CN111996240A - Marine phaeocystis globosa single cell detection method based on TaqMan probe technology - Google Patents
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Abstract
The invention belongs to the technical field of biological method detection of environmental microorganisms, and discloses a TaqMan probe technology-based marine phaeocystis globosa single cell detection method, which comprises the following steps: s1: designing and synthesizing primers and probes; s2: preparing a positive control sample of phaeocystis globosa; s3: negative control quality control; s4: extracting genome DNA of sea area single cell phaeocystis globosa sample and positive control sample; s5: performing fluorescent quantitative PCR detection by using the primers and the probes designed in the step S1; s6: judging a real-time fluorescent quantitative PCR amplification signal; s7: agarose gel electrophoresis analysis of the fluorescent quantitative PCR product; s8: sequencing and analyzing a fluorescent quantitative PCR product; s9: detection and analysis of sea area unicellular phaeocystis globosa. The invention can detect the harmful red tide algae with low density in the early stage of the harmful red tide outbreak, thereby providing an effective early warning tool for early warning and early prevention and control of the red tide outbreak and avoiding economic loss of a large number of breeding and tourism nuclear power industries.
Description
Technical Field
The invention relates to the technical field of detecting environmental microorganisms by a molecular biological method, in particular to a marine phaeocystis globosa single cell detection method based on a TaqMan probe technology.
Background
The phaeocystis fuscus belongs to dinoflagellates of chrysophyta, has wide temperature and salt adaptability, is a harmful red tide cause species commonly existing in offshore China in the global range, and is the most common phaeocystis fuscus. The phaeocystis globosa has unique physiological and ecological characteristics, belongs to marine microalgae with complex and abnormal life history, has two convertible morphological stages of free single cells (3-8 μm) and groups, and mainly forms red tide in the form of groups. The sea area phaeocystis strain capsule body in China is huge, is internationally rare, has the diameter of 3cm, and is obviously larger than the diameter (7-8mm) of the phaeocystis fuscus capsule body in other sea areas in the world. The outer wall of the Zostera marina capsule body contains colloid and polysaccharide, is tough and strong in viscosity, and when the Zostera marina single cells are massively proliferated to form the capsule body red tide, the algae body containing the colloid and the polysaccharide is tightly attached to fish gills to influence the breathing and ingestion of fish, so that the fish die due to asphyxia and oxygen deficiency, and great harm is caused to cage culture.
The red tide outbreak of the phaeocystis globosa will pose a great threat to the tourism industries such as the northern gulf marine aquaculture industry, the natural landscape of the seaside and the like, the marine ecological environment, the coastal nuclear power safety and the human health. Therefore, high attention should be paid to ascosphaera sphaeroides, and monitoring, early warning and treatment research on harmful red tide algae needs to be enhanced urgently. However, the current monitoring means such as microscope technology, flow technology and satellite remote sensing technology are not sensitive enough, time-consuming and labor-consuming, and have poor timeliness, most of which can be perceived after the red tide outbreak (after the capsule is formed), and corresponding treatment measures are taken at this time, but it is too late, so if the dynamics of the red tide algae in the early stage of the red tide can be rapidly monitored before the red tide outbreak, and then corresponding prevention and elimination measures are taken, which can effectively avoid great economic loss, and has great practical significance. Therefore, establishing a rapid detection method for the early stage of red tide outbreak of phaeocystis globosa and applying the method to offshore phaeocystis globosa red tide research become urgent.
The existing research results show that the molecular and genetic methods can relatively accurately and rapidly diagnose and evaluate the red tide species in the water environment, wherein the TaqMan molecular probe detection technology of various red tide algae is successfully established and is successfully applied to the detection of the red tide algae. However, at present, no report for detecting the single-cell phaeocystis globosa based on TaqMan probes exists at home and abroad. The TaqMan probe is one of detection probes of a qPCR technology, and is mainly used for detecting on the basis of the ribosomal gene of a detected species, and the ITS is a high mutation region in the ribosomal gene, so that a pair of primers and an oligonucleotide probe are developed in the most conservative region of the ITS of phaeocystis globosa, the specificity and the sensitivity of the method are further improved, and the method is innovative. Compared with the SYBR dye method detection only using a pair of primers, the TaqMan probe detection method has the advantages that a specific oligonucleotide probe is added, the TaqMan probe detection method has higher specificity, the capability of specifically detecting unicellular phaeocystis globosa in a natural sea area with complex plankton species is greatly improved, and the practicability is good, so that the method for detecting the unicellular phaeocystis globosa based on the TaqMan probe technology is provided.
Disclosure of Invention
Aiming at the problems in the prior art, the invention develops a method for detecting single-cell phaeocystis globosa by using a TaqMan probe, and the single-cell phaeocystis globosa can be detected and identified quickly, accurately and at high flux by using the designed primer and probe.
In order to achieve the purpose, the invention adopts the following technical scheme:
a marine phaeocystis globosa single cell detection method based on TaqMan probe technology comprises the following steps:
s1: design and synthesis of primers and probes:
designing and synthesizing primers and probes in a conserved region of the ITS2 region of the phaeocystis globosa ribosomal gene, wherein the sequence of the designed and synthesized primers is as follows:
upstream primer sequence PGF: 5'-GCGATGCCTCTTTTGCGA-3', annealing temperature 59.6 ℃;
downstream primer sequence PGR: 5'-GCGGGTAGTCTTGCTCCA-3', annealing temperature 58.2 ℃;
sequence PGP of TaqMan probe: 5'-FAM-CGTCTGAACCTCCTCCA-NFQ-MGB-3', annealing temperature 70 ℃;
s2: preparing a positive control sample of phaeocystis globosa;
s3: negative control quality control: sterile water was used as an amplification control for the no DNA template;
s4: extracting genome DNA of sea area single cell phaeocystis globosa sample and positive control sample;
s5: performing fluorescent quantitative PCR detection by using the primers and the probes designed in the step S1;
s6: and (3) judging a real-time fluorescent quantitative PCR amplification signal: negative control should not have amplification signal, otherwise, contamination;
s7: agarose gel electrophoresis analysis of fluorescent quantitative PCR products: carrying out agarose gel electrophoresis analysis on the natural sea area unicellular phaeocystis globosa and the fluorescent quantitative PCR products of the indoor purified and cultured phaeocystis globosa;
s8: sequencing analysis of fluorescent quantitative PCR products: comparing the sequencing results of the fluorescent quantitative PCR products of the unicellular phaeocystis globosa in the natural sea area, finding that the sequence matching degree with a phaeocystis globosa strain (EU077557.1) reaches 96 percent, and confirming that the signals specifically detected by the designed primers and probes from the sea area are phaeocystis globosa;
s9: detection and analysis of sea area unicellular phaeocystis globosa: after the feasibility of the designed primer and the probe is gradually verified through the steps, the primer and the probe can be used for rapid, accurate and high-throughput detection and identification of the unicellular phaeocystis globosa.
Preferably, in the primer sequence of step S1, FAM is a reporter fluorescent group labeled at the 5' end of the probe, NFQ is a non-fluorescent quencher group labeled at the 3' end of the probe, and MGB is a DNA minor groove binder labeled at the 3' end.
Preferably, in step S1, the size of the fluorescent quantitative PCR product fragment of the upstream and downstream primers is 128 bp.
Preferably, in step S2, the positive control sample is obtained by selecting a phaeocystis globosa cell that is separated and purified and cultured in a natural sea area, and the indoor purified and cultured algal cell in a logarithmic growth phase is used as a positive control for detecting the unicellular phaeocystis globosa in the sea area.
Preferably, in step S4, the extraction of genomic DNA is performed by the Takara lysate extraction protocol with the code number of 9770 and with reference to the method steps described in the reagent.
Preferably, in step S5, the reaction system for the fluorescent quantitative PCR detection is 20uL, and probe method reagent available from kanji century corporation is used.
Preferably, in step S5, the reaction procedure of the fluorescence quantitative PCR detection is: pre-denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 15s, annealing and extension at 60 ℃ for 60s, and collecting fluorescence signals for 40 cycles in total.
Preferably, in the step S6, in the determining process of the fluorescence quantitative PCR amplification signal, the phaeocystis globosa cells separated from the natural sea area and purified and cultured are used as positive quality control, an amplification curve appears, and if the positive control has an amplification signal and the unicellular phaeocystis globosa cells in the sea area have no amplification signal, the primers and the probes are redesigned.
Preferably, in step S7, the size of the fluorescent quantitative PCR product fragment is consistent with the size of the expected product fragment when the designed primer and probe are used, so as to preliminarily prove that the signal detected by the designed primer and probe is phaeocystis globosa, and then the product sequencing analysis is continued.
Preferably, in the step S9, in the process of detecting the sample of the sea area unicellular phaeocystis globosa, the sample contains phaeocystis globosa cells when the amplification curve is provided, and the sample does not contain phaeocystis globosa cells when the amplification curve is not provided.
Compared with the prior art, the invention provides a method for detecting marine phaeocystis globosa single cells based on TaqMan probe technology, which has the following beneficial effects:
(1) the invention has simple operation, high detection flux and time and labor saving: the invention saves time and consumables to a great extent, is simple to operate, combines with designed reaction programs, only needs 50min to 1h in the detection process, and can completely complete the whole experiment within 2 h. The invention combines the configuration condition of the instrument, can simultaneously detect 96 samples or 384 samples at one time, can carry out high-throughput detection on the marine unicellular phaeocystis globosa, and improves the detection efficiency;
(2) the invention has strong detection specificity and high sensitivity to the harmful red tide algae phaeocystis globosa: the primer and probe combination designed according to the ITS sequence of the hypervariable region in the phaeocystis globosa ribosome gene has high specificity, and ensures the specific amplification of a template. The designed specific primers and probes are designed from the most conservative region in ITS, so that the sensitivity of the detection of phaeocystis globosa is improved;
(3) the result analysis is simple: compared with a SYBR dye detection method, the method disclosed by the invention is easier to analyze experimental results. Whether the phaeocystis globosa cells exist or not can be judged by observing whether the amplification curve exists or not for each sample without performing melting curve analysis and agarose gel electrophoresis analysis;
the design of the invention can detect the harmful red tide algae with low density in the early stage of the harmful red tide outbreak, thereby providing an effective early warning tool for early warning and early prevention and control of the red tide outbreak and avoiding economic loss of a large number of breeding and tourism nuclear power industries.
Drawings
FIG. 1 is a graph showing the amplification curves of Pachylomyces globosa, Pachylomyces globosa in the natural sea area and negative control, which are provided by the present invention and are purified indoors by using designed primers and probes;
FIG. 2 is the agarose gel electrophoresis analysis of the fluorescent quantitative PCR products of unicellular phaeocystis globosa in the natural sea area and the phaeocystis globosa cultured by indoor purification according to the present invention;
FIG. 3 is the comparison result of the sequencing result of the fluorescent quantitative PCR product of unicellular phaeocystis globosa in the natural sea area provided by the invention;
FIG. 4 shows the result of the fluorescence quantitative PCR detection of 5 samples of marine unicellular phaeocystis globosa according to the present invention;
FIG. 5 shows the results of the detection of 6 cells of phaeocystis globosa with the same number of cells in the chamber.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
A marine phaeocystis globosa single cell detection method based on TaqMan probe technology comprises the following steps:
s1: design and synthesis of primers and probes: designing and synthesizing primers and probes in a conserved region of the ITS2 region of the phaeocystis globosa ribosomal gene, wherein the sequence of the designed and synthesized primers is as follows:
upstream primer sequence PGF: 5'-GCGATGCCTCTTTTGCGA-3', annealing temperature 59.6 ℃;
downstream primer sequence PGR: 5'-GCGGGTAGTCTTGCTCCA-3', annealing temperature 58.2 ℃;
sequence PGP of TaqMan probe: 5'-FAM-CGTCTGAACCTCCTCCA-NFQ-MGB-3', annealing temperature 70 ℃;
wherein FAM is a reporter fluorescent group marked at the 5' end of the probe, NFQ is a non-fluorescent quenching group marked at the 3' end of the probe, and MGB is a DNA minor groove binder marked at the 3' end; the probe designed by the invention is an MGB probe in a TaqMan probe, the TaqMan MGB probe can obviously improve the annealing temperature of the probe, the specificity is better than that of the common TaqMan probe, and the TaqMan MGB probe is easier to design;
the size of the designed fluorescent quantitative PCR product fragment of the upstream primer and the downstream primer is 128 bp;
s2: preparation of a phaeocystis globosa positive control sample: the positive control sample is a spherical phaeocystis globosa cell which is separated from the natural sea area and purified and cultured, and the indoor purified and cultured algal cell in logarithmic growth phase is used as the positive control for the sea area single cell spherical phaeocystis globosa detection;
s3: negative control quality control: sterile water was used as an amplification control for the no DNA template;
s4: extracting genome DNA of sea area single cell phaeocystis globosa sample and positive control sample: the method adopts a Takara lysate extraction scheme (cat # 9770), and the detailed steps are carried out according to the method in the instruction in the reagent;
s5: performing fluorescent quantitative PCR detection by using the primers and the probes designed in the step S1:
the reaction system of the fluorescent quantitative PCR detection in the step S5 is 20uL, and a probe method reagent of Kangji century company is adopted; the components and the volume of each reaction system are shown in a table 1:
TABLE 1 fluorescence quantitative pcr detection system for phaeocystis globosa
The reaction procedure of the fluorescent quantitative PCR detection in the step S5 adopts a two-step method: namely, pre-denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 15s, annealing and extension at 60 ℃ for 60s, wherein fluorescence signals are collected for 40 cycles;
s6: and (3) judging a real-time fluorescent quantitative PCR amplification signal: negative control should not have amplification signal, otherwise, contamination; separating the spherical phaeocystis globosa cells from the natural sea area for positive quality control, wherein an amplification curve must appear, and if the positive control has an amplification signal and the single-cell phaeocystis globosa cells in the sea area have no amplification signal, redesigning a primer and a probe; as shown in figure 1: if both the primers and the probes have amplification signals, the feasibility of the designed primers and probes for detecting the sea area single-cell phaeocystis globosa is preliminarily proved; however, species algae in the natural sea area are abundant, and the specific signal detected in the natural sea area cannot be proved to be the phaeocystis globosa only by the amplification signal; therefore, subsequent agarose gel electrophoresis analysis of the fluorescent quantitative PCR product and sequencing analysis of the fluorescent quantitative PCR product are also needed;
s7: agarose gel electrophoresis analysis of fluorescent quantitative PCR products: as shown in the attached figure 2, agarose gel electrophoresis analysis is carried out on the fluorescent quantitative PCR products of the unicellular phaeocystis globosa in the natural sea area and the phaeocystis globosa cultured in a room in a purifying way, and the size of the product fragment must be consistent with the size of the expected product fragment (128bp) when the primers and the probes are designed, so that the signals detected by the designed primers and the probes can be preliminarily proved to be phaeocystis globosa. But the size of the product fragment is not enough to determine that the detected phaeocystis globosa is completely spherical, and the product sequencing analysis is required to be carried out continuously;
s8: sequencing analysis of fluorescent quantitative PCR products: as shown in figure 3, the sequence matching degree of the natural sea area unicellular phaeocystis globosa fluorescent quantitative PCR product is 96% after comparison with the phaeocystis globosa strain (EU 077557.1); confirming that the signal specifically detected by the designed primer and probe from the sea area is phaeocystis globosa;
s9: the invention can be used for detecting and analyzing the unicellular phaeocystis globosa in the sea area: after the feasibility of the designed primer and the probe is gradually verified through the steps, the primer and the probe can be used for rapid, accurate and high-throughput detection and identification of the unicellular phaeocystis globosa. And the existence of the phaeocystis globosa cells can be known by directly seeing the existence of the amplification curve after each sample detection, and the agarose gel electrophoresis analysis and the sequencing analysis are not needed. The detection result of the invention on the marine single cell phaeocystis globosa is shown in figure 4.
Aiming at the marine phaeocystis globosa single cell detection method based on the TaqMan probe technology, the invention provides the following embodiments:
example 1: detecting marine single-cell phaeocystis globosa by a TaqMan probe method based on real-time fluorescent quantitative PCR:
(1) collecting samples:
in the embodiment, 5 samples are numbered as sample 1, sample 2, sample 3, sample 4 and sample 5, and are collected by the oceanic center of Guangxi academy of sciences in help of red tide voyage survey;
(2) extracting genome DNA:
the method adopts a Takara lysate extraction scheme (cat # 9770), and the detailed steps are carried out according to the method in the instruction in the reagent;
(3) the preparation of a reaction system:
the reaction system of the fluorescent quantitative PCR detection is 20uL, and a probe method reagent of Kangji century company is adopted. The components and the volume of each reaction system are shown in a table 1; the negative control reaction system used 1uL of sterile water as a DNA template, and the addition of the rest of the reaction systems was the same as that in Table 1;
(4) sample detection:
the detection was performed on 5 samples, three in parallel per sample, on a qtowerr 3.0 fluorescent quantitative PCR instrument. Instrument operation reference instrument instructions; the fluorescent quantitative PCR amplification parameters are as follows: pre-denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 15s, annealing and extension at 60 ℃ for 60s, collecting fluorescence signals at the position, and performing 40 cycles in total;
(5) and (3) analyzing an experimental result:
the threshold of the amplification curve in this example is set at the exponential phase of the amplification curve, the set threshold is 5, and the value of the intersection of the threshold and the amplification curve is Ct value; the amplification curves of the 5 samples are shown in figure 4, and the Ct values are shown in Table 2; as can be seen from the results of FIG. 4 and Table 2, the 5 samples all had phaeocystis globosa cells;
table 2: the invention provides a fluorescence quantitative PCR detection result for 5 marine unicellular phaeocystis globosa samples
Example 2: the invention has the following repeatability experiments on the same sample:
in order to detect the repeatability and stability of the detection, the spherical phaeocystis cells purified and cultured in the same number (10000) of chambers are detected, and 6 samples are detected in total and respectively numbered as a repeated sample 1, a repeated sample 2, a repeated sample 3, a repeated sample 4, a repeated sample 5 and a repeated sample 6; each 3 replicates. The detection results are shown in figure 5 and table 3; the result of the repetitive amplification curve shows that the method has good repeatability, and the statistical result shows that the standard deviation of the Ct value of 6 spherical phaeocystis globosa cell DNA templates with the same quantity is 0.19, which indicates that the method has good and stable repeatability;
table 3: the invention provides a detection result of 6 parts of indoor purified and cultured phaeocystis globosa cells with the same quantity.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (10)
1. A marine phaeocystis globosa single cell detection method based on TaqMan probe technology is characterized by comprising the following steps:
s1: design and synthesis of primers and probes:
designing and synthesizing primers and probes in a conserved region of the ITS2 region of the phaeocystis globosa ribosomal gene, wherein the sequence of the designed and synthesized primers is as follows:
upstream primer sequence PGF: 5'-GCGATGCCTCTTTTGCGA-3', annealing temperature 59.6 ℃;
downstream primer sequence PGR: 5'-GCGGGTAGTCTTGCTCCA-3', annealing temperature 58.2 ℃;
sequence PGP of TaqMan probe: 5'-FAM-CGTCTGAACCTCCTCCA-NFQ-MGB-3', annealing temperature 70 ℃;
s2: preparing a positive control sample of phaeocystis globosa;
s3: negative control quality control: sterile water was used as an amplification control for the no DNA template;
s4: extracting genome DNA of sea area single cell phaeocystis globosa sample and positive control sample;
s5: performing fluorescent quantitative PCR detection by using the primers and the probes designed in the step S1;
s6: and (3) judging a real-time fluorescent quantitative PCR amplification signal: negative control should not have amplification signal, otherwise, contamination;
s7: agarose gel electrophoresis analysis of fluorescent quantitative PCR products: carrying out agarose gel electrophoresis analysis on the natural sea area unicellular phaeocystis globosa and the fluorescent quantitative PCR products of the indoor purified and cultured phaeocystis globosa;
s8: sequencing analysis of fluorescent quantitative PCR products: comparing the sequencing results of the fluorescent quantitative PCR products of the unicellular phaeocystis globosa in the natural sea area, finding that the sequence matching degree with a phaeocystis globosa strain (EU077557.1) reaches 96 percent, and confirming that the signals specifically detected by the designed primers and probes from the sea area are phaeocystis globosa;
s9: detection and analysis of sea area unicellular phaeocystis globosa: after the feasibility of the designed primer and the probe is gradually verified through the steps, the primer and the probe can be used for rapid, accurate and high-throughput detection and identification of the unicellular phaeocystis globosa.
2. The method for detecting Zostera marina single cells based on TaqMan probe technology as claimed in claim 1, wherein in the primer sequence of step S1, FAM is a reporter fluorescent cluster labeled at the 5' end of the probe, NFQ is a non-fluorescent quenching cluster labeled at the 3' end of the probe, and MGB is a DNA minor groove binder labeled at the 3' end.
3. The method for detecting phaeocystis globosa single cell based on TaqMan probe technology as claimed in claim 1, wherein in step S1, the sizes of the fluorescent quantitative PCR product fragments of the upstream and downstream primers are 128 bp.
4. The method for detecting phaeocystis globosa single cell based on TaqMan probe technology as claimed in claim 1, wherein in step S2, the positive control sample is selected from phaeocystis globosa cells separated from natural sea area and purified and cultured, and the indoor purified and cultured algal cells in logarithmic growth phase are used as positive control for detecting phaeocystis globosa single cell.
5. The method for detecting phaeocystis globosa single cell based on TaqMan probe technology as claimed in claim 1, wherein the step S4, the genomic DNA is extracted by Takara lysate extraction protocol with a product number of 9770 and with reference to the method steps of the instruction in the reagent.
6. The method for detecting Zostera marina unicell based on TaqMan probe technology according to claim 1, wherein in step S5, the reaction system of the fluorescence quantitative PCR detection is 20uL, and the probe method reagent of Kangji century company is adopted.
7. The method for detecting Zostera marina unicell based on TaqMan probe technology according to claim 1, wherein in step S5, the reaction procedure of the fluorescence quantitative PCR detection is as follows: pre-denaturation at 95 ℃ for 10min, denaturation at 95 ℃ for 15s, annealing and extension at 60 ℃ for 60s, and collecting fluorescence signals for 40 cycles in total.
8. The method for detecting Zostera marina unicells based on TaqMan probe technology according to claim 1, wherein in the step S6, in the process of judging the fluorescence quantitative PCR amplification signals, the Zostera marina cells which are purified and cultured are separated from the natural sea area and used as positive quality control, an amplification curve appears, and if the positive control has an amplification signal and the single-cell Zostera marina in the sea area has no amplification signal, primers and probes are redesigned.
9. The method for detecting Zostera marina unicell based on TaqMan probe technology according to claim 1, wherein in step S7, the size of the fluorescence quantitative PCR product fragment is consistent with the size of the expected product fragment when the designed primer and probe are used, so as to preliminarily prove that the signal detected by the designed primer and probe is Zostera marina, and then the sequencing analysis of the product is continued.
10. The method for detecting phaeocystis globosa single cell based on TaqMan probe technology as claimed in claim 1, wherein in step S9, in the process of detecting the sample of phaeocystis globosa single cell in the sea area, the sample contains phaeocystis globosa cells when the amplification curve is provided, and the sample does not contain phaeocystis globosa cells when the amplification curve is not provided.
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| CN1403590A (en) * | 2002-09-29 | 2003-03-19 | 中山大学 | Nucleotide sequence, nucleic acid molecular probe and method for identifying red tide phaeocystis globosa |
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| CN114574623B (en) * | 2022-03-31 | 2023-12-22 | 广西科学院 | Method for quantitatively analyzing single-cell spherical brown algae in early red tide outbreak |
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