WO2023035334A1 - Method for quantitatively measuring phycocyanin and special standard product therefor - Google Patents

Method for quantitatively measuring phycocyanin and special standard product therefor Download PDF

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WO2023035334A1
WO2023035334A1 PCT/CN2021/120093 CN2021120093W WO2023035334A1 WO 2023035334 A1 WO2023035334 A1 WO 2023035334A1 CN 2021120093 W CN2021120093 W CN 2021120093W WO 2023035334 A1 WO2023035334 A1 WO 2023035334A1
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phycocyanin
standard
quantitative pcr
real
sample
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陈蕾
薛晓杰
姜蕾
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上海城市水资源开发利用国家工程中心有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

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  • the invention relates to the field of molecular biology, in particular to a method for quantitatively detecting phycocyanin and a special standard product thereof.
  • Phycocyanin is one of the important co-photopigment proteins for photosynthesis in algae, and it mainly exists in the phycobilisomes of cyanobacteria, cryptophyta, and some red algae.
  • concentration of phycocyanin can effectively represent the biomass of cyanobacteria, and it is a key indicator of water eutrophication and cyanobacteria blooms. Therefore, it is of great significance to quickly and accurately characterize phycocyanin in water and monitor cyanobacterial blooms.
  • the purpose of the present invention is to provide a method for quantitatively detecting phycocyanin and its special standard, so as to realize the rapid analysis of phycocyanin content in water by using the cpcB gene.
  • the present invention proposes a special standard product for quantitative detection of phycocyanin, said standard product is a recombinant plasmid containing cpcB gene, and the sequence of said cpcB gene is the sequence in Sequence Table 1.
  • the starting plasmid in the recombinant plasmid is pMD-18T.
  • the present invention also proposes a method for quantitative detection of phycocyanin, comprising the following steps:
  • Step 1 using the recombinant plasmid as a standard product template, perform PCR amplification with real-time quantitative PCR, and establish a linear regression curve corresponding to the concentration of the standard product and the critical cycle number to obtain the standard curve;
  • Step 2 replace the standard substance in step 1 with the sample to be tested, perform real-time quantitative PCR amplification, obtain the copy number of the cpcB gene in the sample to be tested according to the critical cycle number of the amplification result and the standard curve, and obtain the sample to be tested The content of phycocyanin in the sample.
  • the amplification system of the real-time quantitative PCR is composed of a real-time quantitative PCR amplification buffer, a primer pair and a template; the concentration of the upstream and downstream primers in the reaction system is 0.2 ⁇ M/L, and the upstream primer is 5'-ATGTTACCTACGCTACCTTCTCTGG - The 3' downstream primer is 5'-GCGGCTTCTTTCATTTTGCT-3'.
  • the concentration of the standard template in the real-time quantitative PCR amplification system is 4.65 ⁇ 10 2 copies/ ⁇ L, 4.65 ⁇ 10 3 copies/ ⁇ L, 4.65 ⁇ 10 4 copies/ ⁇ L, 4.65 ⁇ 10 5 copies / ⁇ L, 4.65 ⁇ 10 6 copies/ ⁇ L, and 4.65 ⁇ 10 7 copies/ ⁇ L.
  • the annealing temperature in the PCR reaction is 60°C.
  • the present invention designs specific primers for the cpcB gene, and adopts real-time quantitative PCR method to quantitatively detect phycocyanin in the water environment.
  • the key technology is to prepare external standards and optimize real-time quantitative PCR reaction conditions.
  • the standard substance for the quantitative detection of phycocyanin of the present invention has the characteristics of combining broad-spectrum recognition and specificity, high sensitivity, simple preparation method, long-term preservation, good purity, wide linear detection range, and can be used in water environment samples Rapid quantitative detection of phycocyanin.
  • Fig. 1 is a flow chart of the steps of a method for quantitatively detecting phycocyanin of the present invention
  • Fig. 2 is the amplification curve of quantitative PCR standard product in the specific embodiment of the present invention.
  • Fig. 3 is the PCR melting curve of the standard substance in the specific embodiment of the present invention.
  • the present invention is a special standard for quantitative detection of phycocyanin, said standard is a recombinant plasmid containing cpcB gene; the sequence of said cpcB gene is the sequence in Sequence List 1.
  • the starting plasmid in the recombinant plasmid is pMD-18T.
  • a method for quantitatively detecting phycocyanin of the present invention as shown in Figure 1, a flow chart of the steps of a method for quantitatively detecting phycocyanin of the present invention, comprising the following steps:
  • Step 101 using the recombinant plasmid as a standard template, performing PCR amplification with real-time quantitative PCR, and establishing a linear regression curve corresponding to the concentration of the standard and the critical cycle number, that is, obtaining the standard curve;
  • Step 102 replace the standard substance in step 1 with the sample to be tested, perform real-time quantitative PCR amplification, obtain the copy number of the cpcB gene in the sample to be tested according to the critical cycle number of the amplification result and the standard curve, that is, obtain the sample to be tested The content of phycocyanin in the sample.
  • the amplification system of the real-time quantitative PCR is composed of a real-time quantitative PCR amplification buffer, a primer pair and a template; the concentration of the upstream and downstream primers in the reaction system is 0.2 ⁇ M/L, and the upstream primer is 5'-ATGTTACCTACGCTACCTTCTCTGG - The 3' downstream primer is 5'-GCGGCTTCTTTCATTTTGCT-3'.
  • the concentration of the standard template in the real-time quantitative PCR amplification system is 4.65 ⁇ 10 2 copies/ ⁇ L, 4.65 ⁇ 10 3 copies/ ⁇ L, 4.65 ⁇ 10 4 copies/ ⁇ L, 4.65 ⁇ 10 5 copies / ⁇ L, 4.65 ⁇ 10 6 copies/ ⁇ L, and 4.65 ⁇ 10 7 copies/ ⁇ L.
  • the annealing temperature in the PCR reaction is 60°C.
  • the recombinant plasmid is a recombinant plasmid containing the cpcB gene; the sequence of the cpcB gene is the sequence in Sequence Listing 1, and the sequence 1 in the Sequence Listing 1 is made up of 139 deoxyribonucleotides.
  • the starting plasmid in the recombinant plasmid is pMD-18T.
  • Embodiment 1 Quantitative detection of preparation of phycocyanin granule standard
  • the cpcB gene sequence was downloaded from GenBank, homology comparative analysis was carried out, specific primers were designed and synthesized, and the length of the PCR product was 139bp.
  • the primer sequences are as follows: the upstream primer is 5'-ATGTTACCTACGCTACCTTCTCTGG-3', and the downstream primer is 5'-GCGGCTTCTTTCATTTTGCT-3'.
  • the primers were prepared into a 100 ⁇ M storage solution with sterile double-distilled water, aliquoted, and stored at -20°C.
  • the total DNA was extracted and purified using a DNA extraction kit to obtain a DNA sample.
  • step 2 Use the DNA sample in step 2 as a template for PCR amplification, and the reaction volume is 50 ⁇ L.
  • the reaction program was: pre-denaturation at 95°C for 5 min, followed by 15 s at 95°C, 45 cycles of annealing at 60°C for 45 s and 40 s at 72°C, and finally 10 min at 72°C.
  • the product was confirmed by 1.5% (wt/vol) agarose gel electrophoresis.
  • Purify the target gene use Axygen’s DNA column gel recovery kit for gel recovery, purify the target gene, and confirm whether the recovery is successful by 1.5% (wt/vol) agarose gel electrophoresis.
  • the above purified PCR product was connected to the plasmid vector pMD18-T, and further transformed into DH5 ⁇ competent E. coli solution, and then spread on the LB solid medium with Amp resistance, and the transformed plasmid was selected by blue-white screening of white colonies.
  • the transformed plasmid was inoculated in the liquid LB medium containing Amp, cultured overnight, and the plasmid was extracted using a plasmid extraction kit to obtain a large number of plasmid standards.
  • the concentration of the plasmid standard was measured using an ultramicro nucleic acid protein analyzer (NanoDrop ND-2000C, USA), and the copy number of the plasmid was calculated. After calculation, it was determined that the copy number of the plasmid standard was 4.65 ⁇ 10 8 copies/ ⁇ L.
  • Embodiment two the establishment of quantitative PCR detection method
  • Example 1 The DNA sample extracted in Example 1 was used as a template. according to The Premix Ex Taq TM kit instructions operate, add the reagents required for real-time quantitative PCR, the reaction system is 20 ⁇ L, 2 ⁇ L DNA sample. Negative and positive controls were established.
  • the Stepone plus Detection System of ABI Company was used for real-time quantitative PCR reaction, and the two-step method was used for PCR amplification.
  • the reaction conditions were: pre-denaturation, 1 cycle, 95°C for 15s; PCR reaction, 45 cycles, 95°C for 5s, 60 °C45s.
  • the experimental results showed that the optimal annealing temperature of gene cpcB primer was 60°C, the optimal primer concentration was 0.2 ⁇ M/L, and the amplification effect was the best.
  • the plasmid standard obtained in Example 1 was serially diluted 10 times, and used as template DNA for quantitative PCR.
  • the PCR reaction system was 20 ⁇ L, and 2 ⁇ L of plasmid standard was added as a reaction template, with a final concentration of 4.65 ⁇ 10 1 -4.65 ⁇ 10 8 copies/ ⁇ L.
  • Quantitative PCR reaction was carried out under the optimal conditions optimized above. Deionized water was used as a negative control.
  • the standard curve of real-time quantitative PCR was established with the logarithmic value of the dilution gradient of the standard as the abscissa and the critical cycle number (Threshold Cycle, Ct) as the ordinate.
  • the PCR amplification curve of the standard product is shown in Figure 2.
  • the exponential amplification phase and plateau phase of the target gene are very obvious. in quantitative analysis.
  • the minimum detection limit is 4.65 ⁇ 10 1 copies/ ⁇ L
  • the quantitative detection range is 4.65 ⁇ 10 2 to 4.65 ⁇ 10 7 copies/ ⁇ L
  • the standard curve slope of the standard curve equation is -3.56
  • R2 was 0.9995
  • the amplification efficiency E was 91%.
  • the PCR melting curve of the standard product is shown in Figure 3. Through the analysis of the melting curve, it was found that the melting curve of the target gene showed a single peak, and the melting temperature was 85.31 ⁇ 1°C, indicating that the gene cpcB can be specifically amplified, indicating that the PCR method is specific. Well, the real-time quantitative PCR results are reliable.
  • Example 3 Using quantitative PCR method to detect phycocyanin in actual environmental water body
  • the total DNA of the sample was extracted and purified according to the operating instructions of the kit, and finally 50 ⁇ L of DNA sample was obtained for each sample.
  • Table 1 is the final result of detecting the copy number of cpcB gene in water by quantitative PCR.
  • a kind of method of the present invention quantitatively detects phycocyanin and special standard product thereof, design specific primers for cpcB gene, adopt real-time quantitative PCR method to quantitatively detect phycocyanin in water environment, its key technology is to prepare external standard product , to optimize the real-time quantitative PCR reaction conditions.
  • the present invention has the advantages of:
  • the standard product and detection method for the quantitative detection of phycocyanin of the present invention have the characteristics of combining broad-spectrum recognition and specificity, high sensitivity, simple preparation method, long-term preservation, good purity, wide linear detection range, and can be used in water environment Rapid quantitative detection of phycocyanin in samples.

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Abstract

The present application relates to the field of molecular biology, and discloses a method for quantitatively measuring phycocyanin, comprising the following steps: step I, taking a recombinant plasmid as a standard product template, performing PCR amplification by using real-time quantitative PCR, and establishing a unary linear regression curve in which the concentration of a standard product corresponds to a critical cycle number, to obtain a standard curve; and step II, replacing the standard product in the step I with a sample to be measured, performing real-time quantitative PCR amplification, and obtaining, according to the critical cycle number of the amplification result and the standard curve, the copy number of cpcB genes in the sample to be measured, to obtain the content of phycocyanin in the sample to be measured.

Description

一种定量检测藻蓝蛋白的方法及其专用标准品A method for quantitatively detecting phycocyanin and its special standard 技术领域technical field
本发明涉及分子生物学领域，特别是涉及一种定量检测藻蓝蛋白的方法及其专用标准品。The invention relates to the field of molecular biology, in particular to a method for quantitatively detecting phycocyanin and a special standard product thereof.
背景技术Background technique
我国70％以上的湖泊和水库水体处于富营养化或超营养化状态，致使水体功能遭到严重破坏。水体富营养化最常见的结果是藻类会大量繁殖，形成蓝藻水华。蓝藻水华爆发时，可在水面形成一层“绿色浮渣”，水体散发异味，降低了水体水质和景观效果，部分蓝藻水华会释放各种天然毒素，严重威胁生态系统安全。More than 70% of the lakes and reservoirs in my country are in the state of eutrophication or hypertrophication, causing serious damage to the function of the water body. The most common result of eutrophication in water bodies is algae blooms, which form cyanobacterial blooms. When cyanobacteria blooms break out, a layer of "green scum" can be formed on the water surface, and the water emits peculiar smell, which reduces the water quality and landscape effect. Some cyanobacteria blooms will release various natural toxins, which seriously threaten the safety of the ecosystem.
藻蓝蛋白是藻类进行光合作用重要的辅光色素蛋白之一，主要存在于蓝藻、隐藻，以及部分红藻的藻胆体中。在环境监测中，特别是在蓝藻水华频繁发生的水域，藻蓝蛋白浓度可以有效地表征蓝藻生物量，是水体富营养化及蓝藻水华重点关注指标。因此快速、准确表征水体中藻蓝蛋白，蓝藻水华监测具有重要意义。Phycocyanin is one of the important co-photopigment proteins for photosynthesis in algae, and it mainly exists in the phycobilisomes of cyanobacteria, cryptophyta, and some red algae. In environmental monitoring, especially in waters where cyanobacteria blooms frequently occur, the concentration of phycocyanin can effectively represent the biomass of cyanobacteria, and it is a key indicator of water eutrophication and cyanobacteria blooms. Therefore, it is of great significance to quickly and accurately characterize phycocyanin in water and monitor cyanobacterial blooms.
发明内容Contents of the invention
为克服上述现有技术存在的不足，本发明之目的在于提供一种定量检测藻蓝蛋白的方法及其专用标准品，以实现运用cpcB基因快速分析水体中藻蓝蛋白含量。In order to overcome the deficiencies in the above-mentioned prior art, the purpose of the present invention is to provide a method for quantitatively detecting phycocyanin and its special standard, so as to realize the rapid analysis of phycocyanin content in water by using the cpcB gene.
为达上述目的，本发明提出一种定量检测藻蓝蛋白的专用标准品，所述标准品为含有cpcB基因的重组质粒，所述cpcB基因的序列是序列表1中的序列。In order to achieve the above purpose, the present invention proposes a special standard product for quantitative detection of phycocyanin, said standard product is a recombinant plasmid containing cpcB gene, and the sequence of said cpcB gene is the sequence in Sequence Table 1.
优选地，所述重组质粒中的出发质粒是pMD-18T。Preferably, the starting plasmid in the recombinant plasmid is pMD-18T.
为达上述目的，本发明还提出一种定量检测藻蓝蛋白的方法，包括以下步骤：In order to achieve the above object, the present invention also proposes a method for quantitative detection of phycocyanin, comprising the following steps:
步骤一，以重组质粒为标准品模板，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一元线性回归曲线，即得到标准曲线； Step 1, using the recombinant plasmid as a standard product template, perform PCR amplification with real-time quantitative PCR, and establish a linear regression curve corresponding to the concentration of the standard product and the critical cycle number to obtain the standard curve;
步骤二，以待测样品替换步骤一中的所述标准品，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cpcB基因的拷贝数，即得到待测样品中藻蓝蛋白的含量。 Step 2, replace the standard substance in step 1 with the sample to be tested, perform real-time quantitative PCR amplification, obtain the copy number of the cpcB gene in the sample to be tested according to the critical cycle number of the amplification result and the standard curve, and obtain the sample to be tested The content of phycocyanin in the sample.
优选地，所述实时定量PCR的扩增体系由实时定量PCR扩增缓冲液、引物对和模板组成；上下游引物在反应体系中的浓度均为0.2μM/L，上游引物为5’-ATGTTACCTACGCTACCTTCTCTGG-3’下游引物为5’-GCGGCTTCTTTCATTTTGCT-3’。Preferably, the amplification system of the real-time quantitative PCR is composed of a real-time quantitative PCR amplification buffer, a primer pair and a template; the concentration of the upstream and downstream primers in the reaction system is 0.2 μM/L, and the upstream primer is 5'-ATGTTACCTACGCTACCTTCTCTGG - The 3' downstream primer is 5'-GCGGCTTCTTTCATTTTGCT-3'.
优选地，所述标准模板在所述实时定量PCR扩增的体系中的浓度为4.65×10 2copies/μL、4.65×10 3copies/μL、4.65×10 4copies/μL、4.65×10 5copies/μL、4.65×10 6copies/μL和4.65×10 7copies/μL中之一。 Preferably, the concentration of the standard template in the real-time quantitative PCR amplification system is 4.65×10 2 copies/μL, 4.65×10 3 copies/μL, 4.65×10 4 copies/μL, 4.65×10 5 copies /μL, 4.65× 10 6 copies/μL, and 4.65×10 7 copies/μL.
优选地，所述PCR反应中的退火温度为60℃。Preferably, the annealing temperature in the PCR reaction is 60°C.
与现有技术相比，本发明针对cpcB基因设计特异性引物，采用实时定量PCR方法定量检测水环境中藻蓝蛋白，其关键的技术是制备外标准品，优化实时定量PCR反应条件。本发明的定量检测藻蓝蛋白的标准品具有广谱识别和特异性相结合的特点，敏感性高，制备方法简便，可以长期保存，纯度好，线性检测范围宽，可以用于水环境样品中藻蓝蛋白的快速定量检测。Compared with the prior art, the present invention designs specific primers for the cpcB gene, and adopts real-time quantitative PCR method to quantitatively detect phycocyanin in the water environment. The key technology is to prepare external standards and optimize real-time quantitative PCR reaction conditions. The standard substance for the quantitative detection of phycocyanin of the present invention has the characteristics of combining broad-spectrum recognition and specificity, high sensitivity, simple preparation method, long-term preservation, good purity, wide linear detection range, and can be used in water environment samples Rapid quantitative detection of phycocyanin.
附图说明Description of drawings
图1为本发明一种定量检测藻蓝蛋白的方法的步骤流程图；Fig. 1 is a flow chart of the steps of a method for quantitatively detecting phycocyanin of the present invention;
图2为本发明具体实施例中定量PCR标准品的扩增曲线；Fig. 2 is the amplification curve of quantitative PCR standard product in the specific embodiment of the present invention;
图3为本发明具体实施例中标准品PCR溶解曲线。Fig. 3 is the PCR melting curve of the standard substance in the specific embodiment of the present invention.
具体实施方式Detailed ways
以下通过特定的具体实例并结合附图说明本发明的实施方式，本领域技术人员可由本说明书所揭示的内容轻易地了解本发明的其它优点与功效。本发明亦可通过其它不同的具体实例加以施行或应用，本说明书中的各项细节亦可基于不同观点与应用，在不背离本发明的精神下进行各种修饰与变更。The implementation of the present invention is described below through specific examples and in conjunction with the accompanying drawings, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific examples, and various modifications and changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention.
在本发明第一个实施例中，本发明一种定量检测藻蓝蛋白的专用标准品，所述标准品为含有cpcB基因的重组质粒；所述cpcB基因的序列是序列表1中的序列。In the first embodiment of the present invention, the present invention is a special standard for quantitative detection of phycocyanin, said standard is a recombinant plasmid containing cpcB gene; the sequence of said cpcB gene is the sequence in Sequence List 1.
优选地，所述重组质粒中的出发质粒是pMD-18T。Preferably, the starting plasmid in the recombinant plasmid is pMD-18T.
在本发明第二个实施例中，本发明一种定量检测藻蓝蛋白的方法，如图1所示，本发明一种定量检测藻蓝蛋白的方法的步骤流程图，包括如下步骤：In the second embodiment of the present invention, a method for quantitatively detecting phycocyanin of the present invention, as shown in Figure 1, a flow chart of the steps of a method for quantitatively detecting phycocyanin of the present invention, comprising the following steps:
步骤101，以重组质粒为标准品模板，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一元线性回归曲线，即得到标准曲线；Step 101, using the recombinant plasmid as a standard template, performing PCR amplification with real-time quantitative PCR, and establishing a linear regression curve corresponding to the concentration of the standard and the critical cycle number, that is, obtaining the standard curve;
步骤102，以待测样品替换步骤一中的所述标准品，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cpcB基因的拷贝数，即得到待测样品中藻蓝蛋白的含量。Step 102, replace the standard substance in step 1 with the sample to be tested, perform real-time quantitative PCR amplification, obtain the copy number of the cpcB gene in the sample to be tested according to the critical cycle number of the amplification result and the standard curve, that is, obtain the sample to be tested The content of phycocyanin in the sample.
优选地，所述实时定量PCR的扩增体系由实时定量PCR扩增缓冲液、引物对和模板组成；上下游引物在反应体系中的浓度均为0.2μM/L，上游引物为5’-ATGTTACCTACGCTACCTTCTCTGG-3’下游引物为5’-GCGGCTTCTTTCATTTTGCT-3’。Preferably, the amplification system of the real-time quantitative PCR is composed of a real-time quantitative PCR amplification buffer, a primer pair and a template; the concentration of the upstream and downstream primers in the reaction system is 0.2 μM/L, and the upstream primer is 5'-ATGTTACCTACGCTACCTTCTCTGG - The 3' downstream primer is 5'-GCGGCTTCTTTCATTTTGCT-3'.
优选地，所述标准模板在所述实时定量PCR扩增的体系中的浓度为4.65×10 2copies/μL、4.65×10 3copies/μL、4.65×10 4copies/μL、4.65×10 5copies/μL、4.65×10 6copies/μL和4.65×10 7copies/μL中之一。 Preferably, the concentration of the standard template in the real-time quantitative PCR amplification system is 4.65×10 2 copies/μL, 4.65×10 3 copies/μL, 4.65×10 4 copies/μL, 4.65×10 5 copies /μL, 4.65× 10 6 copies/μL, and 4.65×10 7 copies/μL.
优选地，所述PCR反应中退火温度为60℃。Preferably, the annealing temperature in the PCR reaction is 60°C.
优选地，所述重组质粒为含有cpcB基因的重组质粒；所述cpcB基因的序 列是序列表1中的序列，所述序列表1中的序列1由139个脱氧核糖核苷酸组成。Preferably, the recombinant plasmid is a recombinant plasmid containing the cpcB gene; the sequence of the cpcB gene is the sequence in Sequence Listing 1, and the sequence 1 in the Sequence Listing 1 is made up of 139 deoxyribonucleotides.
优选地，所述重组质粒中的出发质粒是pMD-18T。Preferably, the starting plasmid in the recombinant plasmid is pMD-18T.
以下将通过具体实施例来进一步说明本发明：Below will further illustrate the present invention by specific embodiment:
实施例一、定量检测藻蓝蛋白质粒标准品的制备 Embodiment 1. Quantitative detection of preparation of phycocyanin granule standard
1、针对cpcB基因设计特异性的引物1. Design specific primers for the cpcB gene
在GenBank中下载cpcB基因序列，进行同源性比较分析，设计和合成特异性的引物，PCR产物长度为139bp。引物序列如下：上游引物为5’-ATGTTACCTACGCTACCTTCTCTGG-3’，下游引物为5’-GCGGCTTCTTTCATTTTGCT-3’。用无菌双蒸水将引物配制成100μM的储存液，分装，于-20℃保存。The cpcB gene sequence was downloaded from GenBank, homology comparative analysis was carried out, specific primers were designed and synthesized, and the length of the PCR product was 139bp. The primer sequences are as follows: the upstream primer is 5'-ATGTTACCTACGCTACCTTCTCTGG-3', and the downstream primer is 5'-GCGGCTTCTTTCATTTTGCT-3'. The primers were prepared into a 100 μM storage solution with sterile double-distilled water, aliquoted, and stored at -20°C.
2、样品中总基因组DNA的提取2. Extraction of total genomic DNA in the sample
利用DNA提取试剂盒进行总DNA提取纯化，得到DNA样本。The total DNA was extracted and purified using a DNA extraction kit to obtain a DNA sample.
3、质粒标准品制备3. Preparation of plasmid standard
1)目的片段的制备1) Preparation of the target fragment
以步骤二中的DNA样本为模板进行PCR扩增，反应体系为50μL。反应程序为：95℃预变性5min，随后95℃15s，退火温度60℃45s和72℃延伸40s，进行45个循环，最终72℃延伸10min。通过1.5％(wt/vol)的琼脂糖凝胶电泳确认产物。Use the DNA sample in step 2 as a template for PCR amplification, and the reaction volume is 50 μL. The reaction program was: pre-denaturation at 95°C for 5 min, followed by 15 s at 95°C, 45 cycles of annealing at 60°C for 45 s and 40 s at 72°C, and finally 10 min at 72°C. The product was confirmed by 1.5% (wt/vol) agarose gel electrophoresis.
2)PCR产物纯化2) PCR product purification
对目标基因进行纯化：利用Axygen的DNA柱式胶回收试剂盒进行胶回收，对目标基因进行纯化，通过1.5％(wt/vol)的琼脂糖凝胶电泳确认回收是否成功。Purify the target gene: use Axygen’s DNA column gel recovery kit for gel recovery, purify the target gene, and confirm whether the recovery is successful by 1.5% (wt/vol) agarose gel electrophoresis.
3)PCR产物与质粒链接3) Linkage of PCR product and plasmid
将上述纯化后的PCR产物与质粒载体pMD18-T连接，并进一步转化到DH5α感受态大肠杆菌溶液，然后涂布到加入Amp抗性的LB固体培养基上，通过蓝白斑筛选，挑选转化了质粒的白色菌落。The above purified PCR product was connected to the plasmid vector pMD18-T, and further transformed into DH5α competent E. coli solution, and then spread on the LB solid medium with Amp resistance, and the transformed plasmid was selected by blue-white screening of white colonies.
4)PCR鉴定阳性克隆和测序分析4) PCR identification of positive clones and sequencing analysis
选取饱满的单菌落接种于含有Amp的液体LB培养液中，于37℃、200转/min振荡培养过夜，用于菌液PCR鉴定和测序分析。Select a plump single colony and inoculate it in the liquid LB culture medium containing Amp, shake and culture overnight at 37°C and 200 rpm for PCR identification and sequencing analysis of the bacterial liquid.
5)质粒标准品的大量获得5) A large number of plasmid standard products are obtained
将转化了质粒接种于含有Amp的液体LB培养液中，过夜培养，利用质粒提取试剂盒提取质粒，获得大量的质粒标准品。The transformed plasmid was inoculated in the liquid LB medium containing Amp, cultured overnight, and the plasmid was extracted using a plasmid extraction kit to obtain a large number of plasmid standards.
6)质粒的检测6) Plasmid detection
将上述获得的大量质粒标准品用pH 8.0的1×TE缓冲液梯度稀释后，利用超微量核酸蛋白测定仪(NanoDrop ND-2000C，美国)测定质粒标准品的浓度，计算质粒的拷贝数。计算后确定质粒标准品的拷贝数为4.65×10 8copies/μL。 After a large number of plasmid standards obtained above were serially diluted with 1×TE buffer solution at pH 8.0, the concentration of the plasmid standard was measured using an ultramicro nucleic acid protein analyzer (NanoDrop ND-2000C, USA), and the copy number of the plasmid was calculated. After calculation, it was determined that the copy number of the plasmid standard was 4.65×10 8 copies/μL.
实施例二、定量PCR检测方法的建立Embodiment two, the establishment of quantitative PCR detection method
1、定量PCR反应条件的优化1. Optimization of quantitative PCR reaction conditions
利用实施例1中提取的DNA样本作为模板。按照
Figure PCTCN2021120093-appb-000001
Premix Ex Taq TM试剂盒说明进行操作，加入实时定量PCR所需试剂，反应体系为20μL，2μLDNA样品。设立阴性对照和阳性对照。 The DNA sample extracted in Example 1 was used as a template. according to
Figure PCTCN2021120093-appb-000001
The Premix Ex Taq TM kit instructions operate, add the reagents required for real-time quantitative PCR, the reaction system is 20 μL, 2 μL DNA sample. Negative and positive controls were established.
应用ABI公司的Stepone plus Detection System进行实时定量PCR反应，采用两步法进行PCR扩增，反应条件为：预变性，1个循环，95℃15s；PCR反应，45个循环，95℃5s，60℃45s。The Stepone plus Detection System of ABI Company was used for real-time quantitative PCR reaction, and the two-step method was used for PCR amplification. The reaction conditions were: pre-denaturation, 1 cycle, 95°C for 15s; PCR reaction, 45 cycles, 95°C for 5s, 60 ℃45s.
实验结果表明，基因cpcB引物的最佳退火温度为60℃，最佳引物浓度为0.2μM/L，扩增效果最好。The experimental results showed that the optimal annealing temperature of gene cpcB primer was 60℃, the optimal primer concentration was 0.2μM/L, and the amplification effect was the best.
2、定量PCR检测限、定量区间及标准曲线2. Quantitative PCR detection limit, quantitative interval and standard curve
将实施例一得到的质粒标准品进行10倍梯度稀释，作为定量PCR模版DNA。PCR反应体系为20μL，加入2μL质粒标准品作为反应模板，最终浓度为4.65×10 1～4.65×10 8copies/μL。以上述优化后的最佳条件进行定量PCR反应。以去离子水作为阴性对照。以标准品稀释梯度的对数值为横坐标，以临界循环数(Threshold Cycle，Ct)为纵坐标建立实时定量PCR的标准曲线。 The plasmid standard obtained in Example 1 was serially diluted 10 times, and used as template DNA for quantitative PCR. The PCR reaction system was 20 μL, and 2 μL of plasmid standard was added as a reaction template, with a final concentration of 4.65×10 1 -4.65×10 8 copies/μL. Quantitative PCR reaction was carried out under the optimal conditions optimized above. Deionized water was used as a negative control. The standard curve of real-time quantitative PCR was established with the logarithmic value of the dilution gradient of the standard as the abscissa and the critical cycle number (Threshold Cycle, Ct) as the ordinate.
标准品PCR扩增曲线如图2所示，目的基因的指数扩增期和平台期都十分明显，荧光定量动力学曲线平滑，扩增曲线平滑，说明本研究PCR方法扩增效果较好，可用于定量分析。The PCR amplification curve of the standard product is shown in Figure 2. The exponential amplification phase and plateau phase of the target gene are very obvious. in quantitative analysis.
利用定量PCR反应条件检测质粒标准品，最低检测限为4.65×10 1copies/μL，定量检测区间为4.65×10 2～4.65×10 7copies/μL，标准曲线方程的标准曲线斜率为-3.56，R 2为0.9995，扩增效率E为91％。 Using quantitative PCR reaction conditions to detect plasmid standards, the minimum detection limit is 4.65×10 1 copies/μL, the quantitative detection range is 4.65×10 2 to 4.65×10 7 copies/μL, and the standard curve slope of the standard curve equation is -3.56, R2 was 0.9995, and the amplification efficiency E was 91%.
以上结果说明本研究的实时定量PCR方法扩增该标准品的效率较高，线性关系良好，检测方法精确可行，符合制备实时定量PCR标准曲线的要求。The above results show that the real-time quantitative PCR method in this study has a high efficiency of 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.
3、定量PCR检测方法的特异性分析3. Specificity analysis of quantitative PCR detection method
标准品PCR溶解曲线如图3所示，通过溶解曲线分析发现，目的基因的溶解曲线呈现单一峰，溶解温度为85.31±1℃，表明基因cpcB可被特异性扩增，说明该PCR方法特异性好，实时定量PCR结果可信。The PCR melting curve of the standard product is shown in Figure 3. Through the analysis of the melting curve, it was found that the melting curve of the target gene showed a single peak, and the melting temperature was 85.31±1°C, indicating that the gene cpcB can be specifically amplified, indicating that the PCR method is specific. Well, the real-time quantitative PCR results are reliable.
实施例三、利用定量PCR方法检测实际环境水体中藻蓝蛋白Example 3: Using quantitative PCR method to detect phycocyanin in actual environmental water body
1、实际水样采集和浓缩1. Actual water sample collection and concentration
在8月，于水面下0.5m处取地表水样。水样经过0.22μm微孔过滤后，将滤膜保存至-20℃，用于后续实验分析。In August, surface water samples were taken at 0.5 m below the water surface. After the water samples were filtered through 0.22 μm micropores, the filter membranes were stored at -20°C for subsequent experimental analysis.
2、DNA的提取2. Extraction of DNA
实验结束后按照试剂盒操作说明进行样品总DNA的提取纯化，最终每个样品获得DNA样本50μL。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.
3、定量PCR检测3. Quantitative PCR detection
取2μL获得的DNA样本为模板，进行定量PCR检测，定量PCR检测体系和方法参考实施例二的1。Take 2 μL of the obtained DNA sample as a template, and perform quantitative PCR detection. For the quantitative PCR detection system and method, refer to 1 of Example 2.
表1为利用定量PCR检测水体中cpcB基因的拷贝数的最终结果。Table 1 is the final result of detecting the copy number of cpcB gene in water by quantitative PCR.
Figure PCTCN2021120093-appb-000002
Figure PCTCN2021120093-appb-000002
可见，本发明一种定量检测藻蓝蛋白的方法及其专用标准品，针对cpcB基因设计特异性引物，采用实时定量PCR方法定量检测水环境中藻蓝蛋白，其关键的技术是制备外标准品，优化实时定量PCR反应条件。Visible, a kind of method of the present invention quantitatively detects phycocyanin and special standard product thereof, design specific primers for cpcB gene, adopt real-time quantitative PCR method to quantitatively detect phycocyanin in water environment, its key technology is to prepare external standard product , to optimize the real-time quantitative PCR reaction conditions.
与现有技术相比，本发明优点在于：Compared with the prior art, the present invention has the advantages of:
本发明定量检测藻蓝蛋白的标准品及检测方法具有广谱识别和特异性相结合的特点，敏感性高，制备方法简便，可以长期保存，纯度好，线性检测范围宽，可以用于水环境样品中藻蓝蛋白的快速定量检测。The standard product and detection method for the quantitative detection of phycocyanin of the present invention have the characteristics of combining broad-spectrum recognition and specificity, high sensitivity, simple preparation method, long-term preservation, good purity, wide linear detection range, and can be used in water environment Rapid quantitative detection of phycocyanin in samples.
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何本领域技术人员均可在不违背本发明的精神及范畴下，对上述实施例进行修饰与改变。因此，本发明的权利保护范围，应如权利要求书所列。The above-mentioned embodiments only illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Any person skilled in the art can modify and change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be listed in the claims.
工业实用性Industrial Applicability
所属领域技术人员根据上文的记载容易得知，本发明技术方案适合在工业中制造并在生产、生活中使用，因此本发明具备工业实用性。Those skilled in the art can easily know from the above description that the technical solution of the present invention is suitable for industrial manufacture and use in production and daily life, so the present invention has industrial applicability.

Claims (6)

  1. 一种定量检测藻蓝蛋白的专用标准品，其特征在于：A special standard substance for quantitative detection of phycocyanin, characterized in that:
    所述标准品为含有cpcB基因的重组质粒，所述cpcB基因的序列是序列表1中的序列。The standard product is a recombinant plasmid containing the cpcB gene, and the sequence of the cpcB gene is the sequence in Sequence Table 1.
  2. 如权利要求1所述的一种定量检测藻蓝蛋白的专用标准品，其特征在于：所述重组质粒中的出发质粒是pMD-18T。A special standard for quantitative detection of phycocyanin according to claim 1, characterized in that: the starting plasmid in the recombinant plasmid is pMD-18T.
  3. 如权利要求1所述的一种定量检测藻蓝蛋白的方法，其特征在于，包括以下步骤：A kind of method for quantitatively detecting phycocyanin as claimed in claim 1, is characterized in that, comprises the following steps:
    步骤一，以重组质粒为标准品模板，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一元线性回归曲线，即得到标准曲线；Step 1, using the recombinant plasmid as a standard product template, perform PCR amplification with real-time quantitative PCR, and establish a linear regression curve corresponding to the concentration of the standard product and the critical cycle number to obtain the standard curve;
    步骤二，以待测样品替换步骤一中的所述标准品，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cpcB基因的拷贝数，即得到待测样品中藻蓝蛋白的含量。Step 2, replace the standard substance in step 1 with the sample to be tested, perform real-time quantitative PCR amplification, obtain the copy number of the cpcB gene in the sample to be tested according to the critical cycle number of the amplification result and the standard curve, and obtain the sample to be tested The content of phycocyanin in the sample.
  4. 如权利要求3所述的一种定量检测藻蓝蛋白的方法，其特征在于：所述实时定量PCR的扩增体系由实时定量PCR扩增缓冲液、引物对和模板组成；上下游引物在反应体系中的浓度均为0.2μM/L，上游引物为5’-ATGTTACCTACGCTACCTTCTCTGG-3’下游引物为5’-GCGGCTTCTTTCATTTTGCT-3’。A kind of method for quantitatively detecting phycocyanin as claimed in claim 3, is characterized in that: the amplification system of described real-time quantitative PCR is made up of real-time quantitative PCR amplification buffer, primer pair and template; The concentration in the system was all 0.2 μM/L, and the upstream primer was 5'-ATGTTACCTACGCTACCTTCTCTGG-3' and the downstream primer was 5'-GCGGCTTCTTTCATTTTGCT-3'.
  5. 如权利要求3所述的一种定量检测藻蓝蛋白的方法，其特征在于：所述标准模板在所述实时定量PCR扩增的体系中的浓度为4.65×10 2copies/μL、4.65×10 3copies/μL、4.65×10 4copies/μL、4.65×10 5copies/μL、4.65×10 6copies/μL和4.65×10 7copies/μL中之一。 A method for quantitatively detecting phycocyanin according to claim 3, wherein the concentration of the standard template in the real-time quantitative PCR amplification system is 4.65×10 2 copies/μL, 4.65×10 One of 3 copies/μL, 4.65×10 4 copies/μL, 4.65×10 5 copies/μL, 4.65×10 6 copies/μL, and 4.65×10 7 copies/μL.
  6. 如权利要求3所述的一种定量检测藻蓝蛋白的方法，其特征在于：所述PCR反应中退火温度为60℃。A method for quantitatively detecting phycocyanin according to claim 3, characterized in that: the annealing temperature in the PCR reaction is 60°C.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101974632A (en) * 2010-10-25 2011-02-16 清华大学 Method for quantitatively detecting toxigenic microcystis and special standard substance thereof
CN101974633A (en) * 2010-10-25 2011-02-16 清华大学 Method for quantitatively detecting microcystin and specific standard product thereof
CN107502678A (en) * 2017-10-24 2017-12-22 上海城市水资源开发利用国家工程中心有限公司 A kind of method and device for detecting blue algae producing microcystic toxins
CN110863033A (en) * 2019-11-27 2020-03-06 上海城市水资源开发利用国家工程中心有限公司 Standard substance for quantitatively detecting microcystis and preparation and detection methods thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101974632A (en) * 2010-10-25 2011-02-16 清华大学 Method for quantitatively detecting toxigenic microcystis and special standard substance thereof
CN101974633A (en) * 2010-10-25 2011-02-16 清华大学 Method for quantitatively detecting microcystin and specific standard product thereof
CN107502678A (en) * 2017-10-24 2017-12-22 上海城市水资源开发利用国家工程中心有限公司 A kind of method and device for detecting blue algae producing microcystic toxins
CN110863033A (en) * 2019-11-27 2020-03-06 上海城市水资源开发利用国家工程中心有限公司 Standard substance for quantitatively detecting microcystis and preparation and detection methods thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"Doctoral Dissertation", 1 May 2016, SHANGHAI JIAOTONG UNIVERSITY, CN, article CHEN, LEI: "Effects of Saltwater Intrusion on Microcystis Aeruginosa Growth and Microcystin Production Mechanism in Estuarine Reservoir", pages: 1 - 126, XP009544137, DOI: 10.27307/d.cnki.gsjtu.2016.003029 *
CHEN LEI, MAO FEIJIAN, KIRUMBA GEORGE CHIRA, JIANG CHENG, MANEFIELD MIKE, HE YILIANG: "Changes in metabolites, antioxidant system, and gene expression in Microcystis aeruginosa under sodium chloride stress", ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY, ACADEMIC PRESS, SAN DIEGO, CA, US, vol. 122, 1 December 2015 (2015-12-01), US , pages 126 - 135, XP093045186, ISSN: 0147-6513, DOI: 10.1016/j.ecoenv.2015.07.011 *
CHEN LEI, YILIANG HE: "Effects of Salinity on Photosynthesis and Microcystin Production in Microcystis aeruginosa with nC 60 stress", WATER PURIFICATION TECHNOLOGY, vol. 37, no. s1, 25 July 2018 (2018-07-25), pages 18 - 24, XP093045179, ISSN: 1009-0177, DOI: 10.15890/j.cnki.jsjs.2018.s1.005 *

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