A rapid detection method for spherical Phaeocystis that can form centimeter-sized cysts

Abstract

The present invention relates to the field of marine environmental protection and provides a rapid detection method for Phaeocystis globosa, a spherical brown cyst algae that can form centimeter-sized cysts in my country's coastal waters. The quantitative detection method provided by the present invention can detect and monitor Phaeocystis globosa, a spherical brown cyst algae that can form centimeter-sized cysts in my country's coastal waters, and has the advantages of high specificity, high sensitivity, rapid detection, and a wide detection range.

Description

Technical Field

[0001] The invention belongs to the field of marine environmental protection and provides a rapid detection method for Phaeocystis globosa, a spherical brown cyst algae that can form centimeter-sized cysts in my country's coastal waters. Background Art

[0002] Phaeocystis globosa is a globally distributed microalgae that frequently causes harmful red tides in tropical and temperate coastal waters. Its life cycle is complex, consisting of two stages: free single cells and gelatinous cysts. The presence of large numbers of cysts in seawater signals a P. globosa bloom. During blooms, P. globosa produces toxic substances that often kill marine life. The gelatinous material on the surface of the cysts can block the respiratory organs of marine fish, causing suffocation. When the bloom subsides, the high biomass of P. globosa cells decomposes, consuming significant amounts of oxygen, causing hypoxia and severely threatening the survival of marine life. Since its first outbreak along my country's southeastern coast in 1997, P. globosa has become the most severe red tide disaster along this coast. A unique characteristic of P. globosa blooms is that they can form centimeter-sized cysts during the bloom. These centimeter-sized cysts often clog coastal nuclear power plant cooling systems, adversely affecting nuclear power plant safety.

[0003] Before the cysts appear in seawater, P. globosa exists as free, single cells. These cells, approximately 3-9 μm in diameter, are morphologically indistinguishable from other algae. Therefore, conventional microscopic observation and counting methods are unsuitable for quantitative detection of free P. globosa cells. Other detection methods, including pigment analysis, flow cytometry, and fluorescence in situ hybridization, are also unsuitable for quantitative detection of free single cells due to their low specificity, high detection limits, and poor sensitivity, limiting their effectiveness in early-stage red tide monitoring and early warning.

[0004] Pseudocystis sphericalensis has complex genetic diversity and diverse morphological characteristics. Through years of research, it was found that among the Pseudocystis sphericalensis distributed in the southeastern coast of my country, some strains can form centimeter-sized cysts, while other strains form cysts smaller than 3 mm (Wang Jinxiu et al., 2019, Study on the pigment composition characteristics of six strains of Pseudocystis sphericalensis, Oceans and Lakes, 50(3):611-620; Hu Xiaokun et al., 2019, Analysis of genetic diversity of Pseudocystis sphericalensis in the Beibu Gulf, Oceans and Lakes, 50(3):601-610). Among them, Pseudocystis sphericalensis that can only form centimeter-sized cysts is the causative species of Pseudocystis sphericalensis red tide in my country's coastal waters. Therefore, a quantitative detection technology with a low detection limit is urgently needed to specifically detect Pseudocystis sphericalensis that can form centimeter-sized cysts, so as to provide technical support for monitoring the occurrence and development process of Pseudocystis sphericalensis red tide in the southeastern coast of my country and for preventing the harm caused by the outbreak of Pseudocystis sphericalensis red tide. Summary of the Invention

[0005] The present invention aims to provide a quantitative method for detecting spherical Phaeocystis that can form centimeter-sized capsules with fast detection speed, high sensitivity and specificity.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A specific primer and probe for detecting Pseudocystis sphericalensis that can form centimeter-sized capsules, wherein the specific primer and probe are:

[0008] Upstream primer: PG-F 5′-GTCGTAGGTTAAGGCTTCAA-3′

[0009] Downstream primer: PG-R 5′-AGTCGCTAACAGTAGAAAGAATAG-3′

[0010] Probe: PG-Pr 5′-TGCGGCCCAAACATTTTCAGCT-3′.

[0011] The 5' end of the probe is linked to Texas Red fluorescent dye.

[0012] The invention discloses an application of a specific primer and a probe, wherein the specific primer and the probe are used in detecting a spherical brown cyst algae capable of forming centimeter-sized cysts.

[0013] A kit for detecting spherical brown cyst algae capable of forming centimeter-sized cysts, the kit comprising the specific primers and probes according to claim 1.

[0014] A method for detecting Phaeocystis globosa, which uses the total algal genomic DNA in a sample as a template, utilizes specific primers and probes for Phaeocystis globosa that can form centimeter-sized cysts, and detects the Phaeocystis globosa that can form centimeter-sized cysts in the sample through fluorescence quantitative PCR amplification and fluorescence signal.

[0015] The fluorescent quantitative PCR system is as follows: the total reaction volume is 10 μL, including 1 μL of genomic DNA, 5 μL of 2×ProbeqPCR Mix Buffer (Takara, Takara commercial product, no Chinese), 0.2 μL each of upstream primer, downstream primer, and probe (concentration 10 μM), and 3.4 μL of sterile deionized water.

[0016] The fluorescent quantitative PCR conditions were as follows: pre-denaturation at 95° C. for 30 s; denaturation at 95° C. for 5 s, annealing / extension at 60° C. for 30 s, 40 cycles, and collection of Texas Red fluorescence.

[0017] The advantages of the present invention are:

[0018] The detection method of the present invention targets the mitochondrial atp8 gene, which has a stable copy number within cells and is capable of distinguishing between Phaeocystis globosa, which can form centimeter-sized cysts, and other Phaeocystis globosa species. Specific primers and Taqman probes are designed for the mitochondrial atp8 sequence of Phaeocystis globosa, which can form centimeter-sized cysts, and detection is performed using fluorescent quantitative PCR.

[0019] The present invention has the following advantages:

[0020] 1. High specificity. The qPCR method of the present invention can be used to establish a quantitative detection method for P. globosa at the infraspecific level, specifically and quantitatively detecting P. globosa, which can form centimeter-sized cysts in my country's coastal waters.

[0021] 2. High sensitivity. The qPCR method of the present invention has a minimum detection limit of 1 cell, which can detect low-abundance Phaeocystis sphericalensis in marine areas, which can form centimeter-sized cysts, thereby facilitating early prediction and early warning of Phaeocystis red tides.

[0022] 3. Wide detection range. The detection limit of the qPCR method of the present invention is in the range of 30 cells / L-3×10 8 cells / L, with a wide detection range of 7 orders of magnitude, meeting the needs of field investigations;

[0023] 4. The detection speed is fast. The qPCR method of the present invention is easy to operate, saves time, and can obtain results in a short time. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A qPCR reaction annealing / extension temperature diagram is provided for the present invention examples.

[0025] Figure 2 This is a working curve provided for an example of the present invention.

[0026] Figure 3 This is a diagram for validating species specificity provided by an embodiment of the present invention.

[0027] Figure 4 This is a diagram for verifying the species specificity provided by an embodiment of the present invention.

[0028] Figure 5 This is a detection limit diagram provided for an embodiment of the present invention. DETAILED DESCRIPTION

[0029] The specific embodiments of the present invention are further described below with reference to examples. It should be noted that the specific embodiments described here are only for illustrating and explaining the present invention, and are not intended to limit the present invention.

[0030] Example 1

[0031] Establishment of a method for detecting Phaeocystis sphericalensis, which can form centimeter-sized cysts:

[0032] 1.1 Design of primers and probes

[0033] Sequence information for mitochondrial atp8 from six dinoflagellates (Chrysochromulina parva NC036938, Chrysochromulina sp. KJ201908, Pavlova lutheri HQ908424, Pavlova sp. MN564259, Phaeocystis antarctica JN131834, and Phaeocystis globosa KC967226) was obtained from Genbank and aligned. Based on the alignment results, primers for amplification of the mitochondrial atp8 sequence were designed using Primer 5.05 software (upstream primer: PG-atp8-F 5'-TGCCTCAATTCGATTTATTTACT-3', downstream primer: PG-atp8-R 5'-GAAGCYYCTAAAGTATTRCCTAA-3').

[0034] 20 mL of algal fluid from Phaeocystis globosa (a strain of Phaeocystis globosa that can form centimeter-sized cysts) and other strains of Phaeocystis globosa that cannot form centimeter-sized cysts during logarithmic growth phase was filtered through a 47 mm diameter, 0.4 μm pore size polycarbonate fiber filter (HTTP04700) at a pressure not exceeding 0.05 MPa and stored at -80°C until DNA extraction. Total DNA was extracted using the conventional CTAB method.

[0035] PCR amplification of the extracted DNA samples was performed using the synthesized primer pair for mitochondrial ATP8 amplification. The reaction system was as follows: a total reaction volume of 20 μL, including 1 μL of total DNA, 8.2 μL of sterile deionized water, 4 μL of 5× rTaq Buffer (Mg 2+ ), 4 μL of 5× PCR solution buffer, 1.6 μL of 2.5 mmol / L dNTP Mix, 0.4 μL of each upstream and downstream primers, and 0.4 μL of ultra-fidelity Taq DNA polymerase (5 U / mL, Beijing Quanshijin Biotechnology Co., Ltd., China). Reaction conditions were: initial denaturation at 98°C for 2 min; 32 cycles of denaturation at 98°C for 20 s, annealing at 58°C for 20 s, and extension at 72°C for 20 s; and extension at 72°C for 5 min.

[0036] The PCR reaction products were electrophoresed on 1% agarose gel, purified, and then sequenced;

[0037] By comparing the sequencing results and based on the comparison results, Beacon Designer 8.14 software was used to design specific primers and probes for P. sphericalus, which can form centimeter-sized cysts.

[0038] Its sequence information is:

[0039] Upstream primer: PG-F 5′-GTCGTAGGTTAAGGCTTCAA-3′

[0040] Downstream primer: PG-R 5′-AGTCGCTAACAGTAGAAAGAATAG-3′

[0041] Probe:PG-Pr 5′-TGCGGCCCAAACATTTTCAGCT-3′

[0042] 1.2 Reaction system optimization

[0043] By setting different primer and probe ratios (400nM:400nM, 400nM:300nM, 400nM:200nM, 400nM:100nM, 300nM:300nM, 300nM:200nM, 300nM:100nM, 200nM:200nM, 200nM:100nM, and 100nM:100nM), the amplification efficiency of qPCR reactions with the same DNA template at different ratios was compared. Each gradient was repeated three times to determine the optimal primer and probe ratio for the reaction.

[0044] The results are shown in Table 1. It can be seen from the results that when the primer-probe ratio is 200 nM:200 nM, the Ct value of the qPCR reaction is the lowest and the amplification efficiency is the highest. Therefore, 200 nM:200 nM is selected as the primer-probe ratio for the reaction.

[0045] Table 1 Ct values ​​of qPCR reaction under different primer-probe ratios

[0046]

[0047] 1.3 Optimization of reaction conditions

[0048] To further improve the amplification efficiency of the reaction, different annealing / extension temperatures (58, 59, 60, 61.2, 62.2, and 63°C) were set. The amplification efficiency of qPCR reactions containing the same DNA template at different temperature conditions in the optimal reaction system was compared, with three replicates per temperature gradient.

[0049] The results showed that at 60°C, the Ct value of the reaction was the lowest and the amplification efficiency was the highest (see Figure 1 ), so 60°C was selected as the annealing / extension temperature of the reaction.

[0050] Example 2

[0051] Take the algae solution of the PG2016 strain of Pseudomonas sphericalis that can form centimeter-sized cysts and the algae solution of Chlorella vulgaris that can form centimeter-sized cysts in the logarithmic growth period from the Institute of Oceanology, Chinese Academy of Sciences, and count them under a microscope. 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 and 10 2 cells / L, and each gradient was set up with 4 parallels, each parallel with 100 mL of mixed algae solution (the density of Chlorella vulgaris was 1×10 8 cells / L, with Chlorella vulgaris added as a protective algae to prevent DNA loss during DNA extraction from the target alga, the spherical Phaeocystis PG2016 strain, which can form centimeter-sized capsules and is beneficial for improving the sensitivity of the method. The sample was filtered through a 47 mm diameter, 0.4 μm pore size polycarbonate fiber filter (HTTP04700) at a pressure not exceeding 0.05 MPa and stored at -80°C until DNA extraction.

[0052] Total DNA was extracted using the traditional CTAB method;

[0053] The qPCR system solution is 10 μL, and the reaction solution composition is as follows: 3.4 μL H2O, 5 μL 2×Probe qPCR MixBuffer, 0.2 μL upstream primer (10 μM), 0.2 μL downstream primer (10 μM), 0.2 μL probe (10 μM), 1 μL DNA dissolution solution; its specific primer and probe sequences are as follows:

[0054] Upstream primer: PG-F 5′-GTCGTAGGTTAAGGCTTCAA-3′,

[0055] Downstream primer: PG-R 5′-AGTCGCTAACAGTAGAAAGAATAG-3′,

[0056] Probe: PG-Pr 5′-TGCGGCCCAAACATTTTCAGCT-3′;

[0057] The qPCR reaction program was as follows: 95°C for 30 s, followed by 40 cycles (95°C for 5 s, 60°C for 30 s);

[0058] The working curve of the method was established based on the obtained Ct values ​​(see Figure 2 The results showed that the rapid quantitative detection kit for Phaeocystis sphericalensis, which can form centimeter-sized cysts, had good linearity (R 2 =0.9997), and the amplification efficiency met the requirements of the qPCR method (E=105%).

[0059] Example 3

[0060] Specificity verification

[0061] Dinoflagellates (Alexandrium pacificum, Gymnodinium chain, Prorocentrum donghaiense, Karenia mikimotoi, Prorocentrum oceanicum, Prorocentrum microphyllum, Toxic algae, Anterior channel algae, Cone-shaped algae, etc.), diatoms (Skeletalum costatum, Triangular brown finger algae, Multi-striped Nitzschia, etc.), needle algae (Cardiophorum, Heterosigma akashiwo), golden algae (Golden ball anoxicula), golden algae (Isochrysis globosum), green algae (Chlorella vulgaris, Platymonas, Dunaliella, Cryptomonas, etc.), and dinoflagellates (Antarctic Phaeocystis, P. rex, P. cordata, P. jahnii) cultivated by the Institute of Oceanology, Chinese Academy of Sciences, were selected as negative controls. Two strains of Phaeocystis spherica isolated from the South China Sea, PG2016 and MEL70, which can form centimeter-sized cysts and are cultivated by the Institute of Oceanology, Chinese Academy of Sciences, were used as positive samples for interspecies specificity verification.

[0062] Several strains of P. sphericalus that cannot form centimeter-sized cysts and are preserved by the Institute of Oceanology, Chinese Academy of Sciences and isolated from coastal waters around the world, including the North Atlantic Caribbean Sea CCMP628 strain, the North Atlantic Gulf of Mexico CCMP629 strain, the Atlantic North American waters CCMP2754 strain, the European North Sea coast RCC736 strain, the English Channel RCC2055 strain, and the South China Sea MEL43 and PG2017 strains, were used as negative controls, and two P. sphericalus strains PG2016 and MEL70 strains isolated from the South China Sea that can form centimeter-sized cysts were used as positive samples to verify the specificity within the species.

[0063] The DNA of each algae was extracted, and then the kit and corresponding method described in the above examples were used to detect each algae through fluorescence quantitative PCR amplification and fluorescence signal.

[0064] The results showed that, whether between different species ( Figure 3 ), or within the species (attached Figure 4 ), the rapid quantitative detection method of the present invention for P. sphericalus that can form centimeter-sized capsules can only detect the fluorescent signals of the target P. sphericalus PG2016 strain and MEL70 strain that can form centimeter-sized capsules, but cannot detect the fluorescent signals of non-target algae, indicating that the method has good specificity.

[0065] Example 4

[0066] Detection limit

[0067] In order to reduce the DNA loss of the Phaeocystis sphericalensis PG2016 strain, which can form centimeter-sized cysts, during DNA extraction, approximately 5×10 7 Chlorella cells were added to a solution containing 1.98 × 10 8 Cells of the spherical Phaeocystis PG2016 strain, which can form centimeter-sized cysts, were mixed in an algal solution (three replicates). These mixed microalgae were then collected for DNA extraction. DNA was extracted using the method described in Example 2 and dissolved in 30 μL of TE buffer. A series of DNA solutions (4, 16, 64, and 6.4 × 10 2 , 6.4×10 3 , 6.4×10 4 , 2.56×10 5 , 1.02×10 6 , 4.10×10 7 and 1.64×10 8 At each concentration, 1 μL of DNA solution (n=3) was used as a template for qPCR reaction, and then amplified using the kit and corresponding method described in the above examples to establish the upper and lower limits of detection (see Figure 5 ).

[0068] The results showed that the detection range was 0.92-9.43×10 6 The cell concentration of the field sample is about 30 cells / L-3×10 8 cells / L), indicating that this method has a wide detection range. Sequence Listing <110> Institute of Oceanology, Chinese Academy of Sciences <120> A rapid detection method for spherical Phaeocystis that can form centimeter-sized cysts <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 gtcgtaggtt aaggcttcaa 20 <210> 2 <211> twenty four <212> DNA <213> Artificial Sequence <400> 2 agtcgctaac agtagaaaga atag 24 <210> 3 <211> twenty two <212> DNA <213> Artificial Sequence <400> 3 tgcggcccaa acattttcag ct 22

Claims

1. A specific primer and probe for detecting Phaeocystis sphericalensis, which can form centimeter-sized cysts in my country's coastal waters, characterized by: The specific primers and probes are: Upstream primer: PG-F5´-GTCGTAGGTTAAGGCTTCAA-3´ Downstream primer: PG-R5´-AGTCGCTAACAGTAGAAAGAATAG-3´ Probe: PG-Pr5´-TGCGGCCCAAACATTTTCAGCT-3´.

2. The specific primers and probe for detecting Phaeocystis sphericalensis that can form centimeter-sized cysts according to claim 1, characterized in that: The 5' end of the probe is linked to TexasRed fluorescent dye.

3. A use of the specific primer and probe according to claim 1, characterized in that: Application of the specific primers and probes in detecting Phaeocystis sphericalensis that can form centimeter-sized capsules; The spherical Phaeocystis is the spherical Phaeocystis PG2016 strain.

4. A kit for detecting Phaeocystis sphericalensis that can form centimeter-sized cysts, characterized in that: The kit comprises the specific primers and probes according to claim 1.

5. A method for detecting Phaeocystis sphericalensis that can form centimeter-sized cysts, characterized in that: The total algal genomic DNA in the sample is used as a template, and the specific primers and probes of the spherical cyst algae that can form centimeter-sized cysts according to claim 1 are used to detect the spherical cyst algae that can form centimeter-sized cysts in the sample through fluorescent quantitative PCR amplification and fluorescent signals; the spherical cyst algae is spherical cyst algae PG2016.

6. The method for detecting Phaeocystis sphericalensis capable of forming centimeter-sized capsules according to claim 5, characterized in that: The fluorescent quantitative PCR system is as follows: the total reaction volume is 10 μL, including 1 μL of genomic DNA solution, 5 μL of 2×Probe qPCR MixBuffer, 0.2 μL each of 10 μM upstream primer, downstream primer, and probe, and 3.4 μL of sterile deionized water.

7. The method for detecting Phaeocystis sphericalensis capable of forming centimeter-sized capsules according to claim 5, characterized in that: The fluorescent quantitative PCR conditions were as follows: pre-denaturation at 95° C. for 30 s; denaturation at 95° C. for 5 s, annealing / extension at 60° C. for 30 s, 40 cycles, and collection of Texas Red fluorescence.

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