A method for ultra-fast virus elimination based on periodate

Abstract

A method for ultra-fast inactivation of viruses based on periodate, which relates to the technical field of waterborne virus disinfection. This method forms a mixed system by adding a periodate oxidant and a homogeneous trivalent ruthenium ion activator of transition metals into the water body containing the virus to be treated, and then rapidly and thoroughly inactivates the virus. The present invention uses a homogeneous transition metal ruthenium ion catalyst to activate periodate, which not only destroys the culturing ability of the virus, but also destroys the nucleic acids encoding protein and RNA replication in the virus, and at the same time affects the functions of attachment and injection into the host during virus proliferation, achieving the complete inactivation of the virus. This method has a wide range of applications, does not require additional energy and instrumentation, saves energy, and is simple to operate.

Description

Technical Field

[0001] The present invention relates to the technical field of waterborne virus disinfection, and particularly to a method for ultra-fast virus disinfection based on periodate. Background Art

[0002] Virus disinfection has received extensive attention. The water environment is an important route for its transmission. Viruses have been detected in urban sewage, industrial wastewater, reclaimed water, etc., seriously threatening human health. The basic chemical structure of viruses mainly consists of the capsid protein of the outer shell and the genetic material nucleic acid inside. Traditional virus disinfection methods include chlorine disinfection, ultraviolet disinfection, chlorine dioxide disinfection, ozone disinfection, etc. It is basically difficult to synchronously and efficiently destroy the culturable ability of viruses and the internal nucleic acid, and there are still problems such as incomplete virus disinfection, the existence of disinfection by-products, the need for complex instruments and equipment, low efficiency, and high energy consumption.

[0003] To solve such problems, people have gradually tended to use advanced oxidation technology for rapid and thorough virus disinfection. Periodate has become a new type of oxidant with strong oxidizing properties due to its chemical stability and easy transportation and activation in the in-situ chemical oxidation process. It has received much attention in the field of water treatment and has become increasingly prominent in improving the degradation efficiency of new pollutants. However, this type of oxidant has not been used in virus disinfection yet. Similar to oxidants such as hydrogen peroxide and persulfate, its ability to independently oxidize pollutants is poor, and it needs to be activated to generate substances with higher oxidizing properties. The current activation methods include homogeneous (ultraviolet / ultrasound / hydroxylamine hydrochloride, etc.), heterogeneous (iron sulfide / activated carbon, etc.), and direct activation (freezing / heating, etc.). These methods more or less have problems such as low reaction rate, easy decomposition of the activator, low energy utilization efficiency, and relatively complex operation. Summary of the Invention

[0004] The purpose of the present invention is to solve the above problems in the prior art, and provide a new method for ultra-fast virus disinfection based on periodate, which has higher efficiency and simpler operation in virus disinfection, can effectively save energy and reduce working equipment.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] A method for ultra-fast virus disinfection based on periodate, adding periodate and a trivalent ruthenium ion activator to the water body containing the virus to be treated to form a mixed system, so as to achieve efficient virus disinfection.

[0007] In the mixed system, the concentration of periodate is not higher than 100.0 μM.

[0008] In the mixed system, the concentration of the trivalent ruthenium ion activator is not higher than 10.0 μM.

[0009] The water body containing the virus to be treated is a water body containing Escherichia coli phage MS2, and the virus concentration is 2×10 6 ~3×10 6 PFU / mL.

[0010] In the present invention, the genes for quantifying the virus are the MP gene and the RR gene; the function causing virus inactivation is the function of attaching and injecting into the Escherichia coli host during proliferation.

[0011] The gene detection method of the present invention is RT-qPCR, and the reaction conditions include: initial denaturation (95°C for 3 min), repeated denaturation (95°C for 15 s), annealing (60°C for 15 s), and extension (72°C for 30 s), for a total of 40 cycles.

[0012] Compared with the prior art, the beneficial effects achieved by the technical solution of the present invention are:

[0013] The present invention first uses homogeneous ruthenium ions to activate periodate for virus disinfection in water bodies; this method can quickly and efficiently eliminate viruses; the chemical reagents required for this method are easily obtainable, the operation is simple, the application value is high, it is easy to promote, and no additional energy and instrument equipment are required. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a schematic diagram of the disinfection of Escherichia coli phage in water bodies by periodates with different concentrations;

[0015] Figure 2 It is a schematic diagram of the disinfection of Escherichia coli phage in water bodies by ruthenium metal ions with different concentrations;

[0016] Figure 3 It is an effect diagram of the removal of the protein-coding gene fragment of Escherichia coli phage in water bodies by the mixed system;

[0017] Figure 4 It is an effect diagram of the removal of the RNA replication gene fragment of Escherichia coli phage in water bodies by the mixed system;

[0018] Figure 5 It is an influence diagram of the mixed system on the attachment of Escherichia coli phage to Escherichia coli hosts in water bodies;

[0019] Figure 6 It is an influence diagram of the mixed system on the injection of Escherichia coli phage into Escherichia coli hosts in water bodies. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0021] Example 1

[0022] Disinfection of Escherichia coli phage in water by periodates with different concentrations.

[0023] In a 20 mL reaction system containing 10.0 mM phosphate buffered saline (PBS), add 2×10 6 Plaque-forming units / mL (PFU / mL) of Escherichia coli phage (MS2, ATCC 15597-B1) to obtain the virus-containing water to be treated. Add sodium periodate to the above water to make its concentrations 0, 10, 50, and 100 μM respectively. At the same time, add ruthenium trichloride to make its concentration 10 μM. Mix well under the action of a magnetic stirrer to obtain a mixed system. The reaction temperature is 20 °C at room temperature, and the solution pH is about 7.4.

[0024] During the reaction process, at fixed reaction time points of 0, 10, 20, 40, and 60 seconds, sample 1 mL and add it to 10 μL of 1.0 M sodium thiosulfate to terminate the reaction to obtain a virus suspension. Dilute the obtained samples by different multiples in turn. Mix 100 μL of Escherichia coli host suspension, 100 μL of diluted sample solution, and 3 mL of semi-solid medium and spread them on the medium. Place the medium in a 37 °C constant temperature incubator for 18 - 24 h. After cultivation, count the number of phage plaques and calculate the logarithm of virus disinfection. The results are shown in Figure 1 .

[0025] Figure 1 It shows that when the concentration of ruthenium(III) ions is 10 μM and the concentration of periodate is 50 μM and 100 μM, complete inactivation of the virus can be achieved at 20 seconds and 10 seconds respectively.

[0026] Example 2

[0027] Disinfection of Escherichia coli phage in water by ruthenium metal ions with different concentrations.

[0028] Repeat Example 1 with the following differences: Keep the concentration of periodate at 50 μM, and adjust the concentration of ruthenium metal ions to 0, 1, 5, and 10 μM respectively. The sampling time points are still 0, 10, 20, 40, and 60 seconds. The results are shown in Figure 2 .

[0029] Figure 2 It shows that when the concentration of ruthenium(III) ions is 10 μM and the concentration of periodate is 50 μM, the disinfection effect is the best, and complete inactivation of the virus can be achieved within 20 seconds.

[0030] Example 3

[0031] Removal of the gene fragment encoding the protein of Escherichia coli phage in the mixed system.

[0032] In a 20 mL reaction system containing 10.0 mM phosphate buffered saline (PBS), add 2×106 ~3×10 6 Escherichia coli phage (MS2) at 6 plaque-forming units per milliliter (PFU / mL) was used to obtain the virus-containing water to be treated. Sodium periodate was added to the above water to a concentration of 50 μM, and ruthenium trichloride was added to a concentration of 10 μM. The mixture was thoroughly mixed under the action of a magnetic stirrer to obtain a mixed system. The reaction temperature was room temperature (20 °C), and the solution pH was approximately 7.4.

[0033] During the reaction, 1 mL of samples was taken at fixed reaction time points of 0, 10, 20, 40, and 60 seconds and added to 10 μL of 1.0 M sodium thiosulfate to terminate the reaction, obtaining virus suspensions. Using the RT-qPCR method, after extracting virus RNA with the Viral DNA / RNA Kit kit, the Uni Reverse Transcriptase kit was used for virus RNA transcription. Then, the SYBR Green PCR Kit kit was used for qPCR quantification. The reaction conditions included: initial denaturation (95 °C for 3 min), repeated denaturation (95 °C for 15 s), annealing (60 °C for 15 s), and extension (72 °C for 30 s), for a total of 40 cycles. The primers for encoding proteins were MP. The results are shown in Figure 3 . The specific primer sequences are as follows:

[0034] MP gene:

[0035] Forward primer MP-F: 5’-AAGGTGCCTACAAGCGAAGT-3’

[0036] Reverse primer MP-R: 5’-TTCGTTTAGGGCAAGGTAGC-3’

[0037] Figure 3 The results showed that the reaction system could achieve a removal rate of more than 99% for the genes encoding viral proteins at 10 seconds and more than 99.9% at 40 seconds.

[0038] Example 4

[0039] Removal of the RNA replication gene fragment of Escherichia coli phage in water by the mixed system.

[0040] Example 3 was repeated with the following difference: The primers for RNA replication were RR. The results are shown in Figure 4 . The specific primer sequences are as follows:

[0041] RR gene:

[0042] Forward primer MP-F: 5’-CTACCGATCGTCGTTGTTTG-3’

[0043] Reverse primer MP-R: 5’-GACCCCTTTCTGGAGGTACA-3’

[0044] Figure 4 It shows that the reaction system can achieve a removal rate of more than 99% for the gene responsible for viral RNA replication at 10 seconds, and more than 99.9% at 40 seconds.

[0045] Example 5

[0046] Effect of the mixed system on the attachment of Escherichia coli phage to Escherichia coli host in water.

[0047] The Escherichia coli host was first cultured for 12 h and then placed on TSA medium. After culturing with constant shaking at 37 °C for 2.5 h, it was centrifuged at 6000 rpm for 10 min. The precipitate was resuspended with 0.1 mM PBS to obtain Escherichia coli host OD 600 = 0.2.

[0048] In a 20 mL reaction system containing 10.0 mM phosphate buffered saline (PBS), 2×10 6 ~3×10 6 plaque-forming units per milliliter (PFU / mL) of Escherichia coli phage (MS2, ATCC 15597-B1) were added to obtain the virus-containing water to be treated. Sodium periodate was added to the above water to a concentration of 50 μM, and ruthenium trichloride was added to a concentration of 10 μM. The mixture was thoroughly mixed under the action of a magnetic stirrer to obtain a mixed system. The reaction temperature was 20 °C at room temperature, and the solution pH was about 7.4. At 20 seconds of the reaction, 100 μL of 1.0 M sodium thiosulfate solution was added to terminate the reaction to obtain the treated virus suspension sample.

[0049] Equal volumes of the virus samples before and after treatment were mixed and cultured with the Escherichia coli host, incubated at 37 °C for 2 h to initiate irreversible host attachment.

[0050] Subsequently, it was centrifuged at 12000 rpm at 4 °C for 10 min. The precipitate was resuspended with an equal volume of the above PBS and repeatedly frozen and thawed 3 times, filtered through a 0.22 μm filter membrane to obtain a virus sample. Finally, by using the RT-qPCR method, after extracting virus RNA with the Viral DNA / RNAKit kit, the virus RNA was transcribed using the Uni Reverse Transcriptase kit, and then Quantitative PCR (qPCR) was performed using the SYBR Green PCR Kit. The reaction conditions included: initial denaturation (95°C for 3 min), repeated denaturation (95°C for 15 s), annealing (60°C for 15 s), and extension (72°C for 30 s), for a total of 40 cycles. The genes used to quantify the virus were the MP gene and the RR gene. The specific primer sequences were the same as above. The results are shown in Figure 5 .

[0051] Figure 5 It was shown that the amount of virus attachment decreased before and after the reaction. Therefore, this reaction system would affect the attachment function of the virus during proliferation on the Escherichia coli host.

[0052] Example 6

[0053] Effect of the mixed system on the injection of Escherichia coli phage in water into the Escherichia coli host.

[0054] Repeat Example 5 with the following differences: After mixing the sample with the Escherichia coli host in equal volume and culturing before and after the reaction, incubate on ice at 4°C for 3 h, then incubate at 37°C for 2 h to allow the virus to inject into the host. Then, wash the sample with 0.005% SDS, centrifuge at 12,000 rpm for 10 min, wash with PBS, and resuspend to obtain the virus sample. Finally, use it for RT-qPCR quantification. The results are shown in Figure 6 .

[0055] Figure 6 It was shown that the amount of virus injection decreased before and after the reaction. Therefore, this reaction system would affect the injection function of the virus during proliferation on the Escherichia coli host.

[0056] In the present invention, a periodate oxidant and a homogeneous trivalent ruthenium ion activator are added to the water body containing the virus to be treated to form a mixed system, thereby rapidly and efficiently disinfecting and killing the virus. The present invention uses a homogeneous transition metal ruthenium ion catalyst to activate the periodate, which not only destroys the culturing ability of the virus but also destroys the nucleic acid encoding protein and RNA replication in the virus, and at the same time affects the functions of attachment and injection into the host during virus proliferation, realizing virus disinfection and killing. This method has a wide range of applications, requires no additional energy and equipment, saves energy, and is simple to operate.

Claims

1. A method for ultra-fast elimination of viruses based on periodate, characterized in that: Add periodate and trivalent ruthenium ion activator into the water body containing the virus to be treated to form a mixed system, so as to achieve efficient disinfection of the virus, and the virus is Escherichia coli phage MS2.

2. The method for ultra-fast virus elimination based on periodate as claimed in claim 1, wherein: In the mixed system, the concentration of periodate is not higher than 100.0 μM.

3. The method for ultra-fast virus elimination based on periodate as claimed in claim 1, wherein: In the mixed system, the concentration of trivalent ruthenium ion activator is not higher than 10.0 μM.

4. A method for ultra-fast virus elimination based on periodate, as described in claim 1, characterized in that: The virus concentration is 2×10 6 ~3×10 6 PFU / mL.

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