Rapid color development detection method for prorocentrum donghaiense
By designing the primer set for East China Sea prodinosa and PCR technology equipped with corresponding kits, the rapid, simplicity and economical problems of East China Sea prodinosa detection in the existing technology are solved, and rapid and accurate detection support is achieved in the red tide event.
Patent Information
- Application Number
- CN202510025098.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-06
- Publication Date
- 2025-03-14
AI Technical Summary
The prior art is difficult to quickly, easily and economically detect the presence and relative concentration of East China Sea prodinosa on-site, especially in the rapid response and prevention and control of red tide events.
A primer set was designed to detect Donghai prodinosa and equipped with a kit, including Bst DNA polymerase, buffer, dNTPs, magnesium sulfate, double distilled water and hydroxynaphthol blue staining agent. The constant temperature amplification was performed at 63°C for 30-40 minutes under PCR technology, and the results were determined by color change.
It realizes rapid color development detection of Donghai prototinoes without complex instruments and easy to operate, which can instantly display the presence and relative concentration of algae, and supports early warning and emergency treatment of red tides.
Smart Images
Figure CN119614740A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biological detection, and particularly relates to a rapid color development detection method for Prorocentrum donghaiense. Background Art
[0002] Prorocentrum donghaiense belongs to the phylum Dinophyceae and family Prorocentrum donghaiense. It is one of the phytoplankton widely distributed in coastal waters around the world, especially along the coast of East Asia, including the East China Sea region of China, where it frequently becomes the main culprit of red tide events. This algae species has not only caused large-scale algal blooms in the waters near coastal cities such as Ningbo, Shanghai, and Fuzhou, damaging water quality and affecting aquaculture and fishery resources, but also posed a serious threat to the marine ecosystem. Therefore, in-depth exploration of the causes and development trends of the Prorocentrum donghaiense red tide and the establishment of an effective monitoring and early warning system have become key research contents in marine environmental protection and management.
[0003] At present, the detection methods for Prorocentrum donghaiense mainly rely on traditional microscopic morphological identification and high-precision molecular biology technology. The former requires the detection personnel to have a high degree of professional classification knowledge and rich practical experience to accurately distinguish between algae species with similar morphology. This process is time-consuming and easily affected by subjective factors. Although the latter has high accuracy, it relies on expensive experimental equipment, complex sample processing procedures and long analysis cycles, which limits its application in the field or in emergency response situations. In view of the rapid response and prevention and control needs of the Prorocentrum donghaiense red tide, it is particularly urgent to develop a field detection method that is fast, intuitive, economical and easy to operate.
[0004] In summary, in order to overcome the limitations of existing detection technologies and meet the needs of real-time and efficient monitoring of Prorocentrum donghaiense, the present invention aims to provide a rapid colorimetric detection method that does not require complex instruments, is easy to operate, and can instantly display the presence and relative concentration of Prorocentrum donghaiense. Summary of the invention
[0005] The purpose of the present invention is to provide a rapid color development detection method for Prorocentrum donghaiense, which does not require complex instruments and is easy to operate. It can instantly display the presence and relative concentration of Prorocentrum donghaiense, and provide strong technical support for early warning and emergency treatment of red tides.
[0006] The objective of the present invention is achieved through the following technical solutions:
[0007] The invention provides a primer set for detecting Prorocentrum donghaiense. The primer set comprises a primer PD3-F3 whose sequence is shown in SEQ ID NO.1, a primer PD3-B3 whose sequence is shown in SEQ ID NO.2, a primer PD3-FIP whose sequence is shown in SEQ ID NO.3, a primer PD3-BIP whose sequence is shown in SEQ ID NO.4, and a primer PD3-LB whose sequence is shown in SEQ ID NO.5.
[0008] The present invention also provides a kit for detecting Prorocentrum donghaiense, and the kit comprises the primer set.
[0009] Furthermore, the kit also includes Bst DNA polymerase, buffer, dNTPs, magnesium sulfate, double distilled water and hydroxynaphthol blue dye (HNB).
[0010] Furthermore, the kit also includes a lysis solution for extracting algae DNA.
[0011] The present invention also provides a rapid color development detection method for Prorocentrum donghaiense, comprising the following steps:
[0012] 1) After the algae sample to be tested is collected by centrifugation, the supernatant is removed and the sample is resuspended in TE buffer; 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 a template for detection;
[0013] 2) adding the template prepared in step 1) to a constant temperature amplification reaction solution containing hydroxynaphthol blue dye and Bst DNA polymerase, using the primer set, reacting at 63° C. for 30-40 minutes, and determining the result by color change;
[0014] 3) The result is determined as follows: if the negative control remains violet and the positive control changes from violet to sky blue with turbidity, the test is normal; if the sample is violet and clear, it is negative; if the sample is sky blue with turbidity, it is positive.
[0015] Furthermore, the diluent is 0.1% Tween20.
[0016] Furthermore, the negative control is prepared by adding double distilled water instead of DNA into the reaction system.
[0017] The present invention also provides an application of the primer set or the kit in detecting Prorocentrum donghaiense.
[0018] Beneficial effects:
[0019] The present invention provides a target sequence designed by using the ITS region sequence of Prorocentrum donghaiense as a primer, designs a set of primers that can be used for specific amplification of target algae genes, explains the binding position of the primers and the target sequence, and analyzes the reasons for the failure of primer design. In addition, the selected primers of Prorocentrum donghaiense were specifically verified using purified genomic DNA. At the same time, a conventional PCR method was used to compare the primers with the primers while ensuring the consistency of the target sequence. The results show that the method is not inferior to the conventional PCR technology in terms of amplification specificity and sensitivity. Moreover, the method only needs to be kept constant at 60-65°C for 40-50 minutes to complete amplification. The product concentration is high and the amplification effect is stable, indicating that the method has good application prospects in the field of rapid detection of red tide algae. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] 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 work.
[0021] Figure 1 This is a diagram showing the electrophoresis results of the ITS region amplification of Prorocentrum donghaiense of the present invention;
[0022] Figure 2 This is a diagram showing the comparison of the ITS region gene sequences of Prorocentrum donghaiense of the present invention;
[0023] Figure 3 This is a diagram showing the alignment results of the ITS-specific segment sequences of Prorocentrum donghaiense of the present invention;
[0024] Figure 4 This is a diagram showing the comparison of the SSU region gene sequences of Prorocentrum donghaiense of the present invention;
[0025] Figure 5 This is a diagram showing the sequence alignment results of the target algae SSU specific segment of Prorocentrum donghaiense of the present invention;
[0026] Figure 6 This is an illustration of typical amplification results in the present invention;
[0027] Figure 7 The figure is a primer validation result diagram of Prorocentrum donghaiense of the present invention, wherein PC: experimental group with added genomic DNA; NTC: control group without template;
[0028] Figure 8 Schematic diagram of primer binding sites of Prorocentrum donghaiense of the present invention;
[0029] Fig. 9The figure is the result of primer specificity verification of Prorocentrum donghaiense of the present invention, wherein A: electrophoresis result; B: color development result; M: DL2000 DNA marker; SC: Skeletonium costatum; PM: Prorocentrum microphylum; PL: Prorocentrum lima; AC: Alexandrium catenella; AP: Alexandrium pacificum; AS: red tide algae; EP: Enteromorpha; PD: Prorocentrum donghaiense; PG: Phaeocystis spherical; KM: Karenia mikimotoi; CC: Chaetoceros spiralis; PT: Phaeochromis triangularis; TP: Thalassiosira pseudomicronaria; NTC: no template control;
[0030] Fig.10 This is a graph showing the primer sensitivity verification result of Prorocentrum donghaiense of the present invention, wherein A: electrophoresis result after detection by the method; B: electrophoresis result of PCR reaction; C: visualization result after detection by the method; 1-6: 4ng / μL, 0.4ng / μL, 0.04ng / μL, 0.004ng / μL, 0.0004ng / μL, 0.00004ng / μL Prorocentrum donghaiense genomic DNA were added as reaction templates, respectively; NTC: no template control. DETAILED DESCRIPTION
[0031] 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 rather should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0032] 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 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.
[0033] 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.
[0034] 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.
[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0036] Unless otherwise specified, the chemical reagents, biochemical reagents and materials used in the present invention can be obtained from commercial sources.
[0037] Example 1
[0038] 1 Primer design and target sequence analysis
[0039] The ITS region gene of Prorocentrum donghaiense was amplified using universal primers of the ITS region of eukaryotic algae. The electrophoresis results of the amplified products are shown in Figure 1 shown.
[0040] The length of the amplified product of the ITS region of Prorocentrum donghaiense is between 600 and 750 bp, which is similar to the length of the ITS region sequence of the target algae in GenBank. The electrophoresis result shows a single band, and the negative control has no amplification, which can confirm that the amplification is successful. The amplified product was sent to the company for sequencing, and the obtained sequence is shown in SEQ ID NO.6.
[0041] The sequencing results were submitted to GenBank for BLASTn comparison analysis. Figure 2 As shown, the sequencing results have a high degree of identity with the Prorocentrum donghaiense sequence in GenBank, both above 98%, so it can be determined that the sequenced algae species is Prorocentrum donghaiense.
[0042] Then, the ITS region sequences of 13 experimental algae were compared using the software Bioedit, and the specific segment of Prorocentrum donghaiense was selected for the design of primers in this method. The sequence comparison results are shown in Figure 3 shown.
[0043] Similarly, the SSU segment sequence was subjected to conservation analysis, and the results were as follows Figure 4 The SSU region sequences of Prorocentrum donghaiense selected in this example have small differences between different strains of the same species, in other words, the target sequence has good conservation.
[0044] The software Bioedit was used to compare the SSU sequences of 13 experimental algae, and the specific segment of Prorocentrum donghaiense was selected for primer design. The sequence comparison results are shown in the figure. Figure 5 shown.
[0045] 2 Primer screening results and final target sequence selection
[0046] The detection results of this method are different from those of conventional PCR reactions. Its multi-primer characteristics and unique amplification mechanism determine the high amplification specificity. The positive amplification product is reflected as a waterfall-like amplification band in the electrophoresis diagram, and the negative product has no amplification band (only primer dimer bands below 100bp). Typical amplification results are as follows: Figure 6 shown.
[0047] For primers that successfully amplified, the products of the experimental group (adding DNA of Prorocentrum donghaiense) showed waterfall-like bands on the electrophoresis graph, while the products of the negative control group (using an equal amount of double-distilled water instead of DNA) showed no amplified bands on the electrophoresis graph, only primer dimer bands with a length of less than 100 bp (such as Figure 6 Primer groups 1, 5, and 6 in the experiment). There are several types of primer groups that fail to amplify: ① No amplification reaction occurs in the experimental group. The reason why this primer is unusable is that it cannot successfully amplify the target sequence (such as primer group 3). ② Amplification bands appear in both the experimental group and the negative control group. This primer group is relatively common. The reason for this phenomenon may be that the primer sequence is improperly selected, resulting in non-specific amplification of the primers as amplification templates, so amplification also occurs in the negative control group, such as Figure 6 As shown in primer set 4 and primer set 7, it is impossible to determine whether the amplified bands in the experimental group are formed by the primers amplifying the target sequence. In other words, it is impossible to ensure the amplification specificity of the primer set for the target sequence. ③ The amplified bands in the experimental group appear sometimes and disappear sometimes, and there are no amplified bands in the negative control. The main reason for this type of failure is that the primers are not stable enough to amplify the target sequence.
[0048] Based on the above content, the detection system of this method for Prorocentrum donghaiense was constructed, and it was placed in a PCR instrument for amplification reaction. The product was detected by 1.5% agarose gel electrophoresis. Since this method requires more primers (generally a set of primers consists of 4 to 6 primers), non-specific amplification is easy to occur. After preliminary verification of primer specificity, the designed multiple sets of primers were used for amplification reaction. The reaction system and conditions are as follows: 0.1μM F3 and B3, 0.8μM FIP and BIP respectively, 0.2μM LB, 1.4mM dNTP, 6mM MgSO4, 1×Bst buffer [10mM KCl, 20mM Tris-HCl (pH8.8), 2mMMgSO4, 10mM (NH4)2SO4, 0.1% Triton X-100], 8U Bst DNA polymerase and 2μL template, the total system is 25μL, and the remaining volume is supplemented with double distilled water. The reaction product was detected by 2% agarose gel electrophoresis. The final detection results of the designed primers are as follows: Figure 7As shown, Figure 7 From left to right in the figure, every three lanes are a set of primers of Prorocentrum donghaiense, and the order is: two parallel groups with added genomic DNA of Prorocentrum donghaiense, and a negative control group with genomic DNA replaced by double distilled water. The criterion for successful amplification is that the first two lanes (i.e., samples with added DNA template) produce ladder-like bands, while the third lane (negative control) only has primer dimer bands below 100bp.
[0049] When screening primers, in addition to ensuring that the primers can be successfully amplified (positive control has amplified bands, negative control has no amplified bands), it is also necessary to ensure that the primers have a high amplification efficiency (high product concentration, that is, brighter electrophoresis bands). Based on the above two aspects, the applicant combined Figure 7 According to the amplification results, PD3 was selected as the best primer for Prorocentrum donghaiense, and the primer sequences are shown in Table 1.
[0050] Table 1 Optimal primer sequence information of Prorocentrum donghaiense
[0051]
[0052] It can be seen that the primers of Prorocentrum donghaiense consist of 5 primer sequences, including 4 regular primers and 1 loop primer. In addition to accelerating the amplification reaction rate, the loop primer can also enhance the amplification specificity of the primer. Figure 8 The ITS region gene consists of three parts: ITS1 region gene, 5.8S region gene, and ITS2 region gene. Among them, the 5.8S region sequence is the most conservative, followed by ITS1, and the ITS2 region has the strongest specificity. The primers for Prorocentrum donghaiense finally selected in this example all use the ITS region as the target sequence, ensuring the specificity of the primers.
[0053] 3 Specificity verification results
[0054] In order to ensure the amplification specificity of the primers of Prorocentrum donghaiense, the primers of Prorocentrum donghaiense were cross-validated using various red tide algae genomic DNA as templates to ensure that the primers of the target algae can only successfully amplify the algae and will not amplify other red tide algae. By adding an appropriate concentration of hydroxynaphthol blue (HNB) dye to the reaction system, the test results can be visualized. The positive amplification product is sky blue with turbidity, and the negative product is violet and clear. The products were detected using 1.5% agarose gel electrophoresis. The verification results of the specificity of the primers of Prorocentrum donghaiense are as follows: Fig. 9 shown.
[0055] Depend on Fig. 9It can be seen that the specific primers of Prorocentrum donghaiense in this method can only achieve amplification with Prorocentrum donghaiense. Waterfall-like step amplification bands appear on the electrophoresis diagram, and the products are mainly concentrated between 100 and 500 bp. The positive amplification product appears sky blue with turbidity, and the negative amplification product appears violet and clear and transparent. The color development results are consistent with the electrophoresis results, which proves that the detection primers of this method for Prorocentrum donghaiense have high interspecies specificity.
[0056] 4 Sensitivity verification results
[0057] The genomic DNA solution of Prorocentrum donghaiense was diluted 10-fold in a series of dilutions (4.0×10 -5 ~4.0×10 0 ng / μL), and then used it as a reaction template for amplification. The product was detected by 1.5% agarose gel electrophoresis, and the amplification results were visualized by adding HNB dye. At the same time, the PCR method was used for horizontal comparison to verify the results. Fig.10 As shown in Table 2.
[0058] Depend on Fig.10 It can be seen that when the initial template concentration is between 4.0×10 -4 ng / μL and 4.0×10 0 ng / μL, this method can amplify the DNA of Prorocentrum donghaiense, producing waterfall-like amplified bands (such as Fig.10 The corresponding reaction tube color is sky blue (as shown in A in Fig.10 Therefore, the primer sensitivity of this method for Prorocentrum donghaiense can reach 4.0×10 - 4 ng / μL genomic DNA. As the initial template concentration in the reaction system decreases, the brightness of the electrophoretic band of the amplified product does not change significantly (e.g. Fig.10 As shown in A, the brightness of the electrophoretic band of the PCR product showed a clear trend of darkening. When the initial template concentration was 4.0×10 -4 ng / μL, the electrophoresis band of the PCR amplification product has become very dim, which means that the stability and sensitivity of this method are not inferior to the PCR method.
[0059] Table 2 Results of primer sensitivity test of Prorocentrum donghaiense
[0060]
[0061] Example 2 Rapid color detection of Prorocentrum donghaiense
[0062] 1. Test materials and equipment
[0063] a) Reagents:
[0064] i Amplification reaction system: Bst DNA polymerase, buffer, dNTPs, magnesium sulfate, double distilled water, hydroxynaphthol blue dye (HNB) and primers SEQ ID NO.1-5 designed in Example 1.
[0065] ii Cracking system:
[0066] Lysis buffer composition: 50mM Tris, 100mM EDTA, 50mM NaCl, 1% (w / v) SDS, 80%% (v / v) formamide, pH 8.0. 1mL of lysis buffer is pre-loaded in the lysis tube.
[0067] Diluent composition: 0.1% Tween 20. 0.5 mL of diluent is pre-filled in each diluent tube.
[0068] b Tenderizer: Constant temperature instrument
[0069] 2. Reaction system
[0070] 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.
[0071] The specific reaction system in the detection process is shown in Table 3:
[0072] Table 3 Reaction system
[0073] Reaction components volume dNTPs(10mM) 3.5μL <![CDATA[MgSO4(100mM)]]> 1.5μL Buffer (10×) 2.5μL F3 (10 μM) 0.5μL B3 (10 μM) 0.5μL FIP (10 μM) 4.0μL BIP (10 μM) 4.0μL LF(10μM) 1.0μL HNB dye (1.875 mM) 2.0μL Bst DNA polymerase 8U / μL 1.0μL Double distilled water 2.5μL template 2.0μL
[0074] Note: The ingredients of the above buffer include: 200mM Tris-HCl, 100mM (NH4)2SO4, 20mM MgSO4, 1% Triton X-100, pH8.8.
[0075] 3. Experimental Procedure
[0076] a) Collection and lysis of algal cells
[0077] The natural seawater containing Prorocentrum donghaiense was filtered with 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 Prorocentrum donghaiense was centrifuged at 12000g for 5 minutes, and the supernatant was removed and resuspended with 250 μL TE buffer (10mM Tris-HCl, 1mM EDTA, pH8.0), fully shaken, vortexed for 30 seconds, 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 fully mixed (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.
[0078] b) Detection
[0079] Take several 200 μL centrifuge tubes and prepare the reaction system according to the reaction system formula in Table 3. 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.
[0080] c) Result determination
[0081] 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.
[0082] In summary, the present invention uses the ITS region sequence of Prorocentrum donghaiense as the primer design target sequence, designs a set of primers that can be used for specific amplification of the target algae gene, explains the binding position of the primers and the target sequence, and analyzes the reasons for the failure of primer design. In addition, the selected set of primers of Prorocentrum donghaiense was specifically verified using purified genomic DNA. At the same time, while ensuring the consistency of the target sequence, it was horizontally compared with the conventional PCR method. The results show that the method is not inferior to the conventional PCR technology in terms of amplification specificity and sensitivity, and the method only needs to be kept constant at 60-65°C for 40-50min to complete the amplification, the product concentration is high, and the amplification effect is stable, indicating that it has good application prospects in the field of rapid detection of red tide algae.
[0083] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.
Claims
1. A primer set for detecting Prorocentrum donghaiense, characterized in that: The primer set includes primer PD3-F3 with a sequence as shown in SEQ ID NO.1, primer PD3-B3 with a sequence as shown in SEQ ID NO.2, primer PD3-FIP with a sequence as shown in SEQ ID NO.3, primer PD3-BIP with a sequence as shown in SEQ ID NO.4, and primer PD3-LB with a sequence as shown in SEQ ID NO.
5.
2. A kit for detecting Prorocentrum donghaiense, characterized in that: The kit comprises the primer set according to claim 1.
3. The kit according to claim 2, characterized in that The kit also includes Bst DNA polymerase, buffer, dNTPs, magnesium sulfate, double distilled water and hydroxynaphthol blue dye (HNB).
4. The kit according to claim 2, characterized in that The kit also includes a lysis solution for extracting algae DNA.
5. A rapid colorimetric detection method for Prorocentrum donghaiense, characterized in that: The following steps are involved: 1) After the algae sample to be tested is collected by centrifugation, the supernatant is removed and the sample is resuspended in TE buffer; The suspension is added to a lysis solution for lysis; the lysis supernatant is added to a diluent for dilution, and the diluent is used as a template for detection; 2) adding the template prepared in step 1) to a constant temperature amplification reaction solution containing hydroxynaphthol blue dye and Bst DNA polymerase, using the primer set of claim 1, reacting at 63° C. for 30-40 minutes, and determining the result by color change; 3) The result is determined as follows: if the negative control remains violet and the positive control changes from violet to sky blue with turbidity, the test is normal; if the sample is violet and clear, it is negative; if the sample is sky blue with turbidity, it is positive.
6. The method according to claim 5, characterized in that The diluent is 0.1% Tween20.
7. The method according to claim 5, characterized in that The negative control is prepared by replacing DNA with double distilled water and adding it into the reaction system.
8. Use of the primer set according to claim 1 or the kit according to any one of claims 2 to 4 in detecting Prorocentrum donghaiense.