Method for quantitatively detecting microcystic toxins

Abstract

The invention provides a method for quantitatively detecting microcystic toxins and a recombinant plasmid, a specific primer is designed aiming at the recombinant plasmid, and the microcystic toxins in a water environment are quantitatively detected by adopting a real-time quantitative PCR (Polymerase Chain Reaction) method. Experiments prove that the method for quantitatively detecting the microcystic toxins and the recombinant patent have the characteristics of combination of broad-spectrum recognition and specificity, are high in sensitivity, simple and convenient in preparation method, capable of being stored for a long time, good in purity and wide in linear detection range, and can be used for rapid quantitative detection of the microcystic toxins in water environment samples.

Description

Technical Field

[0001] The invention belongs to the technical field of water treatment, and in particular relates to a method for quantitatively detecting microcystin and a recombinant plasmid that can be used for quantitatively detecting a microcystin standard product. Background Art

[0002] Blue algae blooms caused by eutrophication of water bodies occur from time to time. Some large lakes and reservoirs, such as Taihu Lake, Dianshan Lake, Dianchi Lake, Xuanwu Lake, and the Three Gorges Reservoir, experience blue algae blooms every summer and autumn. When the blooms occur, the "green scum" formed on the water surface emits a fishy smell, affecting the ecological landscape of recreational water bodies and the drinking water safety of water supply bodies. The dominant species is mainly Microcystis. The large-scale outbreak of blue algae blooms greatly endangers the structure and function of the aquatic ecosystem. One of the main hazards is the production and release of cyanobacterial toxins, with microcystins as the main type.

[0003] Some microcystis, such as Microcystis aeruginosa, Microcystis viridis, Microcystis whitley, etc., can produce and release microcystin toxins, threatening the life safety of wild animals, livestock, and poultry.

[0004] Specifically, the toxin-producing Microcystis aeruginosa can release a large amount of microcystin in the water. Microcystin has been detected in water bodies around the world except in Antarctica. There are more than 100 isomers of microcystin, and its role is still unclear, but it can affect the growth of phytoplankton, invertebrates and vertebrates. Zooplankton ingests the toxin-producing Microcystis aeruginosa, and microcystin accumulates through the food chain. Microcystin can be detected in the bodies of vertebrates and invertebrates living in fresh or salt water, such as mussels, crustaceans, corals and fish. Humans exposed to aerosolized microcystin during recreational activities will experience skin discomfort and temporary breathing difficulties, but generally the harm to the human body is not great. However, if humans eat aquatic products contaminated with microcystin, or accidentally inhale microcystin, they may suffer from severe liver poisoning or neurotoxicity.

[0005] Some zooplankton and fish have great tolerance to microcystins, but microcystins can be enriched in their bodies, eventually posing a potential threat to humans through the ecosystem and food chain. Even non-toxic Microcystis can increase the frequency and severity of blue algae blooms in lakes and reservoirs. Therefore, the detection of microcystins in lakes and reservoirs should be strengthened to provide a scientific basis for protecting the ecological environment and ensuring the safety of drinking water.

[0006] At present, the identification methods of Microcystis are mainly: 1) Physical method, which is mainly based on the spatial layout of morphology and the different configurations of the group. However, this method is time-consuming, labor-intensive, inefficient, requires rich experience, and cannot be carried out by non-professionals; and the environment has a great impact on the morphology of algae, and it is difficult to identify specific species. It is necessary to find direct evidence from the molecule for identification; 2) Biological method, that is, using insects and other animals to conduct toxicity tests on water samples, but the time cost is high, and it involves the use of animals, which does not meet ethical requirements; 3) Chemical method, such as HPLC, GC-MS or spectroscopy, has high sensitivity and good accuracy, but requires expensive professional equipment and technical support. Summary of the invention

[0007] In order to address the deficiencies of the prior art, the present application provides a method for quantitatively detecting microcystins and a standard product thereof.

[0008] One aspect of the present application is to provide a recombinant plasmid that can be used for quantitative detection of a standard substance for microcystin toxins, wherein the recombinant plasmid contains a sequence of SEQ ID No. 1.

[0009] In a preferred embodiment, the starting plasmid in the recombinant plasmid is pMD-18T.

[0010] Preferably, Sequence 1 in the sequence listing consists of 215 deoxyribonucleotides.

[0011] Another aspect of the present application is to provide a method for quantitatively detecting microcystin, comprising:

[0012] Provide the aforementioned standards;

[0013] Using the recombinant plasmid as the standard template, a real-time quantitative PCR amplification was performed using a PCR amplification system to establish a one-dimensional linear regression curve corresponding to the concentration of the standard and the critical cycle number, that is, to obtain a standard curve;

[0014] The standard is replaced by the sample to be tested, and real-time quantitative PCR amplification is performed. According to the critical cycle number of the amplification result and the standard curve, the copy number of the SEQ ID No.1 sequence in the sample to be tested is obtained, that is, the concentration of microcystin in the sample to be tested is obtained;

[0015] The PCR amplification system consists of a real-time quantitative PCR amplification buffer, a primer pair and a template; the concentration of each primer in the primer pair in the system is 0.2 μmol / L, and the primer pair includes an upstream primer and a downstream primer; the upstream primer is sequence 2 in the sequence table, and the downstream primer is sequence 3 in the sequence table.

[0016] In a preferred embodiment, the sample to be tested is a water sample.

[0017] In a preferred embodiment, the concentration of the standard template in the real-time quantitative PCR amplification system is 6.48×10 2 copies / μL, 6.48×10 3 copies / μL, 6.48×10 4 copies / μL, 6.48×10 5 copies / μL, 6.48×10 6 copies / μL, 6.48×10 7 copies / μL or 6.48×10 8 copies / μL.

[0018] In a preferred embodiment, the annealing temperature in the PCR reaction is 50-70°C, preferably 55-65°C, and more preferably 58-60°C.

[0019] The third aspect of the present application is to provide a kit for detecting microcystin, comprising a primer pair, wherein the primer pair comprises an upstream primer and a downstream primer; the upstream primer is sequence 2 in the sequence table, and the downstream primer is sequence 3 in the sequence table.

[0020] The fourth aspect of the present application is to provide a PCR reagent.

[0021] The PCR reagent provided in the present application includes a real-time quantitative PCR amplification buffer and a primer pair; the concentration of each primer in the primer pair in the reagent is 0.2 μM / L; the primer pair includes an upstream primer and a downstream primer; the upstream primer is sequence 2 in the sequence table, and the downstream primer is sequence 3 in the sequence table.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present application provides a method for quantitatively detecting microcystins, a recombinant plasmid, and specific primers designed for the recombinant plasmid, and adopts a real-time quantitative PCR method to quantitatively detect microcystins in a water environment.

[0024] Experiments have shown that the quantitative detection method of microcystins and the recombinant patent of the present application have the characteristics of combining broad-spectrum recognition and specificity, high sensitivity, simple preparation method, long-term storage, good purity, and a wide linear detection range, and can be used for rapid quantitative detection of microcystins in water environment samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the amplification curve of the quantitative PCR standard product of Example 1 of the present invention.

[0026] Figure 2 This is the melting curve of the quantitative PCR in Example 1 of the present invention. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] In the description of the present invention, it should be noted that the orientations or positional relationships indicated by the terms "side", "up", "down", "top", etc. are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0029] In the description of the present invention, it is also necessary to explain that, unless otherwise clearly specified and limited, the terms "set", "install", "connect", and "connect" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0030] Embodiment 1:

[0031] This embodiment provides a method for quantitatively detecting microcystins, the steps comprising:

[0032] (1) Design specific primers for SEQ ID No.1 sequence:

[0033] Specific primers were designed and synthesized for the sequence of SEQ ID No. 1, and the length of the PCR product was 215 bp.

[0034] The primer sequences are as follows: the upstream primer is 5'-ATCGAACCAGGCGAAGGTTT-3' (as shown in sequence 2 in the sequence list), and the downstream primer is 5'-TCGGTCATTTCACGGGTTGT-3' (as shown in sequence 3 in the sequence list). The primers were prepared into a 100 μM storage solution with sterile double distilled water, aliquoted, and stored at -20°C.

[0035] (2) Extraction of total genomic DNA from samples:

[0036] A DNA extraction kit was used to extract and purify total DNA to obtain a DNA sample.

[0037] (3) Preparation of plasmid standards:

[0038] a. Preparation of target fragment

[0039] The DNA sample in step (2) was used as a template for PCR amplification, and the reaction system was 50 μL. The reaction procedure was: 95°C pre-denaturation for 5 min, followed by 95°C for 15 s, annealing at 60°C for 45 s and extension at 72°C for 40 s, for 45 cycles, and finally extension at 72°C for 10 min. The product was confirmed by 1.5% (wt / vol) agarose gel electrophoresis.

[0040] The amplification reaction system is as follows:

[0041]

[0042]

[0043] b. PCR product purification

[0044] Purification of the target gene: gel recovery was performed using Axygen's DNA column gel recovery kit to purify the target gene, and the success of the recovery was confirmed by 1.5% (wt / vol) agarose gel electrophoresis.

[0045] c. Ligation of PCR products and plasmids

[0046] The purified PCR product was connected to the plasmid vector pMD18-T and further transformed into DH5α competent E. coli solution, then spread onto LB solid medium with Amp resistance added, and white colonies transformed with the plasmid were selected through blue-white screening.

[0047] The connection method is as follows: connect the purified DNA fragment to the pMD18-T Vector, 5 μL reaction system, blow gently to mix, and connect at 4°C overnight.

[0048] Ligation reaction components and system:

[0049]

[0050] The ligated product was transferred into DH5α competent cells in full amount under the stimulation of heat shock at 42°C to transform the recombinant plasmid.

[0051] The transformation of the recombinant plasmid includes the following steps:

[0052] 1) Take 10 μL of the ligation product and add it to 50 μL of the competent cell mixture. Stir it gently with a pipette tip. Wash the product in the ligation product tube with bacterial suspension as much as possible and place it in an ice bath for 30 minutes.

[0053] 2) Place the centrifuge tube containing the mixture in a 42°C water bath for 90 seconds without shaking, then quickly transfer the centrifuge tube to an ice bath to cool for 3 minutes.

[0054] 3) Add 940 μL of LB liquid culture medium to the centrifuge tube to make the total volume of the liquid in the centrifuge tube reach 1 mL.

[0055] 4) Incubate at 37°C, 130 rpm, with gentle shaking for 45 min.

[0056] 5) Centrifuge at 5000 r / min for 5 min, discard 900 μL of the supernatant, mix the remaining 100 μL and spread it on the LB plate containing AMP.

[0057] 6) Invert the plate and incubate at 37°C for 12-16 hours.

[0058] d. PCR identification of positive clones and sequencing analysis

[0059] A full single colony was selected and inoculated into liquid LB culture medium containing Amp, and cultured overnight at 37°C and 200 rpm for PCR identification and sequencing analysis.

[0060] e. Obtaining large quantities of plasmid standards

[0061] The transformed plasmid was inoculated into liquid LB culture medium containing Amp, cultured overnight, and the plasmid was extracted using a plasmid extraction kit to obtain a large amount of plasmid standards.

[0062] f. Detection of plasmid

[0063] The large amount of plasmid standards obtained above were diluted with 1×TE buffer at pH 8.0 in a gradient manner, and the concentration of the plasmid standards was measured using an ultra-micro nucleic acid protein analyzer (NanoDrop ND-2000C, USA) to calculate the copy number of the plasmid. The copy number of the plasmid standard was determined to be 6.48×10 8 copies / μL.

[0064] Embodiment 2:

[0065] This embodiment provides a quantitative PCR detection method, the steps comprising:

[0066] (1) Optimization of quantitative PCR reaction conditions:

[0067] The DNA sample extracted in Example 1 was used as a template. Premix Ex Taq TM Follow the instructions of the kit and add the reagents required for real-time quantitative PCR. The reaction system is 20 μL and 2 μL of DNA sample. Set up negative and positive controls.

[0068] The upstream primer is 5'-ATCGAACCAGGCGAAGGTTT-3' (as shown in sequence 2 in the sequence list), and the downstream primer is 5'-TCGGTCATTTCACGGGTTGT-3' (as shown in sequence 3 in the sequence list).

[0069] ABI's Stepone plus Detection System was used for real-time quantitative PCR reaction. A two-step PCR amplification was performed. The reaction conditions for the first step were: pre-denaturation, 1 cycle, 95°C for 15 s; the reaction conditions for the second step were: PCR reaction, 45 cycles, 95°C for 5 s, 60°C for 45 s.

[0070] The experimental results showed that the optimal annealing temperature of the mcyH gene primer was 60℃, the optimal primer concentration was 0.2μM / L, and the amplification effect was the best.

[0071] In this embodiment, the amplification reaction system includes:

[0072]

[0073] (2) Quantitative PCR detection limit, quantitative range and standard curve:

[0074] The plasmid standard obtained in Example 1 was diluted 10 times in a gradient manner and used as a quantitative PCR template DNA. The PCR reaction system was 20 μL, and 2 μL of the plasmid standard was added as a reaction template, with a final concentration of 6.48×10 1 -6.48×10 8 The quantitative PCR reaction was carried out under the above optimized optimal conditions. Deionized water was used as the negative control. The logarithm of the standard dilution gradient was used as the horizontal axis and the critical cycle number (Threshold Cycle, Ct) was used as the vertical axis to establish the standard curve of real-time quantitative PCR.

[0075] ABI's Stepone plus Detection System was used for real-time quantitative PCR. A two-step PCR amplification was performed. The reaction conditions for the first step were: pre-denaturation, 1 cycle, 95°C for 15 s; the reaction conditions for the second step were: PCR reaction, 45 cycles, 95°C for 5 s, 60°C for 45 s.

[0076] The experimental results showed that the optimal annealing temperature of the mcyH gene primer was 60℃, the optimal primer concentration was 0.2μM / L, and the amplification effect was the best.

[0077]

[0078] The PCR amplification curve of the standard product is as follows Figure 1 As shown, the exponential amplification phase and the plateau phase of the target gene are very obvious, the fluorescence quantitative kinetic curve is smooth, and the amplification curve is smooth, indicating that the PCR method provided in this example has a good amplification effect and can be used for quantitative analysis.

[0079] The minimum detection limit of plasmid standards was 6.48×10 1 copies / μL, the quantitative detection range is 6.48×10 2 -6.48×10 8 copies / μL, the slope of the standard curve equation was -3.48, R2 was 0.9991, and the amplification efficiency E was 0.94.

[0080] The above results show that the real-time quantitative PCR method of this embodiment has a high efficiency in amplifying the standard product, a good linear relationship, and the detection method is accurate and feasible, which meets the requirements for preparing a real-time quantitative PCR standard curve.

[0081] (3) Specificity analysis of quantitative PCR detection method:

[0082] The melting curve of the standard PCR product is as follows Figure 2 As shown, through melting curve analysis, it was found that the melting curve of the target gene showed a single peak, and the melting temperature was 81.27±1°C, indicating that the gene mcyH can be specifically amplified, indicating that the PCR method has good specificity and the real-time quantitative PCR results are credible.

[0083] (4) Repeatability of quantitative PCR detection method:

[0084] Five batches of plasmid standards were prepared using the method of Example 1.

[0085] The above-mentioned plasmid standards of different batches were frozen and thawed repeatedly for 6 times, and quantitative PCR was performed using the optimized PCR reaction conditions. The repeatability was evaluated based on the coefficient of variation (CV) of the cycle number.

[0086] The results are shown in Table 1. The coefficient of variation of the cycle number of the five batches of plasmid standards was 0.73%-1.74%, indicating that the standard curve of the plasmid standards had good repeatability.

[0087] Table 1 Repeatability analysis between different batches of plasmid standards

[0088]

[0089] Example 3

[0090] In this example, the quantitative PCR method described in Example 2 is used to detect microcystins in actual water bodies, and the steps include:

[0091] (1) Actual water sample collection and concentration

[0092] In May and August, the present embodiment collected surface water samples at 0.5 m below the water surface. After the water samples were filtered through a 0.22 μm micropore, the filter membrane was stored at -20°C for subsequent experimental analysis.

[0093] (2) DNA extraction

[0094] After the experiment, the total DNA of the sample was extracted and purified according to the operating instructions of the kit, and finally 50 μL of DNA sample was obtained for each sample.

[0095] (3) Quantitative PCR detection

[0096] 2 μL of the obtained DNA sample was taken as a template for quantitative PCR detection. The quantitative PCR detection system and method were consistent with the method steps of Example 2. The experimental results are shown in Table 2.

[0097] Table 2 Detection of the copy number of mcyH gene in water using quantitative PCR

[0098]

[0099] The specific embodiments of the present application are described in detail above, but they are only examples, and the present application is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present application are also within the scope of the present application. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present application should be included in the scope of the present application.

Claims

1. A recombinant plasmid for quantitative detection of a standard substance for microcystin, characterized in that: The recombinant plasmid contains the sequence SEQ ID No.

1.

2. The standard product according to claim 1, characterized in that: The starting plasmid in the recombinant plasmid is pMD-18T.

3. The standard product according to claim 1, characterized in that Sequence 1 in the sequence listing consists of 215 deoxyribonucleotides.

4. A method for quantitatively detecting microcystin, characterized in that: include: Providing a standard according to any one of claims 1 to 3; Using the recombinant plasmid as the standard template, a real-time quantitative PCR amplification was performed using a PCR amplification system to establish a one-dimensional linear regression curve corresponding to the concentration of the standard and the critical cycle number, that is, to obtain a standard curve; The standard is replaced by the sample to be tested, and real-time quantitative PCR amplification is performed. According to the critical cycle number of the amplification result and the standard curve, the copy number of the SEQ ID No.1 sequence in the sample to be tested is obtained, that is, the concentration of microcystin in the sample to be tested is obtained; The PCR amplification system consists of a real-time quantitative PCR amplification buffer, a primer pair and a template; the concentration of each primer in the primer pair in the system is 0.2 μmol / L, and the primer pair includes an upstream primer and a downstream primer; the upstream primer is sequence 2 in the sequence table, and the downstream primer is sequence 3 in the sequence table.

5. The method according to claim 4, characterized in that The sample to be tested is specifically a water sample.

6. The method according to claim 4, characterized in that The concentration of the standard template in the real-time quantitative PCR amplification system is 6.48×10 2 copies / μL, 6.48×10 3 copies / μL, 6.48×10 4 copies / μL, 6.48×10 5 copies / μL, 6.48×10 6 copies / μL, 6.48×10 7 copies / μL or 6.48×10 8 copies / μL.

7. The method according to claim 4, characterized in that The annealing temperature in the PCR reaction is 50-70°C, preferably 55-65°C, more preferably 58-60°C.

8. A kit for detecting microcystin, characterized in that: The kit comprises a primer pair, and the primer pair comprises an upstream primer and a downstream primer; the upstream primer is sequence 2 in the sequence table, and the downstream primer is sequence 3 in the sequence table.

9. A PCR reagent, characterized in that The PCR reagent comprises a real-time quantitative PCR amplification buffer and a primer pair; the concentration of each primer in the primer pair in the reagent is 0.2 μmol / L; the primer pair comprises an upstream primer and a downstream primer; the upstream primer is sequence 2 in the sequence table, and the downstream primer is sequence 3 in the sequence table.

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