Primer group for qualitative and quantitative detection of three types of fish toxicity karenia, detection method and application

Abstract

The invention provides a primer group for qualitative and quantitative detection of three types of fish toxicity karenia, a detection method and application, and belongs to the technical field of marine aquaculture guarantee. The primers as shown in SEQ ID NO.1-6 provided by the invention can be used for respectively identifying the species of the Karenia saddle, the Karenia longgroove and the Karenia butterfly; a plasmid standard curve and a cell standard curve are established in combination with an SYBR Green I fluorescent quantitative PCR technology, and specific identification and quantity determination of the three types of fish-toxic karenia are further realized. The method has the advantages of high specificity, high sensitivity, simplicity in operation, short time consumption and the like, can overcome the limitation of traditional morphological identification, can realize rapid and accurate qualitative identification and quantitative detection of a large number of samples, is time-saving and labor-saving, is relatively low in price, and can become an effective technical means for prediction, early warning and large-area monitoring of the Karenia red tide.

Description

Technical Field

[0001] The present invention relates to the field of marine aquaculture security technology, and in particular to a primer set, a detection method and an application thereof for qualitative and quantitative detection of three types of fish-toxic Karenia. Background Art

[0002] Karenia species belong to the order Gymnodiniales of the class Dinophyceae, the family Kareniaceae, and are a relatively concentrated group of toxic and harmful dinoflagellates. Algae in this family often cause red tides, leading to the mass death of marine life and farmed fish. For example, Karenia mikimotoi in Norway and Japan, Karenia brevis in the United States and New Zealand, Karenia digitata and Karenia longicanalis in Chile have all caused serious red tides. In addition, most species of the family Kareniaceae are toxic, such as Karenia brevis producing brevetoxins and Karenia saddleback producing gymnodimine, which seriously endanger the safety and development of aquaculture.

[0003] Saddle-shaped Karenia, butterfly-shaped Karenia, and long-grooved Karenia are also present in many Karenia red tides. These three types of Karenia are toxic to fish, and when red tides break out, they can cause the death of fish and other marine mammals, and even threaten human health. Therefore, the identification of toxic Karenia is crucial to the development of coastal aquaculture.

[0004] Karenaceae algae are small, have exposed cells, and have no deck structure. The similarity between species within the genus is extremely high. It is difficult to accurately identify this species using traditional species identification methods based on morphological differences under an optical microscope. However, studies have shown that the composition of toxins, toxin content, toxic targets, and mechanisms of action in cells of different Karenaceae species are not the same. Therefore, there is an urgent need for fast and efficient species identification technology to accurately identify the causative species of Karenaceae red tides in order to facilitate early warning of red tide disasters. Secondly, knowing the number of algal cells of the causative species during a Karenaceae red tide outbreak can provide a basis for predicting the occurrence and scale of the red tide. Therefore, quantitative detection of algal cells is also very necessary for the prediction and prevention of Karenaceae red tides.

[0005] There are two main types of detection technologies for the Karenia red tide, namely morphological detection technology and molecular detection technology. Among them, morphological detection technology is mainly based on optical microscopy technology and electron microscopy technology to achieve species identification and quantitative statistics of algae cells. Although electron microscopy technology can identify Karenia species at a higher magnification, the pre-treatment process of algae cells before the machine is complicated, time-consuming and labor-intensive, and easily causes cell deformation, which requires high operator requirements. Some conventional molecular detection technologies, such as molecular fingerprint analysis and fluorescence in situ hybridization, have problems such as poor specificity and low detection sensitivity. They cannot accurately identify different species of Karenia, nor can they achieve quantitative detection of algae cells. Therefore, it is urgent to establish a detection technology that can quickly and accurately identify the three common Karenia species in my country's coastal waters (long-grooved Karenia, saddle-shaped Karenia, butterfly-shaped Karenia), and can achieve quantitative detection of algae cells. Summary of the invention

[0006] The purpose of the present invention is to provide a primer set, a detection method and an application for the qualitative and quantitative detection of three types of fish toxic Karenia, which can realize the species identification of saddle-shaped Karenia, long-grooved Karenia and butterfly-shaped Karenia, and has the advantages of strong specificity, high sensitivity, simple operation and short time consumption.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a primer set for qualitative and quantitative detection of three types of fish toxic Karenia, the primer set comprising:

[0009] Karenia saddleii:

[0010]

[0011] Karenia longituba:

[0012]

[0013] Karenia butterfly:

[0014]

[0015] Preferably, the target gene used for qualitative and quantitative detection of three types of fish-toxic Karenia is the internal transcribed spacer region of ribosomal DNA.

[0016] The present invention also provides a method for qualitatively and quantitatively detecting three types of fish-toxic Karenia, comprising the following steps:

[0017] (1) Extracting DNA from the sample to be tested, using the primer set to perform SYBR Green I qPCR to detect three types of fish toxic Karenia, and measuring their C t value;

[0018] (2) Use the plasmid standard curve and the cell standard curve to obtain the cell number corresponding to the plasmid copy number.

[0019] The plasmid standard curve is as follows:

[0020] Karenia saddleii: y = -3.714x + 42.465;

[0021] Karenia castanea: y = -3.654x + 41.012;

[0022] Karenia longituba: y = -3.515x + 39.402;

[0023] Where y is C t Value, x is the logarithm of the plasmid copy number with base 10;

[0024] The cell standard curve is as follows:

[0025] Karenia saddleii: y = -3.379x + 29.829;

[0026] Karenia butterfly: y = -3.881x + 28.907;

[0027] Karenia longituba: y = -3.646x + 28.168;

[0028] Where x is the logarithm of the number of cells to the base 10, and y is the C t value;

[0029] (3) Using the logarithm of the plasmid copy number to the base 10 as the abscissa and the logarithm of the corresponding cell number to the base 10 as the ordinate, a regression equation of plasmid copy concentration and cell number was generated:

[0030] Karenia saddleii: y = 1.099x-3.740;

[0031] Karenia butterfly: y = 0.942x-3.119;

[0032] Karenia longituba: y = 0.964x-3.081;

[0033] Where y is the logarithm of the cell number with base 10, and x is the logarithm of the plasmid copy number with base 10.

[0034] The present invention also provides a product for qualitative and quantitative detection of three types of fish-toxic Karenia, wherein the product comprises the primer set for qualitative and quantitative detection of three types of fish-toxic Karenia.

[0035] Preferably, the product includes any one of a detection reagent, a detection kit and a detection chip.

[0036] The present invention also provides an application of a product for qualitative and quantitative detection of three types of fish-toxic Karenia to the detection of saddle-shaped Karenia, long-grooved Karenia, and castellated Karenia.

[0037] The beneficial effects of the present invention compared with the prior art are:

[0038] (1) The present invention provides a set of primers for species identification and cell quantity quantification of three types of fish toxic Karenia and a corresponding quantitative detection method. The primers shown in SEQ ID NO. 1 to 6 provided by the present invention can respectively realize species identification of Karenia saddlei, Karenia longifuga, and Karenia castellaniformis; combined with SYBR Green I fluorescent quantitative PCR technology, plasmid standard curves and cell standard curves can be established to further realize specific identification and quantity determination of the three types of fish toxic Karenia.

[0039] (2) The present invention has the advantages of strong specificity, high sensitivity, simple operation, and short time consumption. It can overcome the limitations of traditional morphological identification, save time and effort, and is relatively inexpensive. It can achieve rapid and accurate qualitative identification and quantitative detection of a large number of samples, and can become an effective technical means for the prediction, early warning and large-scale monitoring of Karenia red tides. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0041] Figure 1 Specific amplification curves of the three primer pairs in Example 1 of the present invention, wherein a is the specific amplification curve of the saddle-shaped Karenia primer pair S2Z / S2F qPCR; b is the specific amplification curve of the butterfly-shaped Karenia primer pair P3Z / P3Z qPCR; c is the specific amplification curve of the long-grooved Karenia primer pair L1Z / L1F qPCR; 1: negative control template Karlodinium zhouanum; 2: negative control template Takayama acrotrocha; 3: negative control template Karenia mikimotoi; 4: template butterfly-shaped Karenia; 5: template Karenia selliformis; 6: negative control template Karenia hui; 7: template Karenia longicanalis;

[0042] Figure 2 Specific melting curves of the three primer pairs in Example 1 of the present invention; a is the specific melting curve of the primer pair S2Z / S2F qPCR of Karenia saddleii; b is the specific melting curve of the primer pair P3Z / P3Z qPCR of Karenia castellata; c is the specific melting curve of the primer pair L1Z / L1F qPCR of Karenia longifolia. The red, blue and green curves represent three parallel samples of the same DNA template respectively;

[0043] Figure 3 is the plasmid standard curve of Karenia saddleii in Example 1 of the present invention;

[0044] Figure 4 The standard curve of the Karenia leucoderma plasmid in Example 1 of the present invention;

[0045] Figure 5 The plasmid standard curve of Karenia longituba in Example 1 of the present invention;

[0046] Figure 6 is the standard curve of Karenia saddleii cells in Example 1 of the present invention;

[0047] Figure 7 The standard curve of Karenia leucoderma cells in Example 1 of the present invention;

[0048] Figure 8 This is the standard curve of Karenia longituba cells in Example 1 of the present invention. DETAILED DESCRIPTION

[0049] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0050] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0051] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0052] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0053] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0054] Example 1

[0055] The embodiment of the present invention provides a method for species identification and quantitative detection of Karenia saddlei, Karenia castellani and Karenia longifuga, and the specific steps are as follows:

[0056] (1) DNA extraction

[0057] Take the algae cell culture fluid of Karenia saddle, Karenia butterfly, Karenia longicanalis, Karlodinium zhouanum, Takayama acrotrocha, Karenia mikimotoi, Karenia papilionacea, Karenia selliformis, Karenia hui, Karenia longicanalis, and collect the algae cells on the membrane by filtering with a 0.22 micron mixed cellulose ester filter. Use TaKaRa MiniBEST Universal Genomic DNA ExtractionKit Ver.5.0 DNA kit to extract the algae cell DNA on the membrane, and store the extracted DNA at -20°C for use. The DNA extraction step is carried out according to the kit instructions.

[0058] (2) Preparation of nucleic acid sequences of algae ITS region

[0059] The DNA extracted by the kit was used as the PCR template and the algae ITS region universal amplification primers were used. Know Perform PCR amplification.

[0060] The PCR amplification system consisted of 7 μL RNase Free dH2O, 10 μL 2×Easy Taq PCR super mix, 1 μL template DNA, 1 μL forward primer (10 μM), and 1 μL reverse primer (10 μM).

[0061] PCR reaction program: 94℃ pre-denaturation for 3min, 94℃ denaturation for 30sec, 57℃ annealing for 30sec, 72℃ extension for 1min, 38 cycles, and finally 72℃ extension for 6min. The PCR product was confirmed as the target fragment by gel electrophoresis and sent to the sequencing company for sequencing.

[0062] (3) Construction of recombinant plasmid

[0063] Take 4 μL of the above PCR product for 1.0% agarose gel electrophoresis, cut out the specific amplified fragment, and use the Midi Purification Kit agarose gel DNA recovery kit for gel recovery. Add 1 μL of vector PMD19-T vector and 5 μL of Solution 1 to 4 μL of gel recovery product, place the mixture at 16℃ for 2 hours, and then store it at 4℃.

[0064] Take 10 μL of the above mixture and add it to 50 μL of competent cells, then place it in a -20°C refrigerator for half an hour, warm bath at 42°C for 45 seconds, and let it stand on an ice box for 2 minutes. Take 500 μL of sterile LB culture medium and add it to the mixture, shake and incubate for 1.5 hours (37°C, 200 rpm). Spread the bacterial solution evenly on the blue-white screening culture medium, let it stand for 15 minutes to allow the culture medium to fully absorb the bacterial solution, place it in an incubator and invert it for 15 hours, and set the temperature to 37°C. Add 0.5 mg of ampicillin to a glass tube containing 5 mL of LB liquid culture medium, mix well, then pick up white colonies and add them to the glass tube. At the same time, set a blank group without a single colony for comparison during the culture process, and then place it in a shaker and incubate for 8 hours (225 rpm, 37°C). Use the TIANprep Mini Plasmid Kit for plasmid extraction. Using the extracted plasmid as a template, by Universal primers for amplification of algae ITS region and PCR amplification was performed, and the PCR product was sent to a sequencing company for sequencing. According to the sequencing results, it was further determined that the constructed plasmid contained the standard plasmid of the target Karenia ITS fragment.

[0065] (4) Design and synthesis of specific primers

[0066] According to the ITS region sequences of saddle-shaped Karenia, butterfly-shaped Karenia and long-grooved Karenia obtained in step (3), homology search was performed on NCBI, and sequences of algae species with greater similarity in the same family were searched from Genbank. The ITS region DNA sequences of Karenia species, Karlodinium species and Takayama species with higher sequence similarity and other common dinoflagellates and diatoms were downloaded. CLUSTALX software was used for sequence comparison analysis, and the ITS interval with greater interspecies differences was selected. Primerpremier 5.0 was used to design specific primers for saddle-shaped Karenia, butterfly-shaped Karenia and long-grooved Karenia. Primer BLAST analysis was used to obtain the specificity of the primers, and primer pairs with species specificity for saddle-shaped Karenia, butterfly-shaped Karenia and long-grooved Karenia were obtained. The primer pair sequence information is shown in Table 1, and the primers were synthesized by Gene Company.

[0067] Table 1 Primer sequences

[0068]

[0069]

[0070] (5) Specific primer screening

[0071] Take 2 μL of target algae DNA and control algae DNA respectively, and use the above-designed specific primers to perform PCR amplification with three Karen algae DNA and mixed algae DNA as templates. The PCR amplification system includes 7 μL RNase Free dH2O, 10 μL 2×Easy Taq PCR supermix, 1 μL template DNA, 1 μL forward primer (primer working solution concentration is 10 μM), and 1 μL reverse primer (primer working solution concentration is 10 μM). PCR reaction program: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 15 sec, annealing and extension for 1 min, 38 cycles. Confirm that the PCR product is the only target fragment that meets the amplification length by gel electrophoresis and send it to the sequencing company for sequencing.

[0072] The specificity of primers was determined by sequencing the PCR products, and specific primers for three algae, namely, Karenia saddleii, Karenia butterflyii, and Karenia longigulata, were obtained. and The specific primer pair for Karenia longituba is and The primer pair specific for Karenia sphenopsina is and

[0073] (6) Verification of primer pair effectiveness and specificity

[0074] DNA of three target algae (saddle-shaped Karenia, butterfly-shaped Karenia, and long-grooved Karenia) and control algae Karlodinium zhouanum, Takayama acrotrocha, Karenia mikimotoi, template butterfly-shaped Karenia (Karenia papilionacea); saddle-shaped Karenia (Kareniaselliformis), Karenia hui, and long-grooved Karenia (Karenia longicanalis) were taken and qPCR amplified using FastStart Universal SYBR Green Master (ROX) kit (Roche Ltd..Germany) with three pairs of primers. qPCR reaction was carried out on a fluorescence quantitative PCR instrument.

[0075] The amplification system included 6.8 μL RNase Free dH2O, 10 μL FastStart Universal SYBR Green Master (Rox), 2 μL template DNA, 0.6 μL forward primer (10 μM), and 0.6 μL reverse primer (10 μM).

[0076] Reaction procedure: 95℃ pre-denaturation for 10min, 95℃ denaturation for 15sec, annealing and extension for 1min, 46 cycles. After the reaction, the melting curve analysis of the qPCR reaction confirmed that each target algae was specifically amplified. Figure 1 .

[0077] Figure 1 It was shown that the primer set shown in SEQ ID NO. 1-6 could specifically amplify Karenia saddlei, Karenia butterflyi and Karenia longifuga, while the control algae had no amplification.

[0078] (7) Establishment of plasmid standard curve

[0079] The recombinant plasmids of known concentrations of Karenia saddlei, Karenia castellani, and Karenia longifuga were diluted 10 times in a gradient manner as standard gradient samples, and qPCR was performed on a fluorescent quantitative PCR instrument according to the system and procedure shown below. Three parallel samples were set for each concentration of plasmid.

[0080] The reaction system was 20 μL, including 6.8 μL RNase Free dH2O, 10 μL FastStart UniversalSYBR Green Master (Rox), 2 μL template DNA, 0.6 μL forward primer (primer stock solution concentration was 10 μM), and 0.6 μL reverse primer (primer stock solution concentration was 10 μM); reaction procedure: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 15 sec, annealing and extension for 1 min, and 46 cycles.

[0081] The annealing temperature of the fluorescence quantitative PCR detection reaction is: the annealing temperature of the qPCR reaction of the primer pair S2Z and S2F that specifically amplifies Karenia saddleonis is 55.4℃; the annealing temperature of the qPCR reaction of the primer pair L1Z and L1F that specifically amplifies Karenia longifuga is 57.8℃; the annealing temperature of the qPCR reaction of the primer pair P3Z and P3Z that specifically amplifies Karenia castellaniformis is 56.5℃.

[0082] The plasmid standard curves of Karenia saddlei, Karenia butterflyi and Karenia longigulata were obtained with the logarithm of plasmid copy number as the abscissa and the average Ct of three parallel samples as the ordinate ( Figures 3 to 5 The regression curve equation of Karenia saddlei is y=-3.714x+42.465 (r=0.991, E=85.88%), the regression curve equation of Karenia butterflyi is y=-3.654x+41.012 (r=0.992, E=87.79%), and the regression curve equation of Karenia longifuga is y=-3.515x+39.402 (r=0.990, E=92.53%). t value, x is the logarithmic value of the plasmid copy number with the base of 10; r is the regression coefficient, and the three standard curves have r≥0.99. E is the amplification efficiency.

[0083] (8) Establishment of cell standard curve

[0084] A certain number of cells of Saddle-shaped Karenia, Butterfly-shaped Karenia, and Long-grooved Karenia were collected respectively, and their DNA was extracted according to the steps shown in step (1). The DNA extract was diluted in a 5-fold gradient to obtain cell standard samples of the three algae. qPCR was performed on a fluorescent quantitative PCR instrument using the detection system of step (7). The logarithm of the cell number was used as the horizontal axis, and the C value of the three parallel samples was used as the horizontal axis. tThe average value is the ordinate, and the cell standard curves of Saddle-shaped Karenia, Castellated Karenia and Long-grooved Karenia are obtained respectively: the regression curve equation of Saddle-shaped Karenia is y=-3.379x+29.829 (r=0.997, E=97.67%), the regression curve equation of Butterfly-shaped Karenia is y=-3.881x+28.907 (r=0.990, E=80.99%), and the regression curve equation of Long-grooved Karenia is y=-3.646x+28.168 (r=0.993, E=88.05%). Where x is the logarithm of the number of cells with base 10, and y is the logarithm of C t value; r is the regression coefficient, and the r of the three standard curves is greater than or equal to 0.99; E is the amplification efficiency ( Figure 6-8 ).

[0085] (9) Conversion of cell concentration

[0086] According to the established plasmid standard curve and cell standard curve, the cell number corresponding to the plasmid copy number is obtained. Then, the logarithm of the plasmid copy number (base 10) is used as the abscissa, and the logarithm of the corresponding cell number (base 10) is used as the ordinate to generate the regression equation between the plasmid copy concentration and the cell number: Saddle-shaped Karenia: y = 1.099x-3.740; Butterfly-shaped Karenia: y = 0.942x-3.119; Long-grooved Karenia: y = 0.964x-3.081. Among them, y is the logarithm of the cell number with base 10, and x is the logarithm of the plasmid copy number with base 10.

[0087] (10) Testing system effectiveness test

[0088] 1) Mix three kinds of Karenia algae liquids of Karenia saddlei, Karenia butterflyi and Karenia longifolia with a certain cell concentration, extract DNA according to the steps shown in step (1), use the mixed DNA as a template, and use the detection system of step (7) to perform qPCR on a fluorescent quantitative PCR instrument. Use the plasmid standard curve of the detection system established in step (7), the cell standard curve of step (8) and the regression equation of plasmid copy concentration and cell number in step (9) to calculate the algae cell concentration detected by qPCR to test the effectiveness of the detection system established by the present invention.

[0089] The test results are shown in Table 2. The qPCR test results are close to the actual cell number obtained by microscopic examination, and samples containing only a few algal cells can also be effectively detected. The t-test showed that the P values ​​between the actual cell number and the qPCR detection value were all greater than 0.05, and there was no significant difference. This shows that the detection method has high specificity and effectiveness for Karenia saddlei, Karenia butterflyi and Karenia longifolia.

[0090] Table 2 Comparison of microscopic counts and fluorescence quantitative PCR test results of simulated sample 1

[0091]

[0092] 2) After a certain amount of target algae and control algae liquid are mixed, DNA is extracted according to the steps shown in step (1), and qPCR is performed on a fluorescent quantitative PCR instrument using the mixed DNA as a template and the detection system of step (7). The plasmid standard curve of the detection system established in step (7), the cell standard curve of step (8) and the regression equation of plasmid copy concentration and cell number in step (9) are used to calculate the algae cell concentration detected by qPCR to test the effectiveness of the detection system established by the present invention. The test results are shown in Table 3. The qPCR detection results are similar to the microscopic results. The addition of control algae to the sample has little effect on the detection of target algae, which further proves the specificity and effectiveness of the detection method. The t-test shows that the P values ​​between the actual cell number and the qPCR detection value are all greater than 0.05, and there is no significant difference. Moreover, this method can still be applied to quantitative detection when the algae density is low.

[0093] Table 3 Comparison of microscopic counts and fluorescence quantitative PCR test results of simulated sample 2

[0094]

[0095]

[0096] 3) A certain amount of target algae was mixed with Qingdao offshore seawater to simulate field samples, and further test whether the method can be adapted to the test of field samples. DNA was extracted according to the steps shown in step (1), and the mixed DNA was used as a template, and qPCR was performed on a fluorescent quantitative PCR instrument using the detection system of step (7). The plasmid standard curve of the detection system established in step (7), the cell standard curve of step (8), and the regression equation of plasmid copy concentration and cell number in step (9) were used to calculate the algae cell concentration detected by qPCR to test the effectiveness of the detection system established by the present invention. The test results are shown in Table 4. The qPCR test results are close to the actual cell number, indicating that samples with a lower cell number can also be detected and can be applied to the detection of algae in the field environment. The t-test showed that the P values ​​between the actual cell number and the qPCR detection value were all greater than 0.05, and there was no significant difference. The test results of the simulated seawater environment showed that the present method has good specificity and can be used for the determination of field samples.

[0097] Table 4 Comparison of microscopic counts and fluorescence quantitative PCR test results of simulated sample 3

[0098]

[0099] Example 2

[0100] Example 2 of the present invention uses the primers of Example 1 to quantitatively detect three species of Karenia in 8 seawater samples collected from Beibu Gulf, Guangxi, and the specific steps are as follows:

[0101] 500 mL of surface water was collected on site using a high-density polyethylene bucket, and the larger plankton and visible particles in the water sample were removed by filtering with a 200 μm microporous filter membrane, and then filtered with a 0.22 μm mixed cellulose ester filter membrane to collect the plankton on the membrane. The filter membrane was transferred to a sterile 5 mL centrifuge tube and stored and transported at -80°C. DNA was extracted using the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 DNA kit, and the extracted DNA was stored at -20°C for use. The DNA extraction steps were carried out according to the kit instructions.

[0102] Using the detection system established by the present invention, qPCR detection was performed on a fluorescent quantitative PCR instrument. Using the plasmid standard curve, cell standard curve and the regression equation of plasmid copy concentration and cell number of the detection system established by the present invention, the algae cell concentration detected by qPCR was calculated according to the sampling volume. The results are shown in Table 5.

[0103] Table 5 Results of qPCR detection of algal cell concentrations of three species of Karenia in Beibu Gulf (cells / L)

[0104]

[0105] As shown in Table 5, the specific primers of the present application can specifically detect and quantify the cells of Karenia saddlei, Karenia butterflyi, and Karenia longifuga.

[0106] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A primer set for qualitative and quantitative detection of three types of fish toxic Karenia, characterized in that: The composition of the primer set is: Karenia saddleii: SEQ ID NO.1-S2Z: 5'-TGTTTGGACGATGTGCTACTT-3'; SEQ ID NO.2-S2F: 5'-CACCTTAATCCACAGATACGC-3'; Karenia longituba: SEQ ID NO.3-L1Z: 5'-AATACAACTTTCAGCGACGGA-3'; SEQ ID NO.4-L1F: 5'- ACCAGGCTATCAGAGCACCA-3'; Karenia butterfly: SEQ ID NO.5-P3Z: 5'-ACGATATTGGACCGTCTTCC-3'; SEQ ID NO. 6-P3Z: 5'-CACAAAGCCCAAGCACTAAA-3'.

2. The primer set for qualitative and quantitative detection of three types of fish toxic Karenia according to claim 1, characterized in that: The target gene used for qualitative and quantitative detection of three fish-toxic Karenia species is the internal transcribed spacer region of ribosomal DNA.

3. A method for qualitative and quantitative detection of three types of fish toxicity Karenia, characterized in that: The steps include: (1) Extracting DNA from the sample to be tested, using the primer set described in claim 1 to perform SYBR Green I qPCR to detect three types of fish toxic Karenia, and measuring their C t value; (2) Use the plasmid standard curve and the cell standard curve to obtain the cell number corresponding to the plasmid copy number. The plasmid standard curve is as follows: Saddle-shaped Karenia: y=-3.714x+42.465; Karenia butterfly: y=-3.654x+41.012; Karenia longituba: y=-3.515x+39.402; Where y is C t Value, x is the logarithm of the plasmid copy number with base 10; The cell standard curve is as follows: Saddle-shaped Karenia: y=-3.379x+29.829; Karenia butterfly: y=-3.881x+28.907; Karenia longituba: y=-3.646x+28.168; Where x is the logarithm of the number of cells to the base 10, and y is the C t value; (3) Generate a regression equation between plasmid copy concentration and cell number, using the logarithm of the plasmid copy number to base 10 as the horizontal axis and the logarithm of the corresponding cell number to base 10 as the vertical axis; Saddle-shaped Karenia: y=1.099x-3.740; Karenia butterfly: y=0.942x-3.119; Karenia longituba: y=0.964x-3.081; Where y is the logarithm of the cell number with base 10, and x is the logarithm of the plasmid copy number with base 10.

4. A product for qualitative and quantitative detection of three types of fish toxic Karenia, characterized in that: The product includes the primer set for qualitative and quantitative detection of three types of fish-toxic Karenia as described in claim 1.

5. The product for qualitative and quantitative detection of three types of fish toxicity Karenia according to claim 4, characterized in that: The product includes any one of a detection reagent, a detection kit and a detection chip.

6. Use of the product for qualitative and quantitative detection of three types of fish-toxic Karenia as claimed in any one of claims 4 or 5 in detecting saddle-shaped Karenia, long-grooved Karenia and butterfly-shaped Karenia.

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