Rapid color development detection method for karenia mikimotoi

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a rapid color development detection method of karenia mikimotoi. A primer group in the rapid color development detection method comprises a primer KM3-F3 with a sequence as shown in SEQ ID NO.1, a primer KM3-B3 with a sequence as shown in SEQ ID NO.2, a primer KM3-FIP with a sequence as shown in SEQ ID NO.3, a primer KM3-BIP with a sequence as shown in SEQ ID NO.4 and a primer KM3-LF with a sequence as shown in SEQ ID NO.5. Under the condition of ensuring that target sequences are consistent, compared with a conventional PCR (Polymerase Chain Reaction) method, the method disclosed by the invention has the advantages that the amplification specificity and sensitivity are not lower than those of the conventional PCR technology, the amplification can be completed only at the constant temperature of 60-65 DEG C for 40-50 minutes, the product concentration is high, the amplification effect is stable, and the method is suitable for large-scale popularization and application. Therefore, the method has a good application prospect in the field of rapid detection of red tide algae.

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 Karenia mikimotoi. Background Art

[0002] Karenia mikimotoi belongs to the genus Karenia and is an important phytoplankton widely distributed in coastal waters around the world, especially in temperate to subtropical waters. This algae can proliferate rapidly under specific environmental conditions, forming large-scale algal blooms, which have a significant impact on the marine ecosystem. Especially in the coastal areas of China, including the Yellow Sea and the East China Sea, Karenia mikimotoi frequently becomes one of the main perpetrators of red tide events, posing a potential threat to the marine environment, fishery resources and human health. Its large-scale reproduction will not only lead to water quality deterioration and affect the survival and reproduction of marine organisms, but may also transfer harmful substances through the food chain, posing risks to human health. Therefore, in-depth research on the causes of Karenia mikimotoi red tides, predicting its development trend, and building an effective monitoring and early warning mechanism has become an urgent task in the current marine environmental protection and management work.

[0003] At present, the detection of Karenia mikimotoi mainly relies on traditional microscopic observation for morphological identification and high-precision detection methods based on molecular biology technology. However, the traditional microscopic observation method requires the inspectors to have profound taxonomic knowledge and practical experience to accurately identify different algae species with similar morphology. This process is not only time-consuming and laborious, but also susceptible to subjective judgment. On the other hand, although molecular biology technology has high accuracy and specificity, its expensive experimental equipment, complex sample processing procedures and long detection cycles severely limit the method's ability to respond quickly in field monitoring or emergency situations. Given that the rapid response and effective prevention and control of Karenia mikimotoi red tides are crucial to protecting marine ecological security, fishery resources and human health, it is particularly necessary 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 Karenia mikimotoi, 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 Karenia mikimotoi. Summary of the invention

[0005] The purpose of the present invention is to provide a rapid color development detection method for Karenia mikimotoi, which does not require complex instruments and is easy to operate. It can instantly display the presence and relative concentration of Karenia mikimotoi, providing 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 present invention provides a primer set for detecting Karenia mikimotoi, wherein the primer set comprises a primer KM3-F3 whose sequence is shown in SEQ ID NO.1, a primer KM3-B3 whose sequence is shown in SEQ ID NO.2, a primer KM3-FIP whose sequence is shown in SEQ ID NO.3, a primer KM3-BIP whose sequence is shown in SEQ ID NO.4, and a primer KM3-LF whose sequence is shown in SEQ ID NO.5.

[0008] The present invention also provides a kit for detecting Karenia mikimotoi, 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 Karenia mikimotoi, comprising the following steps:

[0012] 1) After the algae sample to be tested is collected by centrifugation, the supernatant is removed and then 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 Karenia mikimotoi.

[0018] Beneficial effects:

[0019] The present invention provides a target sequence designed by using the ITS region sequence of Karenia mikimotoi 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 Karenia mikimotoi are specifically verified using purified genomic DNA. At the same time, a conventional PCR method is used to compare the primers with the target sequence under the condition of ensuring the consistency of the target sequence. The results show that the P 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 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. 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 result of ITS region amplification of Karenia mikimotoi of the present invention;

[0022] Figure 2 This is a diagram showing the comparison of the ITS region gene sequences of Karenia mikimotoi of the present invention;

[0023] Figure 3 This is a diagram showing the alignment results of the ITS-specific segment sequences of Karenia mikimotoi of the present invention;

[0024] Figure 4 This is a diagram showing the comparison of the SSU region gene sequences of Karenia mikimotoi of the present invention;

[0025] Figure 5 This is a diagram showing the sequence alignment results of the target algae SSU specific segment of Karenia mikimotoi 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 Karenia mikimotoi of the present invention, wherein PC: experimental group with genomic DNA added; NTC: control group without template;

[0028] Figure 8 Schematic diagram of primer binding sites of Karenia mikimotoi of the present invention;

[0029] Fig. 9The figure is a result of LAMP primer specificity verification of Karenia mikimotoi of the present invention, wherein A: electrophoresis result; B: color development result; M: DL2000 DNA marker; SC: Skeletonema costatum; PM: Prorocentrum microphyllum; 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 Karenia mikimoto of the present invention, wherein A: electrophoresis result after detection by the present method; B: electrophoresis result of PCR reaction; C: visualization result after detection by the present method; 1-6: 3.46 ng / μL, 0.346 ng / μL, 0.0346 ng / μL, 0.00346 ng / μL, 0.000346 ng / μL, and 0.0000346 ng / μL of Karenia mikimoto 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 also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[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 those skilled in the art. 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 universal primers for the ITS region of eukaryotic algae were used to amplify the ITS region gene of Karenia mikimotoi. 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 Karenia mikimotoi 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 is 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 Karenia mikimotoi sequence in GenBank, both above 98%, so it can be determined that the sequenced algae species is Karenia mikimotoi.

[0042] Then, the ITS region sequences of 13 experimental algae were compared using the software Bioedit, and the specific segment of Karenia mikimotoi 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 Karenia mikimotoi 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 Karenia mikimotoi 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 Karenia mikimotoi DNA) 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 Karenia mikimotoi was constructed and 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 very likely to occur. After preliminary verification of primer specificity, multiple sets of designed primers were used for amplification reaction. The reaction system and conditions were as follows: 0.1μM F3 and B3, 0.8μM FIP and BIP respectively, 0.2μM LF, 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 was 25μL, and the remaining volume was 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 Karenia mikimoto primers, and the order is: two parallel groups with added Karenia mikimoto genomic DNA, and a negative control group with the genomic DNA replaced by double distilled water. The criterion for successful amplification is that the first two lanes (i.e., samples with added DNA templates) 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, KM3 was selected as the best primer for each target algae, and the primer sequences are shown in Table 1.

[0050] Table 1 Optimal primer sequence information for Karenia mikimotoi

[0051]

[0052] It can be seen that the primers of Karenia mikimotoi 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 Karenia mikimotoi 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 for Karenia mikimotoi, the primers for Karenia mikimotoi were cross-validated using genomic DNA of various red tide algae as templates to ensure that the primers for 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 for Karenia mikimotoi are shown in the figure below. Fig. 9 shown.

[0055] Depend on Fig. 9It can be seen that the Karenia mikimotoi-specific primers of this method can only achieve amplification with Karenia mikimotoi, and 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, proving that the detection primers of this method for Karenia mikimotoi have high interspecies specificity.

[0056] 4 Sensitivity verification results

[0057] The Karenia mikimotoi genomic DNA solution was diluted 10 times in a series of dilutions (3.46×10 -5 ~3.46×10 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 3.46×10 -3 ~3.46×10 0 ng / μL, this method can amplify Karenia mikimotoi DNA and produce waterfall-like amplification bands (such as Fig.10 The corresponding reaction tube is sky blue and turbid (as shown in A). Fig.10 Therefore, the primer sensitivity of Karenia mikimotoi can reach 3.46×10 -3 ng / μL genomic DNA.

[0059] Table 2 Target algae primer sensitivity test results

[0060]

[0061] Example 2 Rapid color detection of Karenia mikimotoi

[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: 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.

[0067] Diluent composition: 0.1% Tween 20. 0.5 mL of diluent is pre-filled in each diluent tube.

[0068] b) Instrument: thermostat

[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]

[0074] Note: The components of the above buffer include: 200mM Tris-HCl, 100mM (NH4)2SO4, 20mM MgSO4, 1% Triton X-100, pH=8.8.

[0075] 3. Experimental Procedure

[0076] a) Collection and lysis of algal cells

[0077] The natural seawater containing Karenia mikimotoi 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 Karenia mikimotoi 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 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.

[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 Karenia mikimotoi as the primer to design the target sequence, designs a set of primers that can be used for the 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 the primer design. In addition, the purified genomic DNA was used to verify the specificity of the set of primers for Karenia mikimotoi. At the same time, the conventional PCR method was used to compare it horizontally while ensuring the consistency of the target sequence. The results showed that the method was not inferior to the conventional PCR technology in terms of amplification specificity and sensitivity, and the method only needed to be kept constant at 60-65°C for 40-50min to complete the amplification, the product concentration was high, and the amplification effect was 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 Karenia mikimotoi, characterized in that: The primer set includes primer KM3-F3 with a sequence as shown in SEQ ID NO.1, primer KM3-B3 with a sequence as shown in SEQ ID NO.2, primer KM3-FIP with a sequence as shown in SEQ ID NO.3, primer KM3-BIP with a sequence as shown in SEQ ID NO.4, and primer KM3-LF with a sequence as shown in SEQ ID NO.

5.

2. A kit for detecting Karenia mikimotoi, 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 color development detection method for Karenia mikimotoi, 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 adding double distilled water instead of DNA 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 Karenia mikimotoi.

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