan qPCR detection with those of optical microscopy counting.

[0064] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A probe and primer composition for TaqMan quantitative PCR detection of *Karenella longspinata* and *Karenella pachyphylla*, characterized in that, The composition of the probe primer composition is as follows: Karenia longifolia: SEQ ID NO.1-L2F:GCTTCAGTGTCAATATGC; SEQ ID NO.2-L2R: CAGACAAAGCAACAGATG; SEQ ID NO.3-L2P: VIC-TATGCCACCGTCAACCTGTCA-MGB; Butterfly-shaped Karenia: SEQ ID NO.4-P-F1: GGTGTTGTCTAATGTGATG; SEQ ID NO.5-P-R1: CCTCAATTATGAACTGACG; SEQ ID NO. 6-P-P1: FAM-CAAGCAACCAGTATCGCATCCAG-BHQ1.

2. The probe and primer composition for TaqMan quantitative PCR detection of *Karenella longiformis* and *Karenella pachymorpha* according to claim 1, characterized in that, The target genes used for TaqMan quantitative PCR detection of Karenia longiformis and Karenia davidii are the ITS region of Karenia longiformis ribosomal DNA and the LSU D1-D3 region of Karenia davidii ribosomal DNA, respectively.

3. A TaqMan fluorescence quantitative detection method for Karenia longiformis and Karenia davidii, characterized in that, Includes the following steps: (1) Extract DNA from the sample to be tested, and use the probe and primer combination shown in SEQ ID NO.1~SEQ ID NO.6 to amplify the extracted DNA by TaqMan qPCR. After the amplification is completed, the cycle threshold (Ct) values ​​of Karenia longiformis and Karenia butterfly are detected respectively. (2) Based on the Ct value obtained in step (1), the cell number of *Karenella longiformis* and *Karenella davidii* is calculated using the plasmid standard curve, cell standard curve, and cell-plasmid standard curve, respectively.

4. The TaqMan fluorescence quantitative detection method for *Karenella longiformis* and *Karenella pachymorpha* according to claim 3, characterized in that, The amplification system for TaqMan qPCR amplification in step (1) is as follows: 10 μL Probe qPCR Super PreMix, 0.4 μL forward primer, 0.4 μL reverse primer, 0.2 μL TaqMan probe, 8 μL ddH2O, and 1 μL DNA template; the amplification program is as follows: 37℃ for 2 min, 95℃ for 10 min, 95℃ for 10 sec, 60℃ for 34 sec, for 40 cycles.

5. The TaqMan fluorescence quantitative detection method for *Karenella longiformis* and *Karenella pachymorpha* according to claim 3, characterized in that, The plasmid standard curve mentioned in step (2) is as follows: Karenia longifolia: y = -3.457x + 38.727; *Karenella pulvinata*: y = -3.27x + 41.015; Where x represents the logarithmic number of the recombinant plasmid base 10, and y represents the Ct value.

6. The TaqMan fluorescence quantitative detection method for *Karenella longspinata* and *Karenella pachyphylla* according to claim 3, characterized in that, The cell standard curve mentioned in step (2) is as follows: Karenia longifolia: y = -3.729x + 29.751; *Carobronchiformes*: y = -3.545x + 26.672; Where x represents the logarithmic number of the recombinant plasmid base 10, and y represents the Ct value.

7. The TaqMan fluorescence quantitative detection method for *Karenella longspinata* and *Karenella pachyphylla* according to claim 3, characterized in that, The cell-plasmid standard curve mentioned in step (2) is as follows: Karenia longifolia: y = 0.927x - 2.407; *Karenella paisleyi*: y = 0.922x - 4.046; Where x represents the logarithmic number of recombinant plasmid copies to the base 10, and y represents the logarithmic number of algal cells to the base 10.

8. A product for the TaqMan fluorescence quantitative detection of Karenia longiformis and Karenia pachyformis, characterized in that, The product comprises the probe and primer composition for TaqMan quantitative fluorescence detection of Karenia longiformis and Karenia davidii as described in claim 1.

9. The product for TaqMan dual fluorescence quantitative detection of *Karenella longifolia* and *Karenella pachyphylla* according to claim 7, characterized in that, The product includes any one of the following: testing reagents, testing kits, and testing chips.

10. The application of a TaqMan fluorescence quantitative detection product for Karenia longiformis and Karenia davidii as described in any one of claims 8 or 9 in the detection of Karenia longiformis and Karenia davidii.

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