Method for evaluating biological safety of water supply system

Abstract

According to the method for evaluating the biological safety of the water supply system through the PCR amplification reaction, quantitative PCR detection is carried out through total DNA, and microorganisms in drinking water can be rapidly and quantitatively detected. According to the primer designed by the invention, DNA of a sample to be detected is extracted, quantitative PCR detection is carried out, the operation is simple and convenient, the operation is easy to master, and the safety of drinking water can be rapidly and accurately determined.

Description

Technical Field

[0001] The invention belongs to the technical field of water treatment, and in particular relates to a method for evaluating the biological safety of a water supply system by using glutathione. Background Art

[0002] Clean drinking water is the main reason for the significant increase in human life expectancy, and the biological safety of drinking water is a basic requirement for water quality. In recent years, water pollution accidents and normal pollution of water sources have occurred frequently, and my country's drinking water safety faces severe challenges.

[0003] Putting biosafety first is a global consensus on drinking water safety, and it is also the core requirement for ensuring the safety of direct drinking water and achieving high-quality drinking water requirements. Worldwide, nearly 20% of the population uses unsafe drinking water, and pathogens in water cause millions of deaths each year. Therefore, ensuring the safety of drinking water and water sources is an important part of protecting public health.

[0004] Patent CN 113718019 A uses superoxide dismutase (SOD) to evaluate the safety of the water supply system, and indirectly evaluates the number and type of microorganisms through specific enzymes. This method is easy to operate and highly sensitive, but it is more susceptible to interference from other factors, such as inhibitors or promoters in the water. Patent CN 111041063B uses ATP (adenosine triphosphate) to quickly evaluate the stability of microorganisms in drinking water. The principle is that ATP is a basic energy unit that only appears in living cells, and its number is proportional to the number of bacteria. However, this method is also more susceptible to interference from other factors, such as ATPase inhibitors or promoters in the water.

[0005] Gene amplification testing (PCR) can specifically amplify a very small amount of target DNA by millions of times, thereby greatly improving the analysis and detection capabilities of DNA molecules. It can detect single-molecule DNA or samples containing only one target DNA molecule per 100,000 cells. Therefore, this method is widely used and rapidly developed in many fields such as molecular biology, microbiology, medicine and genetics. With the development of PCR technology, the application of PCR in the detection of pathogenic microorganisms in drinking water has also made a series of progress. By designing multiple pairs of different primers, different microorganisms can be amplified, thereby realizing rapid detection of pathogenic bacteria. However, when designing multiple primer pairs for PCR detection of multiple microorganisms at the same time, mutual interference is likely to occur. Summary of the invention

[0006] In order to address the deficiencies of the prior art, the present application provides a method for evaluating the biosafety of a water supply system using PCR, which does not require multiple primer pairs.

[0007] In one aspect, the present invention provides a method for evaluating the biological safety of a water supply system, the steps comprising:

[0008] Take a water sample, filter it through a microporous filter, and use the filter membrane to extract and purify the total DNA to obtain the DNA sample to be tested;

[0009] Performing quantitative PCR amplification using the DNA sample to be tested as a template to obtain a Ct value of the sample to be tested;

[0010] The biological safety of the water supply system is evaluated based on the Ct value of the sample to be tested.

[0011] Preferably, a plasmid with a known microbial concentration, or a microbial DNA concentration, or a gene concentration that can be detected by PCR amplification of the method of the present invention is used as a standard, diluted to different concentrations to obtain samples with different concentration gradients, and the quantitative PCR amplification is performed. After the PCR amplification reaction is completed, a PCR standard curve is drawn according to the cycle number threshold (Ct value) and concentration value of the samples of each concentration gradient.

[0012] More preferably, the concentration is the number of gene copies contained in a unit volume, for example, it can be 10 7 -10 2 Copy / L.

[0013] More preferably, the gene copy number in the water sample is calculated according to the Ct value of the sample to be tested to complete the evaluation of the biological safety of the water supply system.

[0014] More preferably, the Ct value of the sample to be tested is compared with the PCR standard curve to calculate the gene copy number in the water sample.

[0015] In a preferred embodiment, the pore size of the micropores is 0.1-0.5 μm, preferably 0.2-0.3 μm, and more preferably one of 0.22, 0.24, 0.25, and 0.27 μm.

[0016] In a preferred embodiment, the total volume of the PCR amplification reaction system is 25 μL.

[0017] Preferably, the upstream primer information is sequence 1 in the sequence listing, and the downstream primer information is sequence 2 in the sequence listing.

[0018] Among them, the total DNA contains the target gene fragment, the upstream and downstream primers can identify the target gene, then amplify it, and finally obtain the target gene copy number. The target gene is GSH.

[0019] Preferably, the reaction procedure of the quantitative PCR amplification comprises the following steps:

[0020] Pre-denaturing the DNA sample to be tested at a first temperature to obtain a pre-denatured DNA sample;

[0021] The pre-denatured DNA is reacted at a second temperature, followed by annealing and extension to complete one cycle.

[0022] Preferably, the reaction procedure of the quantitative PCR amplification includes at least one cycle.

[0023] More preferably, the reaction procedure of the quantitative PCR amplification includes no less than 10 cycles, preferably no less than 30 cycles, and more preferably no less than 40 cycles.

[0024] Preferably, the first temperature is 90-100°C, preferably 92-98°C, more preferably 95-97°C, more preferably 95, 96 or 97°C.

[0025] Preferably, the pre-denaturation time is 2-10 min, preferably 3-8 min, more preferably 5-6 min.

[0026] Preferably, the second temperature is 90-100°C, preferably 92-98°C, more preferably 94-96°C, more preferably 94, 95 or 96°C.

[0027] Preferably, the reaction time of the pre-denatured DNA at the second temperature is 5-50 s, preferably 10-30 s, more preferably 15-25 s.

[0028] Preferably, the annealing temperature is 40-80°C, preferably 50-70°C, more preferably 60-65°C.

[0029] Preferably, the annealing time is 10-120 s, preferably 20-60 s, more preferably 30-45 s.

[0030] Preferably, the stretching temperature is 50-100°C, preferably 60-80°C, more preferably 65-75°C, more preferably 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75°C.

[0031] Preferably, the extension time is 10-120 s, preferably 20-60 s, more preferably 30-40 s.

[0032] Preferably, the PCR amplification is performed for more than one cycle, and the extension time of the last cycle is 2-20 min, preferably 5-15 min, and more preferably 10-12 min.

[0033] More preferably, after the last cycle of PCR amplification is completed, the method further comprises: confirming the product by agarose gel electrophoresis.

[0034] More preferably, the concentration of the agarose gel is 0.4-2.6% (wt / vol), preferably 0.8-1.8% (wt / vol), and more preferably 1-1.2% (wt / vol).

[0035] In this application, the Ct value indicates the number of cycles at which the sample reaction exceeds the fluorescence threshold. During the PCR amplification process, the fluorescence signal increases with the increase in the number of amplification cycles. When the fluorescence signal reaches the set threshold, the corresponding number of cycles is the Ct value. It reflects the initial concentration or copy number of the target nucleic acid.

[0036] The present invention also provides a kit for evaluating the biological safety of drinking water by PCR detection, comprising the forward primer and downstream primer.

[0037] Compared with the prior art, the beneficial effects of this application are:

[0038] The present application provides a method for evaluating the biological safety of a water supply system by using PCR amplification reaction, which can quickly and quantitatively detect microorganisms in drinking water by using total DNA for quantitative PCR detection. The primers designed in the present application are used to extract DNA from samples to be tested and perform quantitative PCR detection. The operation is simple and easy to master, and the safety of drinking water can be quickly and accurately determined. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0040] 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 orientations or positional relationships, which are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply 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.

[0041] 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.

[0042] Embodiment 1:

[0043] This embodiment provides a method for evaluating the biological safety of a water supply system using a GSH gene, the steps comprising:

[0044] 1) Take water samples from a water plant's inlet water, outlet water, municipal pipe network, and secondary water supply. After the water samples are filtered through a 0.22μm micropore filter, the filter membrane is stored at -20°C for subsequent experimental analysis. After the experiment, total DNA is extracted and purified to obtain DNA samples.

[0045] 2) Perform PCR amplification using the DNA sample in step 1) as a template, with a reaction system of 25 μL. The primer information is as follows: upstream primer 5'-CAATCACATCCAATCCGACAA-3', downstream primer 5'-GTGGGTGGACGAGTAGCAAAA-3'.

[0046] 3) The quantitative PCR reaction program is: 95°C pre-denaturation for 5 min, followed by reaction at 94°C for 5 s, annealing at 60°C for 30 s, and extension at 72°C for 40 s to complete one cycle. The above cycle was performed 40 times, and the product was confirmed by 1.2% (wt / vol) agarose gel electrophoresis;

[0047] PCR reaction materials:

[0048]

[0049]

[0050] 4) Evaluate the safety of drinking water based on the Ct value.

[0051] Specifically, the safety of drinking water can be evaluated based on the Ct value. Drinking water with a known microbial concentration can be used as a standard, and the Ct value can be tested by PCR to draw a standard curve. Based on the standard curve, the microbial content corresponding to the Ct of the sample to be tested can be known.

[0052] Example 2

[0053] Preparation of standard curve: Use known concentration of microbial plasmid as standard, and make 10-fold gradient dilution as standard template: dilute the standard to 10 7 -10 2 Quantitative PCR amplification was performed according to the quantitative PCR reaction system step 2) and the reaction procedure step 3) of the above Example 1. After the reaction was completed, a fluorescence quantitative PCR standard curve was drawn according to the Ct value and concentration value of each concentration gradient to obtain a standard curve y=3.45x+12.57, R 2 =0.9996(R 2 is the correlation coefficient);

[0054] Then, according to the method of Example 1, PCR detection of the DNA sample to be tested is performed, and the number of DNA or gene copies in the sample is calculated according to the Ct value of the sample to be tested and the standard curve, thereby evaluating the biological safety of the water supply system.

[0055] 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 method for evaluating the biological safety of a water supply system, characterized in that the steps include: Take a water sample, filter it through a microporous filter, and use the filter membrane to extract and purify the total DNA to obtain the DNA sample to be tested; Performing quantitative PCR amplification using the DNA sample to be tested as a template to obtain a Ct value of the sample to be tested; The biological safety of the water supply system is evaluated based on the Ct value of the sample to be tested.

2. The method according to claim 1, characterized in that The pore size of the micropores is 0.1-0.5 μm, preferably 0.2-0.3 μm, and more preferably one of 0.22, 0.24, 0.25, and 0.27 μm.

3. The method according to claim 1, characterized in that A plasmid with a known microbial concentration, or a microbial DNA concentration, or a gene concentration that can be detected by the PCR amplification is used as a standard, diluted to different concentrations to obtain samples with different concentration gradients, and the quantitative PCR amplification is performed. After the PCR amplification reaction is completed, a PCR standard curve is drawn based on the Ct values ​​and concentration values ​​of the samples in each concentration gradient.

4. The method according to claim 1, characterized in that: The gene copy number in the water sample is calculated according to the Ct value of the sample to be tested, thereby completing the evaluation of the biological safety of the water supply system.

5. The method according to claim 1, characterized in that The reaction procedure of the quantitative PCR amplification comprises the following steps: Pre-denaturing the DNA sample to be tested at a first temperature to obtain a pre-denatured DNA sample; The pre-denatured DNA is reacted at a second temperature, followed by annealing and extension to complete one cycle.

6. The method according to claim 5, characterized in that The reaction procedure of the quantitative PCR amplification includes at least one cycle.

7. The method according to claim 5, characterized in that The first temperature is 90-100°C, preferably 92-98°C, more preferably 95-97°C, more preferably 95, 96 or 97°C.

8. The method according to claim 5, characterized in that The time for the preliminary denaturation is 2-10 min, preferably 3-8 min, and more preferably 5-6 min.

9. The method according to claim 5, characterized in that The stretching temperature is 50-100°C, preferably 60-80°C, more preferably 65-75°C; and / or The extension time is 10-120 s, preferably 20-60 s, more preferably 30-40 s.

10. The method according to claim 5, characterized in that The last cycle of the PCR amplification also includes: confirming the product by agarose gel electrophoresis; The concentration of the agarose gel is 0.4-2.6% (wt / vol), preferably 0.8-1.8% (wt / vol), and more preferably 1-1.2% (wt / vol).

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