CN115961084A - Rapid chromogenic detection method for phaeodactylum acutangulatum - Google Patents

Abstract

The invention provides a rapid chromogenic detection method of rhombohedral spinosyns, wherein a primer group used in the method comprises a primer group with a sequence of SEQ ID NO.1: primer F3 of ATTGCGCTGTACTGGTCT has a sequence of SEQ ID NO.2: the primer B3 of TAGTGCGCAAACCAACA has a sequence of SEQ ID NO.3: the primer FIP of GTAAACGATGGGCACCTCACCTCAATGTTTGGGTGGAA TCTGT has the sequence of SEQ ID NO.4: the primer BIP and the sequence of TTAGAGTGTTCAAAGCAG GCTTATAGGTCCTATATCATTATTCCATGC are SEQ ID NO.5: primer LF of GTACGACAACTTAATGC. The invention provides a rapid detection method of rhombohedral spinelle, which does not need a complicated DNA extraction step in the nucleic acid extraction stage and can realize the crude extraction of algae seed nucleic acid within 30 minutes under the condition of normal temperature; a group of specific primers are designed aiming at a rhombohedral spinosyns 18SrDNA V4 region sequence, amplification and detection of a target sequence can be realized in 30-40 minutes under the constant temperature condition of 63 ℃ by using the primer group and a hydroxynaphthol blue coloring agent, the specificity is good, the sensitivity is high, a complex temperature change process and a precision instrument are not needed, the flow of red tide algae molecule detection is greatly simplified, and the method is suitable for rapid detection of a field sample.

Description

A rapid colorimetric detection method for Pseudo-nitzschia spinulosa

Technical Field

The invention belongs to the technical field of microalgae detection, and particularly relates to a rapid color development detection method for Pseudo-nitzschia spinulosa.

Background Art

Pseudo-nitzschia spinulosa belongs to the phylum Bacillariophyta, family Nitzschiae. It is a common floating algae in coastal waters around the world and a common red tide disaster in my country's waters. There have been reports of algal blooms in Dalian, Qingdao, the Yangtze River Estuary, Xiamen Port and many ports in the northern South China Sea. In addition, Pseudo-nitzschia spinulosa can produce algal toxins, which cause great harm to marine life and humans. Therefore, research on the mechanism, process, monitoring and early warning of red tide outbreaks of Pseudo-nitzschia spinulosa has always been one of the hot topics of concern. Among them, how to accurately and quickly identify Pseudo-nitzschia spinulosa in the wild environment is the primary task.

At present, the methods used to detect Pseudo-nitzschia spinulosa mainly involve observing its morphological characteristics through a microscope or using its nucleic acid information for molecular identification. However, the former requires the observer to have rich classification experience, while the latter requires expensive and sophisticated instruments. These operations are cumbersome, time-consuming and labor-intensive, and are not suitable for rapid detection of on-site samples. Therefore, it is urgent to find a fast, accurate, sensitive and convenient on-site detection method for Pseudo-nitzschia spinulosa.

Summary of the invention

The present invention provides a rapid color development detection method for Pseudo-nitzschia spinulosa, thereby enabling rapid and accurate detection of Pseudo-nitzschia spinulosa.

The present invention first provides a primer set for detecting Pseudo-nitzschia spinulosa, comprising primers with the following sequences:

F3:5′-ATTGCGCTGTACTGGTCT-3′(SEQ ID NO.1),

B3:5′-TAGTGCGCAAACCAACAA-3′(SEQ ID NO.2),

FIP:5′-GTAAACGATGGGCACCCTCACATGTTTGGGTGGAATCTGT-3′

(SEQ ID NO.3),

BIP: 5′-TTAGAGGTGTTCAAAGCAGGCTTATAGGTCCTATATCATTATTC CATGC-3′ (SEQ IDNO.4),

LF: 5′-GTACGACAACTTAATGC-3′ (SEQ ID NO. 5);

The primer set described above was used to detect Pseudo-nitzschia spinulosa.

The present invention further provides a method for rapidly detecting Pseudo-nitzschia spinulosa, comprising the following steps:

1) After the algae sample to be tested is collected by centrifugation, the supernatant is removed and resuspended in TE buffer (10mM Tris-HCl, 1mM EDTA, pH=8.0); the suspension is added to a lysis buffer for lysis; the lysis supernatant is added to a diluent for dilution, and the diluent is used as the test object;

The composition of the lysis solution is as follows: 50 mM Tris, 100 mM EDTA, 50 mM NaCl, 1% (w/v) SDS, 80% (v/v) formamide, pH = 8.0.

The composition of the diluent is as follows: 0.1% Tween 20.

2) Add the test object prepared in step 1) to a constant temperature amplification reaction solution containing hydroxynaphthol blue (HNB) dye and Bst DNA polymerase, use the above primer set, react at 63° C. for 30 to 40 minutes, and determine the result by color change.

3) Result determination:

If the negative control remains violet and the positive control changes from violet to sky blue, the test is normal. If the sample to be tested is violet, it is negative; if the sample to be tested is sky blue, it is positive;

The positive control generally uses the DNA of the target algae species as a reaction template.

For the negative control, double distilled water was added to the reaction system instead of DNA.

The present invention proposes a rapid detection method for Pseudo-nitzschia spinulosa. In the nucleic acid extraction stage, no complicated DNA extraction steps are required. Under normal temperature conditions, the crude extraction of algae nucleic acid can be achieved within 30 minutes. A set of specific primers is designed for the 18SrDNA V4 region sequence of Pseudo-nitzschia spinulosa. The primer set is used in combination with hydroxynaphthol blue (HNB) dye to amplify and complete the detection of the target sequence within 30 to 40 minutes under a constant temperature of 63°C. The method has good specificity and high sensitivity, and does not require a complicated temperature change process and precision instruments. Only a water bath or thermostat can be used to complete the analysis, which greatly simplifies the process of red tide algae molecular detection and is suitable for rapid detection of on-site samples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram showing the comparison results of 18S rDNA (V4) of different strains of Pseudo-nitzschia spinulosa. The 18S rDNA (V4 region) sequences of different strains of Pseudo-nitzschia spinulosa were selected for gene conservation comparison, with the aim of determining the conservation of this gene segment.

FIG2 is a specific comparison result diagram of the target sequence of Pseudo-nitzschia spinulosa, and the 18S rDNA sequences of 12 common red tide algae species such as Prorocentrum microcarpa, Prorocentrum donghaiense, and Karenia mikimotoi were compared to determine that the selected conserved segment has good specificity to ensure the specificity of the designed primers;

Figure 3 is the result of the preliminary screening of primers for Pseudo-nitzschia spinulosa. The V4 region was selected as the target sequence, and 8 sets of primers were designed using PrimerExplorer V5 software. The specificity was verified at the National Center for Biotechnology Information (NCBI) of the United States, and then reacted at 63°C for 50 minutes. After the reaction, the product was detected by 2% agarose gel electrophoresis to select the primer set that can successfully amplify. In the figure, M: DL2000 DNA marker, and each of the subsequent three lanes corresponds to a set of primers. The first two lanes are the experimental groups with added DNA, and the third lane is the negative control group with an equal amount of double distilled water instead of Pseudo-nitzschia spinulosa DNA.

Fig. 4 is a schematic diagram of the position of the reconstructed primer set, in which a loop primer LF is added to the successfully verified conventional primer set (comprising two outer primers F3 and B3, and two inner primers FIP and BIP);

FIG5 is a diagram showing the feasibility test results of the reconstructed primer set, in which M: DL2000 DNA marker, and lanes 1-3 are two parallel groups with added target genomic DNA and a negative control, respectively.

Figure 6 is a graph showing the specificity verification results of the primers for Pseudo-nitzschia spinulosa, wherein Figure a is the electrophoresis detection result, and Figure b is the HNB visualization color development result. In Figure a, lane 1 was added with mixed genomic DNA of 12 common red tide algae species including Pseudo-nitzschia spinulosa (including micro-dinoflagellates, Prorocentrum donghaiense, Karenia mikimoto, red red tide algae, chain-shaped gymnodinium, Prorocentrum lima, Pacific Alexandrium, chain-shaped Alexandrium, mid-rib-stripe algae, Pseudo-nitzschia spinulosa, spherical Phaeocystis, and Enteromorpha), lane 2 was added with mixed genomic DNA of 11 red tide algae except Pseudo-nitzschia spinulosa (including micro-dinoflagellates, Prorocentrum donghaiense, Karenia mikimoto, red red tide algae, chain-shaped gymnodinium, Prorocentrum lima, Pacific Alexandrium, chain-shaped Alexandrium, mid-rib-stripe algae, spherical Phaeocystis, and Enteromorpha), lane 3 was replaced with an equal amount of double distilled water instead of the DNA in lanes 1 and 2 as a negative control; the lanes in Figure b correspond to Figure a.

Figure 7 is a graph showing the sensitivity test results of the primer set for Pseudo-nitzschia spinulosa, where Figure a is the electrophoresis test result; and Figure b is the HNB visualization color development result. The DNA template concentrations in lanes 1-4 are 100pg/μL, 10pg/μL, 1pg/μL, and 0.1pg/μL, respectively, and lane 5 is a negative control; the lanes in Figure b correspond to those in Figure a.

Figure 8 is the electrophoresis diagram a of the rapid detection of Pseudo-nitzschia spinulosa and the HNB visualized color development result diagram b, where M: DL2000 DNA marker, lanes 1, 3, 5, 7, and 9 are respectively the positive sample groups (adding Pseudo-nitzschia spinulosa genomic DNA, if amplification products are produced, the actual image is sky blue, and the corresponding electrophoresis diagram produces ladder-like bands) at 63°C for 20min, 30min, 40min, 50min, and 60min; lanes 2, 4, 6, 8, and 10 are respectively the negative control groups (using an equal amount of double distilled water instead of Pseudo-nitzschia spinulosa genomic DNA, the actual image is violet, and the electrophoresis diagram only has primer dimer bands) at 63°C for 20min, 30min, 40min, 50min, and 60min. Under 63°C conditions, this primer set only takes 30min to 40min to achieve the amplification of the target sequence, the sample tube appears sky blue, and the negative control is violet.

Figure 9 is a visualization of the test results of natural seawater samples using Pseudo-nitzschia spinulosa: the positive control is to directly add the genomic DNA of Pseudo-nitzschia spinulosa to the prepared reaction system, and the negative control is to replace the genomic DNA with an equal amount of double distilled water. Samples 1 and 2 are the products of amplification reactions using crude nucleic acid (sample 1 - natural seawater without Pseudo-nitzschia spinulosa, 2 - natural seawater containing Pseudo-nitzschia spinulosa) as templates.

DETAILED DESCRIPTION

The present invention is described in detail below in conjunction with embodiments and drawings.

Example 1: Screening of gene regions for primer design

1. Target sequence selection

The present invention selects the V4 region of the 18S rDNA gene of Pseudo-nitzschia spinulosa as the target sequence region. First, it is determined that the selected gene segment has a high degree of conservation, so that the designed primers can achieve amplification for different strains of Pseudo-nitzschia spinulosa, and amplification failure will not occur due to strain differences; in addition, the 18SrDNA V4 variable region gene of Pseudo-nitzschia spinulosa has certain specificity, which is convenient for distinguishing from other algae species.

The 18S rDNA (V4 region) sequences of different strains of Pseudo-nitzschia spinulosa were selected for gene conservation comparison, and the comparison results are shown in Figure 1. As shown in Figure 1, the target sequence of Pseudo-nitzschia spinulosa selected by the present invention has good conservation, and there is almost no difference between different strains, and can be applied to the design of specific primers. All gene sequences were downloaded from the National Center for Biotechnology Information (NCBI) of the United States, and sequence alignment was performed using Clustal X software. The GenBank Accession Numbers of the sequences used are as follows: MW336959, MW336967, MW336961, KP709004, GU373968, MZ267111, U18240, JN091724, GU373966, GU373967, and JN091728.

2. Specific verification of gene target segment

The 18S rDNA sequences of 12 common red tide algae were downloaded from the website of the National Center for Biotechnology Information (NCBI) of the United States. The species names and corresponding GenBank Accession Numbers are as follows: Prorocentrum microcarpa (Y16238), Prorocentrum donghaiense (AY551272), Karenia mikimotoi (FJ587220), Red tide algae (AB183672), Gymnodinium catenellae (GU362426), Prorocentrum limae (AB189780), Alexandrium pacificum (KF908800), Alexandrium catenellae (AJ535392), Skeletonema midi (JN676163), Pseudo-nitzschia spinulosa (KP709004), Phaeocystis sphericalensis (AJ278035), Enteromorpha enteromorpha (HQ850569), and the sequences were aligned using Clustal X software to determine the specificity of the target sequence. The alignment results are shown in Figure 2.

The comparison results in FIG2 show that the conserved region of the 18S rDNA V4 region of Pseudo-nitzschia spinulosa selected in the present invention is quite different from the 18S rDNA genes of the other 11 common red tide algae, that is, it has strong specificity and can be used for specific primer design.

Example 2: Primer design

According to the specific target segment selected in Example 1, multiple sets of primers were designed and the primers were initially verified. The designed primers to be screened are as follows:

Table 1: Sequence information of primer sets to be screened

The above designed primer sets were used for specificity verification at the National Center for Biotechnology Information (NCBI). During primer verification, the six segments of the four primers were grouped into groups, F3 and B3 as one group, F2 and B2 as one group, and F1c and B1c as one group. The specificity of the primers was verified separately. The specificity of F2 and B2 was relatively critical. The binding regions of F1c and B1c were compared and verified by Clustal X software. The specificity of F3 and B3 was ensured as much as possible, otherwise light tailing bands may be generated during electrophoresis detection to interfere with the detection. In theory, if one of the six regions of the four primers cannot be matched successfully, successful amplification cannot be achieved.

Taking primer set 4 as an example, the results of the outer primer comparison are shown in Table 2 (F3, B3).

Table 2: BLAST comparison results of external primers

Table 2 shows the results of the comparison of the outer primers (F3, B3) in primer set 4 designed using Pseudo-nitzschia spinulosa in the gene library of the 12 common red tide algae mentioned above (micro-dinoflagellates, Prorocentrum donghaiense, Mikimoto's Kailan, red red tide algae, chain-shaped gymnodinium, Lima Prorocentrum, Pacific Alexandrium, chain-shaped Alexandrium, mid-rib-stripe algae, Pseudo-nitzschia spinulosa, spherical cysts, and Enteromorpha). The table only shows the gene segments with relatively high similarity to the target primers, and those with particularly large differences are not shown in the table. It can be seen that the outer primers have good specificity, can completely match the corresponding segments of Pseudo-nitzschia spinulosa, can match the chloroplast gene of spherical cysts partially, but the difference is large, and the theoretical PCR product length is as long as 4000bp, which almost does not cause amplification of spherical cysts, which once again confirms the high specificity of the outer primers.

The results of the internal primer alignment are shown in Table 3 (F2, B2).

Table 3: BLAST comparison results of inner primers

The comparison results in Table 3 show that F2 and B2 have very high specificity. The BLAST results show that this primer segment can only match the 18S rDNA gene of various strains of Pseudo-nitzschia spinulosa, and has significant differences with the genes of the other 11 target algae, which further ensures the specificity of primer set 4.

After preliminary verification of primer specificity, the designed primer sets were used for amplification reactions. The reaction system and conditions were as follows: 0.1 μM F3 and B3, 0.8 μM FIP and BIP respectively, 1.4 mM dNTP, 6 mM MgSO ₄ , 1×Bst buffer [10 mM KCl, 20 mM Tris-HCl (pH=8.8), 2 mM MgSO ₄ , 10 mM (NH ₄ ) ₂ SO ₄ , 0.1% Triton X-100], 8 U Bst DNA polymerase and 2 μL template, in a total volume of 25 μL, and constant temperature at 63°C for 50 min. The reaction product was detected by 2% agarose gel electrophoresis, and the detection results are shown in Figure 3: from left to right, every three lanes are a set of Pseudo-nitzschia spinulosa primers, and the order is: two parallel groups with added Pseudo-nitzschia spinulosa genomic DNA, and a negative control group with the genomic DNA replaced by double distilled water. The standard for successful amplification is that the first two lanes (i.e., samples with added DNA templates) produce ladder-like bands, while at the same time, the third lane (negative control) only has primer dimer bands below 100 bp. Only the primer set that meets both conditions is considered to be successfully amplified.

Due to the large number of primers in the amplification reaction, the negative control is prone to produce background amplification signals. A primer set should be selected that has no background signal generated in the negative control for a long time and can amplify the target band in the sample group. As shown in Figure 3, primer set 4 was successfully amplified.

For the selected primer set 4, in order to enhance its amplification efficiency, a loop primer LF was added on the basis of four conventional primers (F3, B3, FIP, BIP). The positions of the relevant primers are shown in FIG4 .

The addition of loop primers is a double-edged sword. While increasing the amplification rate, it may also cause nonspecific amplification. Therefore, the reconstructed primer set needs to be retested. The reaction system and conditions are as follows: 0.1 μM F3 and B3, 0.8 μM FIP and BIP, 0.2 μM LF, 1.4 mM dNTP, 6 mM MgSO ₄ , 1×Bst buffer [10 mM KCl, 20 mM Tris-HCl (pH = 8.8), 2 mM MgSO ₄ , 10 mM (NH ₄ ) ₂ SO ₄ , 0.1% Triton X-100], 8 U Bst DNA polymerase and 2 μL template. The total system is 25 μL, and the remaining volume is supplemented with double distilled water. Keep the temperature at 63°C for 30 to 40 minutes. The reaction product is detected by 2% agarose gel electrophoresis, and the detection results are shown in Figure 5.

No ladder-like bands were produced in the negative control group, which proved that the strip-loop primer had a good compatibility with the selected conventional primer set (primer set 4 in Table 1) and did not cause nonspecific amplification. The reconstructed primer set was usable.

Example 3: Detection effect of the screened primers

1. Primer specificity

The genomic DNA of 12 target algae species were mixed in equal volumes as template 1, and the genomic DNA of 11 red tide algae species other than Pseudo-nitzschia spinulosa was mixed in equal volumes as template 2. In the experiment, three reaction solutions were prepared according to the following system (0.1 μM F3 and B3, 0.8 μM FIP and BIP respectively, 0.2 μM LF, 1.4 mM dNTP, 6 mM MgSO ₄ , 1×Bst buffer [10 mM KCl, 20 mM Tris-HCl (pH=8.8), 2 mM MgSO ₄ , 10 mM (NH ₄ ) ₂ SO ₄ , 0.1% Triton X-100], 8 U Bst DNA polymerase), and then 2 μL template 1, 2 μL template 2, and 2 μL double distilled water (negative control) were added in sequence, and amplification was carried out at 63°C. After completion, the product was detected by agarose gel electrophoresis, and the results showed that the primers had good specificity. The test results are shown in Figure 6: the electrophoresis test results of the sample with template 1 showed ladder-like bands, which were blue, indicating a positive result; the sample tube with template 2 and the negative control group had no bands, which were violet, indicating a negative result. This proves that the primer set has good specificity for the amplification of Pseudo-nitzschia spinulosa.

2. Primer sensitivity

Using the standard positive clone plasmid of Pseudo-nitzschia spinulosa as a template, the initial concentration was 100 pg/μL, and it was diluted tenfold, 100 times, 100 times, and 1000 times respectively, amplified according to the reaction system and conditions of Example 3-1, and detected by 2% agarose gel electrophoresis, the sensitivity can reach 1 pg/μL positive clone plasmid.

As can be seen from Figure 7, when plasmids with concentrations of 100 pg/μL, 10 pg/μL, and 1 pg/μL were used as reaction templates, the electrophoresis detection results showed that amplified bands were generated, and the colorimetric method showed that the first three tubes were sky blue, which was consistent with the electrophoresis results and was positive. When the plasmid with a concentration of 0.1 pg/μL was used as a template, the electrophoresis results showed that no amplified bands were generated and the colorimetric result was violet, which was negative. That is, the detection sensitivity of this primer set can reach 1 pg/μL of Pseudo-nitzschia spinosa plasmid DNA.

3. Reaction time

Using the genomic DNA of Pseudo-nitzschia spinulosa as a template, the amplification reaction was carried out at 63°C according to the system of Example 3-1, and the reaction time gradient was set: 20, 30, 40, 50, 60 min. In order to prevent the occurrence of nonspecific amplification, a negative control was added to each gradient sample (DNA was replaced with an equal amount of double distilled water), and the product was detected by 2% agarose gel electrophoresis.

The test results are shown in Figure 8. When the amplification time is 30 minutes, lane 3 in Figure 8 (a), which is the reaction group with DNA added, begins to show amplification product bands, and the corresponding physical picture is sky blue; lane 4 in Figure 8 (a), which is the negative control group with an amplification time of 30 minutes, does not produce amplification bands, and the corresponding physical picture is violet. When the reaction time is 40, 50, and 60 minutes, the reaction groups with DNA added all produce ladder-like bands of similar brightness, and the corresponding physical pictures begin to show sky blue. The negative controls all have no background signals, and the physical pictures are violet. Therefore, after 40 minutes of amplification reaction, the product can be detected.

Example 4: Basic process of detection

1. Test materials and equipment

a) Reagents:

iAmplification reaction system: Bst DNA polymerase, buffer, dNTPs, magnesium sulfate, primers, double distilled water and hydroxynaphthol blue dye (HNB); 5 primers were designed and synthesized according to the 18S rDNA (V4) gene sequence of Pseudo-nitzschia spinulosa, which are a pair of outer primers (F3 and B3), a pair of inner primers (FIP and BIP) and a loop primer (LF). The above primer sequences are shown in Table 4.

Table 4: Primer sequence information of Pseudo-nitzschia spinulosa

ii Cracking system:

Lysis buffer composition: 50 mM Tris, 100 mM EDTA, 50 mM NaCl, 1% (w/v) SDS, 80% (v/v) formamide pH = 8.0. 1 mL of lysis buffer is pre-loaded in a lysis tube.

Diluent composition: 0.1% Tween 20. 0.5 mL of diluent is pre-filled in each diluent tube.

b) Instruments:

Thermostat

2. Reaction system

The components of the detection system and the concentrations of each component are shown in Table 5, and the total reaction system is 25 μL.

Table 5: Reaction system table

*The components of the above buffer include: 200 mM Tris-HCl, 100 mM (NH ₄ ) ₂ SO ₄ , 20 mM MgSO ₄ , 1% Triton X-100, pH=8.8.

3. Experimental Procedure

a) Collection and lysis of algal cells

The natural seawater containing Pseudo-nitzschia spinulosa was filtered through a 0.45 μm pore size filter membrane, and then the filter membrane was rinsed with sterilized seawater to achieve the effect of concentrating and enriching algal cells; the seawater containing Pseudo-nitzschia spinulosa was centrifuged at 12000×g for 5 min, and the supernatant was removed and resuspended with 250 μL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH=8.0), fully shaken, vortexed for 30 s, and 200 μL of the suspension was added to the lysis solution tube for lysis (1 mL of lysis solution was pre-loaded in the lysis tube); 10 μL of the above lysis supernatant was added to the dilution tube and mixed thoroughly (0.5 mL of dilution solution was pre-loaded in the dilution tube). At this time, the liquid in the dilution tube can be used as the detection object.

b) Detection

Take several 200 μL centrifuge tubes and prepare the reaction system according to the reaction system formula in Table 5. After the system is prepared, place the reaction system after adding the sample in a thermostat and react at 63°C for 30 to 40 minutes. The result is determined by the color change.

c) Result determination

When the negative control is violet and the positive control changes from violet to sky blue, the test process is normal. If the sample to be tested is violet, it is negative; if the sample to be tested is sky blue, it is positive.

Typical detection results are shown in Figure 9: the positive control (directly adding Pseudo-nitzschia spinulosa DNA to the system for amplification) and sample 2 (using crude nucleic acid extracted from natural seawater containing Pseudo-nitzschia spinulosa as a template for amplification) are sky blue; the negative control (using an equal amount of double distilled water instead of DNA as a template for amplification) and sample 1 (using crude nucleic acid extracted from natural seawater without Pseudo-nitzschia spinulosa as a template for amplification) are violet.

In summary, the present invention designs and screens a group of specific primers (a total of five, including four conventional primers and one loop primer). By using the primer group in combination with hydroxynaphthol blue (HNB) dye, the target sequence can be amplified and detected in 30 to 40 minutes under a constant temperature of 63°C. It has good specificity and high sensitivity, and does not require a complicated temperature change process and precision instruments. Only a water bath or a thermostat is required to complete the analysis, which greatly simplifies the process of red tide algae molecular detection and is suitable for rapid detection of on-site samples.

Claims (10)

1. The primer group for detecting the rhombohedral spinosynaptera is characterized by comprising the following primers:

the sequence is SEQ ID NO.1: primer F3 of ATTGCGCTGTACTGGTCT,

The sequence is SEQ ID NO.2: primer B3 of TAGTGCGCGCAAACCAACA,

The sequence is SEQ ID NO.3: the primer FIP of GTAAACGATGGGCACCTCACACTATGTTGGGTG GAATCTGTT,

The sequence is SEQ ID NO.4: primer BIP of TTAGTGTTCAAAGCAGGCTTATAGGTCCTATATATCATTCCATGC,

The sequence is SEQ ID NO.5: primer LF of GTACGACAACTTAATGC.

2. The primer set of claim 1, wherein the primer set is used for detecting rhombohedral acus poinarius.

3. A detection kit for detecting phaeodactylum acutangatum, which is characterized by comprising the primer group of claim 1.

4. The kit of claim 1, wherein the kit comprises isothermal amplification reaction solution of Bst DNA polymerase and hydroxynaphthol blue dye.

5. The kit of claim 4, wherein the kit further comprises a lysis solution for extraction of algae DNA.

6. The kit of claim 5, wherein the lysate comprises the following components:

50mM Tris,100mM EDTA,50mM NaCl,1%, SDS,80%, formamide, pH 8.0.

7. A method for rapidly detecting phaeodactylum acutum, which is characterized in that the primer group of claim 1 is used for detection.

8. The method of claim 7, wherein the method comprises the steps of:

1) Centrifuging and collecting an algae sample to be detected, removing supernatant, and then re-suspending with TE buffer solution; adding the suspension into a lysis solution for lysis; adding the cracking supernatant into a diluent for dilution, wherein the diluent is used as a template for detection;

2) Adding the template prepared in the step 1) into a constant-temperature amplification reaction solution containing hydroxynaphthol blue dye and Bst DNA polymerase, reacting at 63 ℃ for 30 to 40 minutes by using the primer group of claim 1, and judging the result through color change;

3) And (4) judging a result:

the violet color of the negative control is unchanged, and the sky blue color of the positive control is changed from the violet color, so that the test process is normal; if the sample to be detected is violet, the sample is negative; and if the sample to be detected is sky blue, the sample to be detected is positive.

9. The method of claim 8, wherein the diluent is 0.1% Tween20.

10. The method of claim 8, wherein the negative control is double distilled water instead of DNA.

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