Probe primer composition, method and product for quantitative detection of karenia longgroove and karenia butterfly

Abstract

The invention provides a probe primer composition, a method and a product for quantitative detection of karenia longgroove and karenia butterfly, and belongs to the technical field of microbiological detection. The invention provides a group of probe primer compositions which are shown in SEQ ID NO.1-6 and are used for quantitative detection of the karenia longgroove and the karenia butterfly. The composition can be used for quantitative detection of the karenia longgroove and the karenia butterfly respectively, and is strong in specificity and high in sensitivity. The method overcomes the limitation of traditional morphological identification, is time-saving and labor-saving, is simple, and can realize rapid and accurate qualitative identification and quantitative detection of a large number of samples. The method can be used for accurately identifying the karenia longguichanensis and the karenia butterfly, provides accurate and effective technical support for detection of harmful red tide, and also can provide a new technical means for early warning and large-area monitoring of the red tide of the karenia.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial detection, and in particular to a probe primer composition, method and product for quantitative detection of Karenia longituba and Karenia leucophylla. Background Art

[0002] At present, red tide has become a serious ecological disaster problem in many sea areas around the world, especially in coastal areas. Dinoflagellates are the main group that causes marine disasters. Among them, the red tide caused by Karenia has attracted widespread attention from all walks of life due to its wide distribution range, high frequency of occurrence and large disaster losses. Among them, Karenia longituba can produce polyunsaturated fatty acids in its body, which makes it show hemolytic activity, while Karenia butterfly can produce short toxin PbTx-2 in its cells, and it is believed that the algae may be potentially toxic. The harmful red tides caused by both may pose a threat to marine ecological security and endanger human health. Both types of Karenia have records of red tide outbreaks in my country's coastal waters and have caused the death of farmed fish. Therefore, in order to better prevent and control harmful red tide events caused by Karenia longifolia and Karenia butterfly, first of all, a highly specific method is needed to accurately identify Karenia longifolia and Karenia castellata; secondly, in order to understand the occurrence mechanism of red tides caused by Karenia longifolia and Karenia butterfly, it is also necessary to establish a technology that can quantitatively detect the number of algae cells of Karenia longifolia and Karenia butterfly.

[0003] At present, the detection technologies for these two types of algae mainly include morphological detection technology, cytochrome-based detection technology and high-throughput detection technology based on molecular biology. The most commonly used method for identifying red tide algae in my country's local monitoring stations is to use optical microscopy to identify and count algae species.

[0004] Although the morphological analysis method using an optical microscope is the most intuitive and commonly used method for detecting red tide species, morphological classification requires extremely high professional knowledge of the identification personnel, and the identification takes a long time, making it difficult to conduct rapid and large-scale detection of algae species. In addition, when the target algae in the environmental sample are at low density, the detection capacity of the microscope is limited. Karenia is a type of naked dinoflagellate with individuals only about 20μm in size, and it is difficult to accurately identify a specific species under an optical microscope. However, studies have found that different types of Karenia have different toxin compositions and toxicity levels. Therefore, it is necessary to develop simpler and more sensitive detection methods to assist traditional microscopy detection methods in order to achieve accurate identification of toxic and harmful Karenia at the species level.

[0005] PCR detection technology is one of the fastest-growing and most widely used technologies in molecular biology. However, ordinary PCR technology can only identify the target species, but cannot perform quantitative analysis on its abundance. Fluorescence quantitative PCR (quantitative real-time PCR, qPCR) technology is a molecular biology technology developed on the basis of PCR. According to the different principles of fluorescent group binding, quantitative PCR can be divided into SYBR Green I dye method and TaqMan probe method. The SYBR Green I dye method has strong versatility, but because the dye has no specificity, it may bind to the amplification product or non-target sequence during the reaction process, resulting in a false positive experimental result. Therefore, it is urgent to find a faster, more sensitive and more specific quantitative detection method. Summary of the invention

[0006] The object of the present invention is to provide a probe primer composition, method and product for the quantitative detection of Karenia longifolia and Karenia pellucida, which can realize the quantitative detection of Karenia longifolia and Karenia pellucida, have good specificity and high sensitivity, and provide accurate and effective technical support for the detection of red tide.

[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 probe primer composition for TaqMan fluorescence quantitative detection of Karenia longituba and Karenia castellata, characterized in that the probe primer composition is composed of:

[0009] Karenia longituba:

[0010]

[0011] Karenia butterfly:

[0012]

[0013] Preferably, the target genes for TaqMan fluorescence quantitative detection of Karenia longifuga and Karenia pellucida are the ITS region of the ribosomal DNA of Karenia longifuga and the LSUD1-D3 region of the ribosomal DNA of Karenia pellucida, respectively.

[0014] The present invention also provides a TaqMan fluorescence quantitative detection method for Karenia longifolia and Karenia pellucida, comprising the following steps:

[0015] (1) extracting DNA from a sample to be tested, performing TaqMan qPCR amplification on the extracted DNA using a probe primer combination shown in SEQ ID NO.1 to SEQ ID NO.6, and after the amplification, detecting the Ct values ​​of Karenia longisulcata and Karenia pellucida respectively;

[0016] (2) According to the Ct value obtained in step (1), the cell numbers of Karenia longifuga and Karenia pellucida are calculated using the plasmid standard curve, the cell standard curve, and the cell-plasmid standard curve. The method is to substitute the Ct values ​​obtained by amplification of Karenia longifuga and Karenia pellucida into the regression equations of the plasmid standard curves of Karenia longifuga and Karenia pellucida to calculate the recombinant plasmid DNA copy numbers of Karenia longifuga and Karenia pellucida in the sample; and then calculate the cell numbers of Karenia longifuga and Karenia pellucida by 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.

[0017] The plasmid standard curve was established because the recombinant plasmid is stable even after long-term storage, and its quantity is easy to calculate by measuring the optical density. In addition, it can be used as a positive template to calculate the stability of the qPCR assay. We used two standard curves instead of the cell-based standard curve to improve the accuracy of cell counting caused by changes in DNA copy number in cells.

[0018] Preferably, the amplification system of TaqMan qPCR amplification in step (1) is: 10 μL Probe qPCR SuperPreMix, 0.4 μL forward primer, 0.4 μL reverse primer, 0.2 μL TaqMan probe, 8 μL ddH 2 O, 1 μL DNA template; amplification program: 37°C for 2 min, 95°C for 10 min, 95°C for 10 sec, 60°C for 34 sec, 40 cycles.

[0019] Preferably, the plasmid standard curve in step (2) is:

[0020] Karenia longituba: y = -3.457x + 38.727;

[0021] Karenia castanea: y = -3.27x + 41.015.

[0022] Wherein, x represents the logarithmic value (base 10) of the recombinant plasmid copy number, and y represents the Ct value.

[0023] Preferably, the cell standard curve in step (2) is:

[0024] Karenia longituba: y = -3.729x + 29.751;

[0025] Butterfly Karenia: y = -3.545x + 26.672.

[0026] Wherein, x represents the logarithmic value (base 10) of the recombinant plasmid copy number, and y represents the Ct value.

[0027] Preferably, the cell-plasmid standard curve in step (2) is:

[0028] Karenia longituba: y = 0.927x-2.407;

[0029] Butterfly Karenia: y = 0.922x-4.046.

[0030] Wherein, x represents the logarithmic value (base 10) of the recombinant plasmid copy number, and y represents the logarithmic value (base 10) of the algal cell number.

[0031] The present invention also provides a product for TaqMan fluorescence quantitative detection of Karenia longisulcata and Karenia sphenodonta, wherein the product comprises the probe primer composition for TaqMan fluorescence quantitative detection of Karenia longisulcata and Karenia castellaniformis according to claim 1.

[0032] Preferably, the product includes any one of a detection reagent, a detection kit and a detection chip.

[0033] The present invention also provides a product for TaqMan fluorescence quantitative detection of Karenia longisulcata and Karenia peltatum, and its application in detecting Karenia longisulcata and Karenia castellaniformis.

[0034] The beneficial effects of the present invention compared with the prior art are:

[0035] The present invention provides a group of probe primer compositions for quantitative detection of Karenia longifuga and Karenia pelargoniformis as shown in SEQ ID NOs. 1 to 6. The composition can perform quantitative detection on Karenia longifuga and Karenia castellata, has strong specificity, high sensitivity and low detection limit of algal cells, overcomes the limitations of traditional morphological identification, saves time and labor, and has a simple method. It can realize rapid and accurate qualitative identification and quantitative detection of a large number of samples, can be used for accurate identification of Karenia longifuga and Karenia pelargoniformis, provides relatively accurate and effective technical support for the detection of red tide, and also provides new technical means for early warning and large-area monitoring of Karenia algae red tide. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 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.

[0037] Figure 1The specific amplification result of Karenia longischii in Example 1 of the present invention, wherein 1 represents Karenia longischii DNA, 2 represents Karenia saddleii DNA, 3 represents Karenia mikimotoi DNA, 4 represents Karenia butterfly DNA, 5 represents control algae mixed DNA, and 6 represents a negative control;

[0038] Figure 2 is the specific amplification result of Karenia pellucida in Example 1 of the present invention, wherein 1 represents Karenia pellucida DNA, 2 represents Karenia saddleii DNA, 3 represents Karenia mikimotoi DNA, 4 represents Karenia longisulcata DNA, 5 represents control algae mixed DNA, and 6 represents a negative control;

[0039] Figure 3 This is a standard curve diagram of the Karenia longituba plasmid in Example 2 of the present invention;

[0040] Figure 4 This is a standard curve of the Karenia leucoderma plasmid in Example 2 of the present invention;

[0041] Figure 5 This is a standard curve diagram of Karenia longituba cells in Example 2 of the present invention;

[0042] Figure 6 This is a standard curve diagram of the butterfly Karenia cells in Example 2 of the present invention;

[0043] Figure 7 This is a standard curve of Karenia longituba cells-plasmids in Example 2 of the present invention;

[0044] Figure 8 This is a standard curve of the butterfly Karenia cell-plasmid in Example 2 of the present invention. DETAILED DESCRIPTION

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0050] Example 1

[0051] In Example 1 of the present invention, a probe primer combination for quantitative detection of Karenia longituba and Karenia castellata was prepared, and the detection effect of the probe primer combination was detected. The specific steps are as follows:

[0052] The ribosomal RNA (rRNA) of the genus Karenia is encoded by the ribosomal DNA (rDNA) in the nucleolus organizing region. The rDNA can be divided into the non-transcribed region (NTS), the external transcribed spacer (ETS), the 18SrDNA (SSU rDNA), the internal transcribed spacer (ITS, including ITS1, 5.8S rDNA, ITS2) and the 28SrDNA (LSU rDNA). Among them, the 18S rDNA sequence has a high sequence conservation in biological evolution and is usually used for species analysis at the phylum, class and order levels. The LSU and ITS sequences evolve faster than the 18S rDNA and have obvious sequence polymorphisms among different species, which can be used for species identification of different genera and species. Therefore, designing primers for these two segments can make species identification more specific. The present invention targets Karenia longituba and Karenia butterfly, searches for specific sites according to the ITS region of Karenia longituba and the LSU region of Karenia butterfly, designs corresponding specific primers and TaqMan probes, and thus constructs TaqMan qPCR detection technology for algae species, aiming to accurately identify them, thereby providing targeted technical support for the monitoring of the causes of harmful algal blooms.

[0053] (1) Take 1 mL of Karenia longituba and Karenia butterfly algae cells grown to the exponential phase, add Lugo reagent to fix and mix, take 100 μL of sample and observe under a microscope in a plankton counting frame, then filter 300 mL of algae liquid 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 in a -80°C refrigerator.

[0054] The filter membrane obtained by filtration was cut into pieces with sterilized scissors, and the algae DNA was extracted according to the instructions of the TaKaRa MiniBEST Universal GenomicDNA Extraction Kit Ver.5.0 (Takara Biotechnology Co., Ltd. (Dalian)). The DNA was dissolved in 40 μL of elution buffer and stored at -20°C for later use.

[0055] (2) Use universal primers for the ITS region of eukaryotic algae:

[0056]

[0057] PCR amplification of DNA from Karenia longituba

[0058] Use universal primers for LSU D1-D3 region

[0059]

[0060] PCR amplification of Karenia sphenops.

[0061] The PCR reaction system was as follows: 10 μL Super Mix (Quanshijin Biotechnology Co., Ltd. (Beijing)), 7 μL ddHO 2 O, 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 1 μL DNA sample. The amplification program for the ITS region primers is: 94°C 3min, 94°C 30sec, 57°C 30sec, 72°C 1min, 38 cycles, 72°C 6min; the amplification program for the LSU region primers is: 94°C 3min, 94°C 1min, 55°C 1.5min, 72°C 1min, 35 cycles, 72°C 10min. The PCR product was initially observed by 1% agarose gel electrophoresis to meet the target fragment length and then sent to BGI (Shenzhen) for sequencing.

[0062] Clustal software was used to analyze the sequence differences between the ITS or LSU region sequences of the target algae and the control algae to find the specific interval of the target algae. Specific primers and TaqMan probes were designed in Beacon Designer 7, and the specificity of primers and probes was preliminarily verified using NCBI-PrimerPlast. The successfully verified primers and probes were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis, and the fluorescent reporter group and fluorescent quencher group were labeled at the 5′ and 3′ ends of the TaqMan probe, respectively.

[0063] (3) Primer and TaqMan probe specificity test

[0064] Karenia longigulata and Karenia sphenoides were used as target algae, and Karenia hui, Coscinodiscusradiatus, Gymnodinium impudicum, Karlodinium australe, Karlodiniumdecipien, Karlodinium digitatum, Karlodinium elegans, Karlodinium veneficum, Karlodiniumzhouanum, and Takayama acrotrocha were used as control algae. L1 medium (salinity of about 33 ± 2, pH of about 8.0 ± 2) was used in a light incubator (temperature 20 ± 1 ° C, light intensity 80-100 μmol·s -1 ·m -2 , light-dark ratio of 12h:12h) for algae cultivation.

[0065] Take 1 mL of algal cells grown to the exponential phase, add Luger's reagent to fix and mix, take 100 μL of sample and observe it under a microscope in a plankton counting frame, filter 300 mL of algal liquid 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°C refrigerator.

[0066] The filter membrane obtained by filtration was cut into pieces with sterilized scissors, and the algae DNA was extracted according to the instructions of the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 kit. The DNA was dissolved in 40 μL of elution buffer and stored at -20°C for later use.

[0067] Take 5 μL of each control algae DNA solution and mix them in equal amounts to make a control algae DNA solution. Use the TaqMan qPCR reaction system to amplify the DNA of Karenia longifuga, Karenia butterfly, control algae DNA and deionized water (negative control) to verify the specificity of the primers and TaqMan probes of the two algae. The target algae amplification products were sent to BGI (Shenzhen) for sequencing, and the amplification product sequences were homologously compared on NCBI-Blast to further determine the specificity of the primers and probes.

[0068] (4) Primer and TaqMan probe specificity test results

[0069] Primers L2F, L2R and probe L2P only amplified the DNA of Karenia longituba, and no amplification signal was found in the control algae and negative control (results are shown in Figure 1 The sequencing results of qPCR products were compared with the species in NCBI-B1ast, and the specific amplification product was found to be Karenia longissipes, which further confirmed that the qPCR had species-level specific amplification for Karenia longissipes. P-F1, P-R1, and P-P1 produced positive amplification signals for Karenia castellata DNA, while the control algae and negative control had no amplification curve (the results are shown in Figure 2 The sequencing results of qPCR products were compared in NCBI-Blast, and the results showed that the amplified products were all from Butterfly Karenia, further confirming that the qPCR had species-specific amplification of Butterfly Karenia.

[0070] Depend on Figure 1 , 2 It can be seen that the Karenia longifolia specific primers and probes / Karenia pellucida specific primers and probes designed by the present invention only amplify the Karenia longifolia DNA / Karenia pellucida DNA, further confirming that the qPCR has species-level specific amplification for Karenia longifolia / Karenia pellucida. It can be seen that the primer sets and probes designed by the present invention for Karenia longifolia and Karenia pellucida are highly specific, can be used for the accurate identification of Karenia longifolia and Karenia pellucida, and provide more accurate and effective technical support for the detection of red tide.

[0071] Example 2

[0072] Example 2 of the present invention established plasmid standard curves, cell standard curves, and cell-plasmid standard curves for Karenia longituba and Karenia castellata, respectively, and the specific steps are as follows:

[0073] (1) Establishment of plasmid standard curve:

[0074] A. Preparation of recombinant plasmid: DNA of Karenia longissima 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.

[0075] PCR amplification of Karenia castellaniformis was performed using universal primers D1R (SEQ ID NO.9: 5'-ACCCGCTGAATITAAGCATA) and D3Ca (SEQ ID NO.10: 5'-ACGAACGATTTGCACGTCAG) in the LSU D1-D3 region.

[0076] The PCR reaction system was as follows: 10 μL SuperMix, 7 μL ddH 2 O, 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 1 μL DNA sample. Among them, the amplification program corresponding to the primers in the ITS region is: 94℃3min, 94℃30sec, 57℃30sec, 72℃1min, 38 cycles, 72℃6min; the amplification program corresponding to the primers in the LSU region is: 94℃3min, 94℃1min, 55℃1.5min, 72℃1min, 35 cycles, 72℃10min. The PCR product was initially observed by 1% agarose gel electrophoresis to meet the target fragment length and then sent to BGI (Shenzhen) for sequencing. After the PCR amplified sequence was compared on the NCBI website to determine the required algae species, BGI synthesized the recombinant plasmid. The mass concentration of the recombinant plasmid was measured by Picodrop ultra-micro UV spectrophotometer, according to the formula: copy number concentration (copies / μL) = [mass concentration (ng / μL) × 10 -9 ×6.02×10 23 ] / (number of base pairs × 660), and the mass concentration of the recombinant plasmid was converted into the copy number concentration.

[0077] The recombinant plasmid was diluted 10-fold and 2 ~10 8 Six consecutive concentrations of recombinant plasmids were selected as templates for TaqMan qPCR amplification of Karenia longifuga and Karenia pelargoniformis. The amplification systems of Karenia longifuga and Karenia castellata were: 10μL Probe qPCR Super PreMix, 0.4μL forward primer (10μM), 0.4μL reverse primer (10μM), 0.2μL TaqMan probe (10μM), 8μL ddH 2O, 1μL DNA template. The amplification program is: 37℃2min, 95℃10min, 95℃10sec, 60℃34sec, 40 cycles. The amplification curves of different algae species were obtained with the number of reaction cycles as the horizontal axis and the real-time fluorescence signal intensity as the vertical axis. The logarithm of the recombinant plasmid copy number (1gX) was used as the horizontal axis and the cycle threshold (Ct) as the vertical axis to obtain the plasmid standard curves of Karenia longuli and Karenia butterfly, and the amplification efficiency (efficiency, E) and correlation coefficient r were calculated. The results are as follows:

[0078] Karenia longituba: Its plasmid standard curve regression equation is: y = -3.457x + 38.727 (where: x represents the logarithmic value of the recombinant plasmid copy number (base 10), y represents the Ct value), the correlation coefficient r = -0.997. The amplification efficiency is 94.76%. Figure 3 shown.

[0079] Karenia leucoderma: Its plasmid standard curve regression equation is: y = -3.27x + 41.015 (where: x represents the logarithmic value of the recombinant plasmid copy number (base 10), y represents the Ct value), the correlation coefficient r = -0.998, and the amplification efficiency is 102.20%. Figure 4 shown.

[0080] (2) Establishment of cell standard curve:

[0081] Using an optical microscope, count the algal cells of Karenia longituba and Karenia castellanii that have grown to the exponential phase, and filter 30 mL of algal solution to extract its DNA, and calculate the number of algal cells corresponding to each microliter of DNA solution. Dilute the DNA solution 10 times in a gradient to obtain a target algal density of 10 0 -10 4 cells / μL DNA standard sample. TaqMan qPCR amplification was performed using the DNA solution as a template. The cell number logarithm (1gX) was used as the horizontal axis and the Ct value was used as the vertical axis to obtain the cell standard curve, and its amplification efficiency and correlation coefficient r were calculated. The results are as follows:

[0082] Karenia longituba: Its cell standard curve regression equation is: y = -3.729x + 29.751 (where x represents the logarithmic value of the number of algae cells (base 10), y represents the Ct value), the correlation coefficient r = -0.999, and the amplification efficiency is 85.43%. Figure 5 shown.

[0083] Karenia leucoderma: The cell standard curve regression equation is: y = -3.545x + 26.672 (where x represents the logarithmic value of the number of algae cells (base 10), y represents the Ct value), the correlation coefficient r = -0.997, and the amplification efficiency is 91.45%. Figure 6 shown.

[0084] (3) Establishment of cell-plasmid standard curve

[0085] Combining the above plasmid standard curve and cell standard curve, the horizontal axis is the logarithmic value of the recombinant plasmid copy number with a base of 10, and the vertical axis is the logarithmic value of the number of cells corresponding to each microliter of DNA solution with a base of 10. The curve equation of the linear relationship between the plasmid copy number and the algal cell number is obtained, and the results are as follows:

[0086] Karenia longituba: The regression equation of the Karenia longituba cell-plasmid standard curve is y=0.927x-2.407. Figure 7 As shown in the figure, x represents the logarithmic value of the recombinant plasmid copy number with a base of 10, and y represents the logarithmic value of the cell number with a base of 10.

[0087] Karenia castellaniformis: The regression equation of the Karenia sphenopsella cell-plasmid standard curve is y=0.922x-4.046. Figure 8 As shown in the figure, x represents the logarithmic value of the recombinant plasmid copy number with a base of 10, and y represents the logarithmic value of the cell number with a base of 10.

[0088] (4) Sensitivity test:

[0089] 10 0 ~10 8 copies / μL recombinant plasmid, 10 -2 ~10 4 TaqMan qPCR amplification was performed using DNA extracted from cells / μL algal cells as a template to calculate the plasmid copy numbers of the four Karenia species and the minimum detection concentration of the cells.

[0090] Karenia longituba: The minimum detection limit of Karenia longituba recombinant plasmid was 2.67 copies / μL, and the detection sensitivity was high. The minimum number of cells detected per microliter of DNA solution was 0.039 cells. According to the formula: Algal cells in water sample (cells / L) = [Number of cells corresponding to DNA solution (cells / μL) × Total DNA volume (μL)] / water sample volume (L), the minimum algal density corresponding to Karenia longituba in the water body was 5 cells / L.

[0091] Butterfly Karenia: The minimum detection limit of the recombinant plasmid of Butterfly Karenia is 1.73 copies / μL, and the detection sensitivity is high. The minimum number of cells detected per microliter of DNA solution is 0.044 cells. According to 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 algae density corresponding to Butterfly Karenia in water is 5 cells / L.

[0092] (5) Repeatability test

[0093] Take 10 4 ~10 6 copies / μL of recombinant plasmid, 10 2 ~10 4 The DNA extracted from cells / μL was used as a template to perform intra-group repeatability and inter-group repeatability tests. Intra-group repeatability: 3 concentrations of recombinant plasmid and DNA solution were used as templates, each in triplicate, and the Ct values ​​were obtained and substituted into the following formula to calculate the intra-group coefficient of variation. Inter-group repeatability: 3 independent experiments were performed with 3 concentrations of recombinant plasmid and DNA solution as a 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%, it can be considered that the repeatability is good.

[0094] Karenia longituba: Select the concentration range of 2.67×10 4 -2.67×10 6 The recombinant plasmid of Karenia longituba at 1000 copies / μL was used as a template to test the intra-group repeatability and inter-group repeatability. The intra-group and inter-group coefficients of variation of the recombinant plasmid of Karenia longituba at different concentration gradients were both less than 2%, indicating that the experimental repeatability was good.

[0095] Butterfly Karenia: Select the concentration range of 1.73×10 4 -1.73×10 6 The recombinant plasmid of Karenia sphenodontii with 100 copies / μL was used as a template to test the intra-group repeatability and inter-group repeatability. The intra-group and inter-group coefficients of variation of the recombinant plasmid of Karenia sphenodontii at different concentration gradients were both less than 2%, indicating that the experimental repeatability was good.

[0096] Example 3

[0097] Example 3 of the present invention detects the number of Karenia longituba and Karenia butterfly in field simulation samples, and the specific steps are as follows:

[0098] (1) Preparation of field simulation samples:

[0099] Take 30mL of each algae with a density of 2.4×104 cells / mL、4.5×10 4 cells / mL、5.2×10 4 cells / mL、5.8×10 4 cells / mL of Karenia mikimotoi, Karenia saddlei, Karenia longifuga, and Karenia butterfly were mixed with 1000mL of Qingdao Shazikou seawater (unsterilized) to prepare laboratory field simulation samples. The simulated sample DNA was extracted and diluted into three groups so that the number of cells contained in each group of DNA was different.

[0100] (2) The field simulated sample DNA obtained above was used as a template, and the probe primer combination shown in SEQ ID NO.1 to SEQ ID NO.6 was used. The amplification system and amplification program of Karenia longifuga and Karenia pellucida in step (1) of Example 2 were adopted to perform TaqMan qPCR fluorescence quantitative amplification to obtain Ct values. The Ct values ​​obtained by amplification of Karenia longifuga and Karenia pellucida were respectively substituted into the regression equation of the plasmid standard curve of Karenia longifuga and Karenia castellata in Example 2 to calculate the copy number of the recombinant plasmid DNA of Karenia longifuga and Karenia pellucida in the sample; then, the cell number of Karenia longifuga and Karenia pellucida was calculated by 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 (Table 2), and the differences in the microscopic cell density of the four algae and the algae cell density obtained by fluorescence quantitative PCR were compared using the SPSS t-test.

[0101] The results of the single-sample t-test showed that there was no significant difference between the TaqMan qPCR detection results of the two algae and the number of algal cells under microscopic examination (P>0.05), and the detection rate was 100%, indicating that this method has a good detection rate for the target algae species, and the TaqMan qPCR detection method established in the present invention can be further used for on-site sample detection.

[0102] Table 2 Comparison of the results of TaqMan qPCR detection and optical microscopy counting

[0103]

[0104] 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 probe primer composition for TaqMan fluorescence quantitative detection of Karenia longituba and Karenia sphenodon, characterized in that: The probe primer composition comprises: Karenia longituba: SEQ ID NO.1-L2F: GTGGAAGTGTATAATTCG; SEQ ID NO.2-L2R: CAGATTAGCTAGACGAAG; SEQ ID NO.3-L2P: VIC-TATCAGATCGTGGCTCGTTCT-MGB; Karenia butterfly: SEQ ID NO.4-P-F1: GGCACATTATAACGTGAGG; SEQ ID NO.5-P-R1: CCTATTTAAGACTAGGACG; SEQ ID NO. 6-P-P1: FAM-GGACCCTTCATGCTGGAAGCAG-BHQ1.

2. The probe primer composition for TaqMan fluorescent quantitative detection of Karenia longituba and Karenia sphenodon according to claim 1, characterized in that: The target genes used for TaqMan fluorescence quantitative detection of Karenia longifolia and Karenia pellucida are the ITS region of the ribosomal DNA of Karenia longifolia and the LSU D1-D3 region of the ribosomal DNA of Karenia pellucida, respectively.

3. A TaqMan fluorescence quantitative detection method for Karenia longituba and Karenia butterfly, characterized in that: The steps include: (1) extracting DNA from the sample to be tested, performing TaqMan qPCR amplification on the extracted DNA using the probe primer combination shown in SEQ ID NO.1 to SEQ ID NO.6, and after the amplification, detecting the cycle threshold (Ct) values ​​of Karenia longisulcata and Karenia pellucida respectively; (2) According to the Ct value obtained in step (1), the cell numbers of Karenia longituba and Karenia butterfly were calculated using the plasmid standard curve, cell standard curve, and cell-plasmid standard curve, respectively.

4. The TaqMan fluorescence quantitative detection method of Karenia longituba and Karenia butterfly according to claim 3, characterized in that: The amplification system of TaqMan qPCR amplification described in step (1) is: 10 μL Probe qPCR Super PreMix, 0.4 μL forward primer, 0.4 μL reverse primer, 0.2 μL TaqMan probe, 8 μL ddH2O, 1 μL DNA template; the amplification program is: 37°C 2 min, 95°C 10 min, 95°C 10 sec, 60°C 34 sec, 40 cycles.

5. The TaqMan fluorescence quantitative detection method of Karenia longituba and Karenia butterfly according to claim 3, characterized in that: The plasmid standard curve in step (2) is: Karenia longituba: y=-3.457x+38.727; Karenia butterfly: y=-3.27x+41.015; Wherein, x represents the logarithmic value of the recombinant plasmid copy number with base 10, and y represents the Ct value.

6. The TaqMan fluorescence quantitative detection method of Karenia longituba and Karenia butterfly according to claim 3, characterized in that: The cell standard curve in step (2) is: Karenia longituba: y=-3.729x+29.751; Karenia butterfly: y=-3.545x+26.672; Wherein, x represents the logarithmic value of the recombinant plasmid copy number with base 10, and y represents the Ct value.

7. The TaqMan fluorescence quantitative detection method of Karenia longituba and Karenia butterfly according to claim 3, characterized in that: The cell-plasmid standard curve in step (2) is: Karenia longituba: y=0.927x-2.407; Karenia butterfly: y=0.922x-4.046; Wherein, x represents the logarithmic value of the recombinant plasmid copy number with base 10, and y represents the logarithmic value of the algal cell number with base 10.

8. A product for TaqMan fluorescence quantitative detection of Karenia longituba and Karenia pellucida, characterized in that: The product comprises the probe primer composition for TaqMan fluorescent quantitative detection of Karenia longisulcata and Karenia sphenodon as described in claim 1.

9. The product for TaqMan dual fluorescence quantitative detection of Karenia longituba and Karenia leucoderma according to claim 7, characterized in that: The product includes any one of a detection reagent, a detection kit and a detection chip.

10. Use of the product for TaqMan fluorescence quantitative detection of Karenia longifuga and Karenia pellucida as claimed in any one of claims 8 or 9 in detecting Karenia longifuga and Karenia pellucida.

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