WO2023029126A1 - 一种用sodB基因评价供水系统生物安全性的方法 - Google Patents

一种用sodB基因评价供水系统生物安全性的方法 Download PDF

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WO2023029126A1
WO2023029126A1 PCT/CN2021/120103 CN2021120103W WO2023029126A1 WO 2023029126 A1 WO2023029126 A1 WO 2023029126A1 CN 2021120103 W CN2021120103 W CN 2021120103W WO 2023029126 A1 WO2023029126 A1 WO 2023029126A1
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陈蕾
姜巍巍
黄佳
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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  • the invention relates to the field of drinking water safety bio-safety, in particular to a method for evaluating the bio-safety of a water supply system by using sodB gene.
  • Drinking unclean water is one of the top four causes of diseases in the world. Ensuring the microbiological safety of drinking water is both an important component of the public health sector and a primary goal of water workers. In recent years, it has been found that the drinking water that has been disinfected and qualified in urban water purification plants, under the condition of ensuring a certain amount of residual chlorine, the phenomenon of heterotrophic bacteria in the pipe network water is still increasing, which will threaten the biological safety and water quality of drinking water. .
  • Superoxide dismutase (SOD), widely present in animals, plants and microorganisms, catalyzes the disproportionation reaction of superoxide anion free radicals, thereby achieving the effect of removing superoxide anion free radicals, and maintaining superoxide anion in organisms It plays an important role in the dynamic balance of free radical production and elimination.
  • SOD Superoxide dismutase
  • Superoxide dismutase is a kind of acidic white matter. Due to the covalent binding of metal ions, it shows good stability in terms of heat, pH and certain physical and chemical properties. Therefore, the concentration of superoxide dismutase can be detected Analyze the presence of microorganisms in drinking water.
  • superoxide dismutase interacts with superoxide anion free radicals very quickly, it brings certain difficulties to the detection of superoxide dismutase enzyme activity.
  • Traditional superoxide dismutase detection methods mainly include chemiluminescence method, pyrogallol method, epinephrine method, NBT method, polarographic oxygen electrode method, etc.
  • Traditional methods are relatively tedious, time-consuming, and have certain detection limits.
  • the object of the present invention is to provide a method for evaluating the biological safety of water supply system with sodB gene, participate in encoding and synthesizing superoxide dismutase by sodB, carry out quantitative PCR detection, and can quickly quantitatively detect Superoxide dismutase concentration.
  • the present invention proposes a kind of method with sodB gene evaluation water supply system biological safety, comprises the steps:
  • Step 1 Take a water sample, filter through a 0.22 ⁇ m micropore, and extract and purify the total DNA from the filter membrane to obtain a DNA sample;
  • Step 2 performing quantitative PCR amplification using the DNA sample as a template
  • Step 3 use the plasmid containing the sodB gene of known concentration as a standard product, and use 10-fold gradient dilution as a standard template: 107 to 102 copies/L, and perform quantitative PCR amplification on the standard template and the sample to be tested simultaneously according to step 2 reaction;
  • Step 4 after the reaction is finished, draw a fluorescence quantitative PCR standard curve according to the Ct value and concentration of each concentration gradient;
  • Step five calculate the sodB gene copy number in the sample according to the Ct value of the sample to be tested and the standard curve.
  • the water sample is one or more of the incoming raw water of the water plant, the outgoing water, the municipal pipe network, and the secondary water supply.
  • the reaction system of the quantitative PCR amplification reaction is 25 ⁇ L, and the primer information is as follows: upstream primer 5'-TCTTGACTCTTTACCCGCCG-3', downstream primer 5'-ATTGCCGCCCCTATTTCTCC-3'.
  • the reaction program of quantitative PCR amplification is: pre-denaturation at 95°C for 5min, followed by extension at 94°C for 15s, annealing at 60°C for 45s and 40s at 72°C for 40 cycles, and finally extension at 72°C 10 min, and confirm the product by 1.2% (wt/vol) agarose gel electrophoresis.
  • the present invention uses the sodB gene to evaluate the biosafety method of the water supply system, uses software to design the primers of the gene sodB, extracts the DNA of the sample to be tested, performs quantitative PCR detection, and calculates the amount to be tested according to the standard curve.
  • the copy number of the sodB gene of the sample and the safety of the water supply system can be analyzed, and the concentration of the sodB gene in the water supply system can be quickly and accurately determined, and the operation is simple and easy to master.
  • Fig. 1 is a flow chart of the steps of a method for evaluating the biological safety of a water supply system using the sodB gene of the present invention.
  • Fig. 1 is a flow chart of the steps of a method for evaluating the biological safety of a water supply system using the sodB gene of the present invention.
  • a kind of method of sodB gene evaluation water supply system biological security of the present invention comprises the steps:
  • Step S1 taking a water sample, and after filtering through a 0.22 ⁇ m micropore, extracting and purifying the total DNA from the filter membrane to obtain a DNA sample;
  • Step S2 performing quantitative PCR amplification using the DNA sample as a template
  • Step S3 using a known concentration of the plasmid containing the sodB gene as a standard product, 10-fold serial dilution as a standard template: 107 to 102 copies/L, performing quantitative PCR amplification on the standard template and the sample to be tested simultaneously according to step 2 reaction;
  • Step S4 after the reaction is finished, draw a fluorescence quantitative PCR standard curve according to the Ct value and concentration of each concentration gradient;
  • Step S5 calculating the sodB gene copy number in the sample according to the Ct value of the sample to be tested and the standard curve.
  • the water sample is one or more of raw water entering a water plant, outgoing water, municipal pipe network, and secondary water supply.
  • the reaction system of the quantitative PCR amplification reaction is 25 ⁇ L, and the primer information is as follows: upstream primer 5'-TCTTGACTCTTTACCCGCCG-3', downstream primer 5'-ATTGCCGCCCCTATTTTCTCC-3'.
  • the reaction program of quantitative PCR amplification is: pre-denaturation at 95°C for 5min, followed by 15s at 94°C, annealing at 60°C for 45s and 40s at 72°C for 40 cycles, and finally extension at 72°C 10 min, and confirm the product by 1.2% (wt/vol) agarose gel electrophoresis.
  • step 2) Perform PCR amplification using the DNA sample in step 1) as a template, and the reaction volume is 25 ⁇ L.
  • the primer information is as follows: upstream primer 5'-TCTTGACTCTTTACCCGCCG-3', downstream primer 5'-ATTGCCGCCCCTATTTTCTCC-3'.
  • the quantitative PCR reaction program was: pre-denaturation at 95°C for 5min, followed by 15s at 94°C, annealing at 60°C for 45s and extension at 72°C for 40s, for 40 cycles, and finally at 72°C for 10min, and passed through 1.2% (wt/vol) agar Sugar gel electrophoresis to confirm the product;
  • Table 1 is the copy number of sodB gene in the different water bodies:
  • the present invention has following advantage:
  • the present invention is a method for evaluating the biological safety of a water supply system with the sodB gene, using software to design the primers of the gene sodB, extracting the DNA of the sample to be tested, performing quantitative PCR detection, and calculating the copy number of the sodB gene of the sample to be tested according to the standard curve And analyze the biological safety of the water supply system, can quickly and accurately determine the concentration of sodB gene in the water supply system, and has the advantages of simple operation and easy mastery.

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Abstract

一种用sodB基因评价供水系统生物安全性的方法，包括以下步骤：步骤一，取水样，经过0.22μm微孔过滤后，将滤膜进行总DNA提取纯化，得到DNA样本；步骤二，以所述DNA样本为模板进行定量PCR扩增；步骤三，用已知浓度的含有sodB基因的质粒为标准品，10倍梯度稀释作为标准模板：107～102拷贝/L，依照步骤二将所述标准模板与待测样品同时进行定量PCR扩增反应；步骤四，反应结束后，根据各个浓度梯度的Ct值和浓度绘制荧光定量PCR标准曲线；步骤五，根据待测样品的Ct值和标准曲线计算样品中sodB基因拷贝数。

Description

一种用sodB基因评价供水系统生物安全性的方法 技术领域
本发明涉及饮用水安全生物安全领域，特别是涉及一种用sodB基因评价供水系统生物安全性的方法。
背景技术
饮用不清洁的水是全球四大疾病诱因之一。保障饮用水的微生物安全既是公共卫生部门的重要组成部分，也是水务工作者的首要目标。近年发现在城市净水厂中经过消毒合格的饮用水，在保证一定余氯量的情况下，管网水中的异养细菌仍存在增多的现象，将威胁饮用水的生物安全性及其水质安全。超氧化物歧化酶(SOD)，广泛存在于动、植物和微生物中，它催化超氧化物阴离子自由基发生歧化反应，从而达到清除超氧化物阴离子自由基的效果，在维持生物体内超氧阴离子自由基产生与消除的动态平衡中起着重要的作用。超氧化物歧化酶是一种酸性质白质，由于共价结合了金属离子，所以在热、pH以及某些理化性质方面都表现出了良好的稳定性，所以可通过检超氧化物歧化酶浓度分析饮用水中微生物存在情况。
由于超氧化物歧化酶与超氧化物阴离子自由基的作用相当迅速，给超氧化物歧化酶的酶活检测工作带来了一定的困难。传统超氧化物歧化酶检测方法，主要有化学发光法，邻苯三酚法，肾上腺素法，NBT法，极谱氧电极法等。传 统方法相对繁琐、耗时长，并且具有一定的检测限。
发明内容
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用sodB基因评价供水系统生物安全性的方法，通过sodB参与编码合成超氧化物歧化酶，进行定量PCR检测，可快速定量检测超氧化合物歧化酶浓度。
为达上述目的，本发明提出一种用sodB基因评价供水系统生物安全性的方法，包括如下步骤：
步骤一，取水样，经过0.22μm微孔过滤后，将滤膜进行总DNA提取纯化，得到DNA样本；
步骤二，以所述DNA样本为模板进行定量PCR扩增；
步骤三，用已知浓度的含有sodB基因的质粒为标准品，10倍梯度稀释作为标准模板：107～102拷贝/L，依照步骤二将所述标准模板与待测样品同时进行定量PCR扩增反应；
步骤四，反应结束后，根据各个浓度梯度的Ct值和浓度绘制荧光定量PCR标准曲线；
步骤五，根据待测样品的Ct值和标准曲线计算样品中sodB基因拷贝数。
优选地，于步骤一中，所述水样为水厂进厂原水、出厂水、市政管网、二次供水水样中的一种或几种。
优选地，于步骤二中，所述定量PCR扩增反应的反应体系为25μL，引物信息如下：上游引物5’-TCTTGACTCTTTACCCGCCG-3’、下游引物5’-ATTGCCGCCCCTATTTCTCC-3’。
优选地，于步骤二中，定量PCR扩增的反应程序为：于95℃预变性5min，随后于94℃15s，退火温度60℃45s和72℃延伸40s，进行40个循环，最终72℃延伸10min，并通过1.2％(wt/vol)的琼脂糖凝胶电泳确认产物。
与现有技术相比，本发明一种用sodB基因评价供水系统生物安全性的方法，利用软件设计基因sodB的引物，提取待检测样本的DNA，进行定量PCR检测，根据标准曲线计算出待测样本的sodB基因的拷贝数和分析供水系统的安全性，可以实现快速准确测定供水系统中sodB基因的浓度，操作简便，易于掌握。
附图说明
图1为本发明一种用sodB基因评价供水系统生物安全性的方法的步骤流程图。
具体实施方式
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。
图1为本发明一种用sodB基因评价供水系统生物安全性的方法的步骤流程图。如图1所示，本发明一种用sodB基因评价供水系统生物安全性的方法，包括如下步骤：
步骤S1，取水样，经过0.22μm微孔过滤后，将滤膜进行总DNA提取纯化，得到DNA样本；
步骤S2，以所述DNA样本为模板进行定量PCR扩增；
步骤S3，用已知浓度的含有sodB基因的质粒为标准品，10倍梯度稀释作为标准模板：107～102拷贝/L，依照步骤二将所述标准模板与待测样品同时进行定量PCR扩增反应；
步骤S4，反应结束后，根据各个浓度梯度的Ct值和浓度绘制荧光定量PCR标准曲线；
步骤S5，根据待测样品的Ct值和标准曲线计算样品中sodB基因拷贝数。
优选地，于步骤S1中，所述水样为水厂进厂原水、出厂水、市政管网、二次供水水样中的一种或几种。
优选地，于步骤S2中，所述定量PCR扩增反应的反应体系为25μL，引物信息如下：上游引物5’-TCTTGACTCTTTACCCGCCG-3’、下游引物5’-ATTGCCGCCCCTATTTCTCC-3’。
优选地，于步骤S2中，定量PCR扩增的反应程序为：于95℃预变性5min，随后于94℃15s，退火温度60℃45s和72℃延伸40s，进行40个循环，最终72℃延伸10min，并通过1.2％(wt/vol)的琼脂糖凝胶电泳确认产物。
通过一个优选的实施例具体说明：
1)取某水厂进厂原水、出厂水、市政管网和二次供水水样，进行sodB基因的定量分析。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。实验结束后进行总DNA提取纯化，得到DNA样本。
2)以步骤1)中的DNA样本为模板进行PCR扩增，反应体系为25μL。
引物信息如下：上游引物5’-TCTTGACTCTTTACCCGCCG-3’、下游引物5’-ATTGCCGCCCCTATTTCTCC-3’。
定量PCR反应程序为：95℃预变性5min，随后94℃15s，退火温度60℃45s和72℃延伸40s，进行40个循环，最终72℃延伸10min，并通过1.2％(wt/vol)的琼脂糖凝胶电泳确认产物；
3)制作标准曲线：用已知浓度的含有sodB基因的质粒为标准品，10倍梯度稀释作为标准模板：107～102拷贝/L，依照步骤2)将标准模板与待测样品同时进行定量PCR扩增，反应结束后，根据各个浓度梯度的Ct值和浓度值绘制荧光定量PCR标准曲线，即得到标准曲线y＝-3.41x+14.79，R2＝0.9998；
4)根据待测样品的Ct值和标准曲线计算样品中sodB基因拷贝数，进而评价供水系统生物安全性。
表1为所述不同水体中sodB基因的拷贝数：
表1：不同水体中sodB基因的拷贝数
Figure PCTCN2021120103-appb-000001
根据表1可见，净水工艺后，sodB基因浓度比进厂水与原水大量减少，说明现阶段处理工艺可有效保障饮用水生物安全。
与现有技术相比，本发明具有如下优点：
本发明一种用sodB基因评价供水系统生物安全性的方法，利用软件设计基因sodB的引物，提取待检测样本的DNA，进行定量PCR检测，根据标准曲线计算出待测样本的sodB基因的拷贝数和分析供水系统生物安全性，可以快速准确的测定供水系统中sodB基因的浓度，并且具有操作简便、易于掌握的优点。
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。
工业实用性
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。

Claims (4)

  1. 一种用sodB基因评价供水系统生物安全性的方法，其特征在于，包括如下步骤：
    步骤一，取水样，经过0.22μm微孔过滤后，将滤膜进行总DNA提取纯化，得到DNA样本；
    步骤二，以所述DNA样本为模板进行定量PCR扩增；
    步骤三，用已知浓度的含有sodB基因的质粒为标准品，10倍梯度稀释作为标准模板：107～102拷贝/L，依照步骤二将所述标准模板与待测样品同时进行定量PCR扩增反应；
    步骤四，反应结束后，根据各个浓度梯度的Ct值和浓度绘制荧光定量PCR标准曲线；
    步骤五，根据待测样品的Ct值和标准曲线计算样品中sodB基因拷贝数。
  2. 如权利要求1所述的一种用sodB基因评价供水系统生物安全性的方法，其特征在于：于步骤一中，所述水样为水厂进厂原水、出厂水、市政管网、二次供水水样中的一种或几种。
  3. 如权利要求1所述的一种用sodB基因评价供水系统生物安全性的方法，其特征在于：于步骤二中，所述定量PCR扩增反应的反应体系为25μL，引物信息如下：上游引物5’-TCTTGACTCTTTACCCGCCG-3’、下游引物5’-ATTGCCGCCCCTATTTCTCC-3’。
  4. 如权利要求1所述的一种用sodB基因评价供水系统生物安全性的方法，其特征在于：于步骤二中，定量PCR扩增的反应程序为：于95℃预变性5min，随后于94℃15s，退火温度60℃45s和72℃延伸40s，进行40个循环，最终72℃延伸10min，并通过1.2％(wt/vol)的琼脂糖凝胶电泳确认产物。
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MAJE MPHO DEFNEY, KAPTCHOUANG TCHATCHOUANG CHRIST DONALD, MANGANYI MADIRA COUTLYNE, FRI JUSTINE, ATEBA COLLINS NJIE: "Characterisation of Vibrio Species from Surface and Drinking Water Sources and Assessment of Biocontrol Potentials of Their Bacteriophages", INTERNATIONAL JOURNAL OF MICROBIOLOGY, HINDAWI PUBLISHING CORPORATION, vol. 2020, 4 August 2020 (2020-08-04), pages 1 - 15, XP093042879, ISSN: 1687-918X, DOI: 10.1155/2020/8863370 *
MOROPENG RESOKETSWE CHARLOTTE, BUDELI PHUMUDZO, MOMBA MAGGY NDOMBO BENTEKE: "An Integrated Approach to Hygiene, Sanitation, and Storage Practices for Improving Microbial Quality of Drinking Water Treated at Point of Use: A Case Study in Makwane Village, South Africa", INTERNATIONAL JOURNAL OF ENVIRONMENTAL RESEARCH AND PUBLIC HEALTH, vol. 18, no. 12, 1 January 2021 (2021-01-01), pages 6313, XP093042876, DOI: 10.3390/ijerph18126313 *
NTEMA V. M., POTGIETER N., BARNARD T. G.: "Detection of Vibrio cholerae and Vibrio parahaemolyticus by molecular and culture based methods from source water to household container-stored water at the point-of-use in South African rural communities", WATER SCIENCE & TECHNOLOGY, vol. 61, no. 12, 1 June 2010 (2010-06-01), pages 3091 - 3101, XP093042875, ISSN: 0273-1223, DOI: 10.2166/wst.2010.222 *
QUROLLO BARBARA A., RIGGINS DANA, COMYN ALAIRE, ZEWDE MERONE T., BREITSCHWERDT EDWARD B.: "Development and Validation of a Sensitive and Specific sodB -Based Quantitative PCR Assay for Molecular Detection of Ehrlichia Species", JOURNAL OF CLINICAL MICROBIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 52, no. 11, 1 November 2014 (2014-11-01), US , pages 4030 - 4032, XP093042881, ISSN: 0095-1137, DOI: 10.1128/JCM.02340-14 *

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