阅读说明：本技术 一种定量检测微囊藻的标准品及制备、检测方法 (Standard substance for quantitatively detecting microcystis and preparation and detection methods thereof ) 是由 陈蕾 姜巍巍 姜蕾 于 2019-11-27 设计创作，主要内容包括：本发明公开了一种定量检测微囊藻的标准品及制备、检测方法,该检测方法包括：步骤一,根据实验优化定量PCR反应条件；步骤二,以重组质粒为标准品模板,根据优化后的定量PCR反应条件用实时定量PCR进行PCR扩增,建立标准品的浓度与临界循环数对应的标准曲线,该重组质粒为包含M16基因的重组质粒或重组细胞,该M16基因的序列由97个脱氧核糖核苷酸组成,为序列表SEQ IDNO.1中的序列；步骤三,用待测样品替换步骤二中的标准品,根据优化后的定量PCR反应条件进行实时定量PCR扩增,根据扩增结果的临界循环数和标准曲线,得到待测样品中微囊藻的数量,通过本发明,可实现水环境样品中微囊藻的快速定量检测。(The invention discloses a standard substance for quantitatively detecting microcystis and a preparation and detection method thereof, wherein the detection method comprises the following steps: step one, optimizing quantitative PCR reaction conditions according to experiments; secondly, using the recombinant plasmid as a standard template, performing PCR amplification by using real-time quantitative PCR according to optimized quantitative PCR reaction conditions, and establishing a standard curve corresponding to the concentration of the standard and the critical cycle number, wherein the recombinant plasmid is a recombinant plasmid or a recombinant cell containing M16 gene, and the sequence of the M16 gene consists of 97 deoxyribonucleotides and is a sequence in a sequence table SEQ ID NO. 1; and step three, replacing the standard substance in the step two with the sample to be detected, carrying out real-time quantitative PCR amplification according to the optimized quantitative PCR reaction conditions, and obtaining the quantity of the microcystis in the sample to be detected according to the critical cycle number and the standard curve of the amplification result.)

1. A standard substance for quantitatively detecting microcystis is characterized in that: the standard substance is a recombinant plasmid or a recombinant cell containing M16 gene, and the sequence of the M16 gene consists of 97 deoxyribonucleotides and is a sequence in a sequence table SEQ ID NO. 1.

2. The standard substance for quantitatively detecting microcystis as claimed in claim 1, wherein: the starting plasmid in the recombinant plasmid is pMD-18T.

3. A method of preparing the standard of claim 1, comprising the steps of:

designing specific primers aiming at an M16 gene, wherein the primers comprise an upstream primer and a downstream primer, the upstream primer is SEQ ID NO.2, and the downstream primer is SEQ ID NO. 3;

step two, taking a surface water sample, and extracting and purifying total DNA by using a DNA extraction kit to obtain a DNA sample;

step three, performing PCR amplification by taking the DNA sample in the step two as a template;

step four, purifying the PCR amplification product;

step five, linking the purified PCR amplification product with a plasmid vector pMD18-T to obtain a white colony transformed with the plasmid;

and sixthly, selecting a full single colony, inoculating the full single colony in a culture solution, culturing overnight, and extracting plasmids by using a plasmid extraction kit to obtain a large number of plasmid standard products for PCR identification and sequencing analysis of the bacterial liquid.

4. The method of preparing a standard according to claim 3, wherein after step six, the method further comprises:

diluting the obtained large amount of plasmid standard substance with 1 × TE buffer solution with pH of 8.0, measuring the concentration of plasmid standard substance with ultramicro nucleic acid protein analyzer, calculating the copy number of plasmid, and determining the copy number of plasmid standard substance to be 5.88 × 10⁸copies/μL。

5. The method of preparing a standard according to claim 4, wherein: in step three, the reaction system is 50 μ L, and the primer information is: the upstream primer is SEQ ID NO.2, the downstream primer is SEQ ID NO.3, and the reaction program is as follows: pre-denaturation at 95 ℃ for 5min, followed by 95 ℃ for 15 sec, annealing temperature 60 ℃ for 45 sec and 72 ℃ extension for 40 sec, were performed for 45 cycles, and final extension at 72 ℃ for 10min, and the product was confirmed by 1.5% (wt/vol) agarose gel electrophoresis.

6. A method for quantitatively detecting microcystis comprises the following steps:

step one, optimizing quantitative PCR reaction conditions according to experiments;

secondly, performing PCR amplification by using a real-time quantitative PCR (polymerase chain reaction) according to optimized quantitative PCR reaction conditions by using a recombinant plasmid as a standard template, and establishing a standard curve corresponding to the concentration of the standard and the critical cycle number, wherein the recombinant plasmid is a recombinant plasmid or a recombinant cell containing M16 gene, and the sequence of the M16 gene consists of 97 deoxyribonucleotides and is the sequence in a sequence table SEQID NO. 1;

and step three, replacing the standard substance in the step two with the sample to be detected, carrying out real-time quantitative PCR amplification according to the optimized quantitative PCR reaction conditions, and obtaining the copy number of the M16 gene in the sample to be detected according to the critical cycle number and the standard curve of the amplification result, so as to obtain the quantity of the microcystis in the sample to be detected.

7. The method according to claim 6, wherein the method comprises the following steps: the real-time quantitative PCR amplification system comprises a real-time quantitative PCR amplification buffer solution, a primer pair and a template, wherein the primer pair comprises: the upstream primer is SEQ ID NO.2, the downstream primer is SEQ ID NO.3, and the concentrations of the upstream primer and the downstream primer in a reaction system are both 0.1-0.3 mu M/L.

8. The method according to claim 7, wherein the method comprises the following steps: in the second step, the plasmid standard is diluted by a plurality of times of gradients to be used as a quantitative PCR template DNA for real-time quantitative PCR amplification, the operation is carried out according to the instruction of the kit, a real-time quantitative PCR reagent is added, the PCR reagent comprises a real-time quantitative PCR amplification buffer solution and a primer pair, the PCR reaction system is 20 mu L, 2 mu L of the plasmid standard is added to be used as a reaction template for real-time quantitative PCR reaction, and a two-step method is adopted for PCR amplification, wherein the reaction conditions are as follows: pre-denaturation, 1 cycle, 15 seconds at 95 ℃; and (3) carrying out PCR reaction for 45 cycles, wherein the temperature is 95 ℃ for 5 seconds, the temperature is 60 ℃ for 45 seconds, deionized water is used as a negative control, the logarithm value of the dilution gradient of the standard substance is used as an abscissa, and the critical cycle number is used as an ordinate to establish a standard curve of real-time quantitative PCR.

9. The method according to claim 8, wherein the method comprises the following steps: the concentration of the standard template in the real-time quantitative PCR amplification system is 5.88 multiplied by 10^1-5.88×10⁸copies/μL。

10. The method according to claim 7, wherein the method comprises the following steps: in the first step, the optimal annealing temperature of the gene M16 primer is determined to be 60 ℃, and the optimal primer concentration is determined to be 0.2 mu M/L.

Technical Field

The invention relates to the field of molecular biology, in particular to a standard substance for quantitatively detecting microcystis and a preparation and detection method thereof.

Background

The cyanobacterial bloom caused by water eutrophication is becoming more and more serious in China. Blue algae blooms are generated in large lakes and reservoirs, such as Taihu lake, lake hilly lake, Dian lake, basalt lake, Sanxia reservoir and the like, every summer and autumn. The 'green scum' formed on the water surface during the outbreak of the water bloom gives off fishy smell, influences the ecological landscape of the entertainment water body and influences the drinking water safety of the water supply water body.

In many eutrophic lakes and reservoirs, microcystis is an important dominant population and is also a water-blooming cyanobacteria with wide distribution, large scale and lasting growth in lake water bodies all over the world. Some microcystis, such as microcystis aeruginosa, green microcystis, microcystis huiwei and the like, can generate and release microcystin, and threaten the life safety of wild animals, livestock and poultry. Some zooplankton and fish have great tolerance to microcystins, but the microcystins can be enriched in the body of the zooplankton and fish, and finally, potential threats are caused to human beings through an ecological system and a food chain. Some non-toxic microcystis can increase the frequency and the severity of the blue algae blooms in the lakes and reservoirs. Therefore, it is necessary to enhance the detection of microcystis in water bodies of lakes and reservoirs, and provide scientific basis for protecting ecological environment and ensuring drinking water safety.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a standard product for quantitatively detecting microcystis, a preparation method and a detection method thereof, so as to realize the rapid quantitative detection of the microcystis in a water environment sample and provide scientific basis for protecting the ecological environment and ensuring the safety of drinking water.

In order to achieve the purpose, the invention provides a standard substance for quantitatively detecting microcystis, wherein the standard substance is a recombinant plasmid or a recombinant cell containing M16 gene, and the sequence of the M16 gene consists of 97 deoxyribonucleotides and is a sequence in a sequence table SEQ ID NO. 1.

Preferably, the starting plasmid in the recombinant plasmid is pMD-18T.

In order to achieve the purpose, the preparation method of the standard substance comprises the following steps:

designing specific primers aiming at an M16 gene, wherein the primers comprise an upstream primer and a downstream primer, the upstream primer is SEQ ID NO.2, and the downstream primer is SEQ ID NO. 3;

step two, taking a surface water sample, and extracting and purifying total DNA by using a DNA extraction kit to obtain a DNA sample;

step three, performing PCR amplification by taking the DNA sample in the step two as a template;

step four, purifying the PCR amplification product;

step five, linking the purified PCR amplification product with a plasmid vector pMD18-T to obtain a white colony transformed with the plasmid;

and sixthly, selecting a full single colony, inoculating the full single colony in a culture solution, culturing overnight, and extracting plasmids by using a plasmid extraction kit to obtain a large number of plasmid standard products for PCR identification and sequencing analysis of the bacterial liquid.

Preferably, after the sixth step, the method further comprises:

diluting the obtained large amount of plasmid standard substance with 1 × TE buffer solution with pH of 8.0, measuring the concentration of plasmid standard substance with ultramicro nucleic acid protein analyzer, calculating the copy number of plasmid, and determining the copy number of plasmid standard substance to be 5.88 × 10⁸copies/μL。

Preferably, in step three, the reaction system is 50 μ L, and the primer information is: the upstream primer is SEQ ID NO.2, the downstream primer is SEQ ID NO.3, and the reaction program is as follows: pre-denaturation at 95 ℃ for 5min, followed by 95 ℃ for 15 sec, annealing temperature 60 ℃ for 45 sec and 72 ℃ extension for 40 sec, were performed for 45 cycles, and final extension at 72 ℃ for 10min, and the product was confirmed by 1.5% (wt/vol) agarose gel electrophoresis.

In order to achieve the above object, the present invention further provides a method for quantitatively detecting microcystis, comprising the following steps:

step one, optimizing quantitative PCR reaction conditions according to experiments;

secondly, performing PCR amplification by using a real-time quantitative PCR according to optimized quantitative PCR reaction conditions by using a recombinant plasmid as a standard template, and establishing a standard curve corresponding to the concentration of the standard and the critical cycle number, wherein the recombinant plasmid is a recombinant plasmid or a recombinant cell containing M16 gene, and the sequence of the M16 gene consists of 97 deoxyribonucleotides and is the sequence in a sequence table SEQ ID NO. 1;

and step three, replacing the standard substance in the step two with the sample to be detected, carrying out real-time quantitative PCR amplification according to the optimized quantitative PCR reaction conditions, and obtaining the copy number of the M16 gene in the sample to be detected according to the critical cycle number and the standard curve of the amplification result, so as to obtain the quantity of the microcystis in the sample to be detected.

Preferably, the real-time quantitative PCR amplification system comprises a real-time quantitative PCR amplification buffer, a primer pair and a template, wherein the primer pair comprises: the upstream primer is SEQ ID NO.2, the downstream primer is SEQ ID NO.3, and the concentrations of the upstream primer and the downstream primer in a reaction system are both 0.1-0.3 mu M/L.

Preferably, in the second step, the plasmid standard is diluted by several times of gradient to be used as a quantitative PCR template DNA, real-time quantitative PCR amplification is performed, the operation is performed according to the kit instructions, a real-time quantitative PCR reagent is added, the PCR reagent comprises the real-time quantitative PCR amplification buffer solution and a primer pair, the PCR reaction system is 20 μ L, 2 μ L of the plasmid standard is added to be used as a reaction template, real-time quantitative PCR reaction is performed, a two-step method is used for PCR amplification, and the reaction conditions are as follows: pre-denaturation, 1 cycle, 15 seconds at 95 ℃; and (3) carrying out PCR reaction for 45 cycles, wherein the temperature is 95 ℃ for 5 seconds, the temperature is 60 ℃ for 45 seconds, deionized water is used as a negative control, the logarithm value of the dilution gradient of the standard substance is used as an abscissa, and the critical cycle number is used as an ordinate to establish a standard curve of real-time quantitative PCR.

Preferably, the concentration of the standard template in the real-time quantitative PCR amplification system is 5.88 × 10^1-5.88×10⁸copies/μL。

Preferably, in the first step, the optimal annealing temperature of the primer M16 gene is determined to be 60 ℃, and the optimal primer concentration is determined to be 0.2 μ M/L.

Compared with the prior art, the standard product for quantitatively detecting microcystis and the preparation and detection methods of the standard product design specific primers aiming at the M16 gene, the microcystis in water environment is quantitatively detected by preparing the external standard product and optimizing the real-time quantitative PCR reaction condition and adopting the real-time quantitative PCR method.

Drawings

FIG. 1 is a flowchart illustrating the steps of a method for preparing a standard substance for quantitatively detecting microcystis according to a second embodiment of the present invention;

FIG. 2 is a flowchart illustrating the steps of a method for quantitatively detecting microcystis according to a third embodiment of the present invention;

FIG. 3 is an amplification curve of a quantitative PCR standard in an embodiment of the present invention;

FIG. 4 is a dissolution curve of a standard in an embodiment of the present invention.

Detailed Description

Other advantages and capabilities of the present invention will be readily apparent to those skilled in the art from the present disclosure by describing the embodiments of the present invention with specific embodiments thereof in conjunction with the accompanying drawings. The invention is capable of other and different embodiments and its several details are capable of modification in various other respects, all without departing from the spirit and scope of the present invention.

In the first embodiment of the invention, the standard substance for quantitatively detecting microcystis is a recombinant plasmid containing M16 gene; the sequence of the M16 gene is a sequence in a sequence table SEQ ID NO.1, and the starting plasmid in the recombinant plasmid is pMD-18T.

FIG. 1 is a flowchart illustrating the steps of a method for quantitatively detecting a standard substance of microcystis according to a second embodiment of the present invention. As shown in figure 1, the preparation method of the standard substance for quantitatively detecting microcystis comprises the following steps:

in step 101, specific primers are designed for the M16 gene.

Specifically, the M16 gene sequence was downloaded in GenBank (National Center for Biotechnology Information, NCBI, National Center for Biotechnology Information, USA) and subjected to homology comparison analysis to design and synthesize specific primers, the PCR product length was 97bp (see SEQ ID NO. 1). The primer sequences are as follows: the upstream primer is 5'-AGTTGGTGGGGTAAGAGCCTA-3' (SEQ ID NO.2), and the downstream primer is 5'-CGTAGGAGTCTGGGCCGTG-3' (SEQ ID NO. 3). The primers were prepared into 100. mu.M stock solutions with sterile double distilled water, subpackaged, and stored at-20 ℃.

And 102, obtaining a surface water sample, and extracting and purifying total DNA by using a DNA extraction kit to obtain a DNA sample.

And 103, performing PCR amplification by using the DNA sample in the step 102 as a template, wherein the reaction system is 50 mu L. The reaction procedure is as follows: pre-denaturation at 95 ℃ for 5min, followed by 95 ℃ for 15s, annealing temperature 60 ℃ for 45s and 72 ℃ extension for 40s, for 45 cycles, and final extension at 72 ℃ for 10min, and confirmation of the product by 1.5% (wt/vol) agarose gel electrophoresis.

And 104, purifying the PCR amplification product. Specifically, gel recovery can be carried out by using DNA column gel recovery kit of Axygen, PCR amplification products are purified, and whether recovery is successful or not is confirmed by agarose gel electrophoresis of 1.5% (wt/vol).

And step 105, linking the purified PCR amplification product with the plasmid to obtain a white colony transformed with the plasmid, specifically, connecting the purified PCR amplification product with a plasmid vector pMD18-T, further transforming into DH5 α competent escherichia coli solution, then coating the solution on an LB solid culture medium added with Amp resistance, and selecting the white colony transformed with the plasmid through blue-white screening.

And step 106, selecting a full single colony (namely, selecting the single white colony transformed in the step 105) to inoculate in a liquid LB culture solution containing Amp, carrying out shaking culture at 37 ℃ for 200 r/min overnight, and extracting plasmids by using a plasmid extraction kit to obtain a large number of plasmid standard products for PCR identification and sequencing analysis of bacterial liquid.

Step 107, detection of plasmids: after diluting the large number of plasmid standards obtained above with a gradient of 1 XTE buffer at pH 8.0,measuring the concentration of plasmid standard substance with ultramicro nucleic acid protein analyzer (NanoDrop ND-2000C, USA), calculating the copy number of plasmid, and determining the copy number of plasmid standard substance to be 5.88 × 10⁸copies/μL。

FIG. 2 is a flowchart illustrating the steps of a method for quantitatively detecting microcystis according to a third embodiment of the present invention. As shown in FIG. 2, the method for quantitatively detecting microcystis of the invention comprises the following steps:

step 201, according to the experiment, optimizing the quantitative PCR reaction condition.

The DNA sample extracted in the second embodiment was used as a template. According to

Premix Ex Taq^TMThe kit is indicated to be operated, reagents required by real-time quantitative PCR are added, a reaction system is 20 mu L, 2 mu LDNA samples are used, and a negative control and a positive control are set.

The real-time quantitative PCR reaction is carried out by using a Stebone plus Detection System of ABI company, and the PCR amplification is carried out by adopting a two-step method, wherein the reaction conditions are as follows: pre-denaturation, 1 cycle, 15s at 95 ℃; PCR reaction, 45 cycles, 95 ℃ 5s, 60 ℃ 45 s.

The experimental result shows that the optimal annealing temperature of the gene M16 primer is 60 ℃, the optimal primer concentration is 0.2 mu M/L, and the amplification effect is best.

And step 202, performing PCR amplification by using the recombinant plasmid as a standard template and using real-time quantitative PCR according to optimized quantitative PCR reaction conditions, and establishing a unitary linear regression curve corresponding to the concentration of the standard and the critical cycle number to obtain a standard curve.

The real-time quantitative PCR amplification system consists of a real-time quantitative PCR amplification buffer solution, a primer pair and a template; the primer pair comprises an upstream primer and a downstream primer, wherein the concentrations of the upstream primer and the downstream primer in a reaction system are both 0.1-0.3 mu M/L (optimally 0.2 mu M/L), the upstream primer is 5'-AGTTGGTGGGGTAAGAGCCTA-3' (SEQ ID NO.2), and the downstream primer is 5'-CGTAGGAGTCTGGGCCGTG-3' (SEQ ID NO. 3).

Specifically, the plasmid standard obtained in the second example of the present invention was subjected to a 10-fold operationDiluting in gradient, and performing real-time quantitative PCR amplification as quantitative PCR template DNA according to kit (e.g. kit)

Premix Ex Taq^TM)) The operation is illustrated, a real-time quantitative PCR reagent is added, the PCR reagent comprises a real-time light quantitative PCR amplification buffer solution and a primer pair (the concentrations of the upstream primer and the downstream primer in the primer pair in the reagent are both 0.2 mu M/L), the PCR reaction system is 20 mu L, 2 mu L of plasmid standard substance is added as a reaction template, and the final concentration is 5.88 multiplied by 10^1-5.88×10⁸And (3) performing real-time quantitative PCR reaction by using copies/mu L, and performing PCR amplification by using a two-step method under the reaction conditions of: pre-denaturation, 1 cycle, 15 seconds at 95 ℃; and (3) carrying out PCR reaction for 45 cycles, wherein the reaction temperature is 95 ℃ for 5 seconds, the reaction temperature is 60 ℃ for 45 seconds, deionized water is used as a negative control, the logarithmic value of the dilution gradient of the standard substance is used as an abscissa, and the critical Cycle (Ct) is used as an ordinate to establish a standard curve of real-time quantitative PCR.

In a specific embodiment of the present invention, the concentration of the standard template in the real-time quantitative PCR amplification system is 5.88 × 10²copies/μL、5.88×10³copies/μL、5.88×10⁴copies/μL、5.88×10⁵copies/μL、5.88×10⁶copies/. mu.L and 5.88X 10⁷copies/μL。

And 203, replacing the standard product in the step 202 with the sample to be detected, carrying out real-time quantitative PCR amplification according to the optimized quantitative PCR reaction conditions, and obtaining the copy number of the M16 gene in the sample to be detected according to the critical cycle number and the standard curve of the amplification result, namely obtaining the quantity of the microcystis in the sample to be detected.

Specifically, a surface water sample is taken, total DNA extraction and purification are carried out by utilizing a DNA extraction kit to obtain a DNA sample as a sample to be detected, the sample to be detected is used for replacing the standard substance in the step 202, and real-time quantitative PCR amplification is carried out, namely, according to the kit (such as

Premix Ex Taq^TM)) The operation is instructed, and the reagent required by real-time quantitative PCR is addedThe PCR reagent comprises a real-time light quantitative PCR amplification buffer solution and a primer pair (the concentration of the upstream and downstream primers in the primer pair in the reagent is 0.2 mu M/L), a reaction system is 20 mu L, a 2 mu LDNA sample is used, a negative control and a positive control are set, the real-time quantitative PCR reaction is carried out, a two-step method is adopted for PCR amplification, and the reaction conditions are as follows: pre-denaturation, 1 cycle, 15 seconds at 95 ℃; and carrying out PCR reaction for 45 cycles, at 95 ℃ for 5 seconds and at 60 ℃ for 45 seconds, and obtaining the copy number of the M16 gene in the sample to be detected according to the critical cycle number of the amplification result and the standard curve, so as to obtain the quantity of the microcystis in the sample to be detected.

The invention will be further illustrated by the following specific examples: