Application of slightly acidic hypochlorous acid in preparation of disinfectant for preventing and controlling African swine fever

Abstract

The invention provides application of slightly acidic hypochlorous acid in preparation of a disinfectant for preventing and controlling African swine fever. The invention also provides a disinfectant for preventing and controlling African swine fever. The disinfectant contains a slightly acidic hypochlorous acid aqueous solution. Specifically, in the disinfectant, the content of available chlorine is greater than or equal to 600mg / L, and the pH is 6.4. The slightly acidic hypochlorous acid aqueous solution is used for preventing and controlling the African swine fever in the livestock farm for the first time, and compared with a sodium hypochlorite disinfectant, the slightly acidic hypochlorous acid aqueous solution has the advantages of low irritation, low use concentration and capability of disinfecting with livestock, and has a good killing effect on pathogenic microorganisms.

Description

Technical Field

[0001] The present invention belongs to the field of chemistry and relates to a disinfectant, specifically to the use of slightly acidic hypochlorous acid in the preparation of a disinfectant for preventing and controlling African swine fever. Background Art

[0002] African swine fever (ASF) is an acute, highly virulent and highly contagious disease caused by the African swine fever virus (ASFV), which infects domestic pigs and wild boars. It will cause serious economic losses and pose a serious threat to the development of the global pig industry. ASF has a history of over a hundred years, and there are still no commercially available vaccines and antiviral drugs that can be promoted globally. Strict disinfection procedures are currently the most basic and effective means of preventing and controlling African swine fever.

[0003] In the prior art, the following several types of disinfectants are commonly used for preventing and controlling African swine fever during the breeding process: one is alkaline disinfectants, mainly including sodium hydroxide and quicklime, etc.; the second is aldehyde disinfectants, mainly including formaldehyde, glutaraldehyde, etc., which are mostly used for fumigation or immersion disinfection; the third is iodine preparations, mainly including iodine and iodine tincture solution; the fourth is phenolic disinfectants, mainly including lysol, compound phenol, phenol, etc. The fifth is chlorine preparation disinfectants, mainly including bleaching powder, sodium hypochlorite and sodium dichloroisocyanurate, etc. The sixth is quaternary ammonium salts, mainly including benzalkonium bromide (the active ingredient is benzalkonium bromide, bromobenzylalkylamine), and baidusha (the active ingredients are glutaraldehyde and decamethonium bromide). The seventh is peroxide-based, mainly including potassium permanganate, peracetic acid, hydrogen peroxide, ozone, etc. These disinfectants have the disadvantages of limited use scenarios, poor effects on viruses, poor stability, difficulty in storage, and corrosiveness, etc.

[0004] Compared with the above several types of disinfectants, slightly acidic hypochlorous acid disinfectant has the advantages of high efficiency, rapidity, broad spectrum, safety and environmental protection. The main component of its available chlorine is HClO (hypochlorous acid), which is reduced to water after the bactericidal action, and has no irritation or pollution to humans, animals and the environment. It is convenient to use and has a wide range of applications. It can be used for disinfection of livestock, humans, air, skin, oral cavity, body surface and object surfaces, drinking water and immersion disinfection. At the same time, its production process is simple and the cost is cheap. The total cost is only one-third of that of the cheapest conventional chemical disinfectants. There is currently no report on its application in the prevention and control of African swine fever in pig farms. Summary of the Invention

[0005] In view of the above technical problems in the prior art, the present invention provides the use of slightly acidic hypochlorous acid in the preparation of a disinfectant for preventing and controlling African swine fever. The use of such slightly acidic hypochlorous acid in the preparation of a disinfectant for preventing and controlling African swine fever aims to solve the technical problems in the prior art that the disinfectants for preventing and controlling African swine fever have limited usage scenarios, poor effects on viruses, poor stability, are not easy to store, and are corrosive.

[0006] The present invention provides the use of slightly acidic hypochlorous acid in the preparation of a disinfectant for preventing and controlling African swine fever.

[0007] The present invention also provides a disinfectant for preventing and controlling African swine fever, which contains slightly acidic hypochlorous acid.

[0008] Specifically, in the said disinfectant, the available chlorine content is greater than or equal to 600 mg / L, and the pH is 6.4.

[0009] The slightly acidic hypochlorous acid disinfectant of the present invention is prepared by using an electrochemical technique. An aqueous solution with an available chlorine content of 600 mg / L and a pH of 6.4 is produced by electrolyzing a sodium chloride solution in an electrolysis chamber between an anode and a cathode.

[0010] In the said application method, the results of qualitative and quantitative killing tests show that the slightly acidic hypochlorous acid solution with effective concentrations of 300 and 200 mg / L can completely kill African swine fever virus ASFV after acting on the virus for 5 minutes, and the average inactivation logarithm value of the disinfectant against the virus is 6.5 (>2).

[0011] The results of on-site disinfection tests show that the 400 mg / L and 200 mg / L slightly acidic hypochlorous acid solutions act on the floor and walls of the pigsty by spraying for 5 minutes, and the bactericidal effect is qualified. While the 200 mg / L and 100 mg / L slightly acidic hypochlorous acid solutions are used to rinse the slotted floor, ASFV nucleic acid cannot be detected.

[0012] When slightly acidic hypochlorous acid is used as a disinfectant for African swine fever, it can be used for the environment of the farm, disinfection of animals with livestock, etc.

[0013] When slightly acidic hypochlorous acid is used as a disinfectant, the usage method is usually as follows: Fill the slightly acidic hypochlorous acid disinfectant into a sprayer and directly spray it on the surface of an object or the body surface of an animal, or atomize and spray it on the environment, space, etc.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1. The hypochlorous acid solution disinfectant of the present invention is prepared by using an electrochemical technique. It is produced by electrolyzing a sodium chloride solution in an electrolysis chamber between an anode and a cathode. The raw materials for production are only water and sodium chloride, and the total cost is only one-third of that of the cheapest conventional chemical disinfectant, significantly reducing the usage cost.

[0016] 2. The efficacy of the present invention was evaluated at the National African Swine Fever Regional Laboratory (Veterinary Drug Research and Evaluation Center of South China Agricultural University), and qualitative and quantitative tests, carrier disinfection tests, and on-site disinfection test evaluations were carried out. The evaluation tests showed that the product was qualified in killing African swine fever virus and was a green and environmentally friendly disinfectant with good killing effect on African swine fever virus and safe to use among existing disinfectants.

[0017] 3. The present invention first applied slightly acidic hypochlorous acid aqueous solution to the prevention and control of African swine fever in farms. Compared with sodium hypochlorite disinfectant, it had the advantages of low irritation, low use concentration, and the ability to disinfect livestock, and had good killing effects on pathogenic microorganisms. Description of the Drawings

[0018] Figure 1 It is a picture of hemadsorption when measuring the titer by microscope observation. Detailed Embodiments

[0019] Example 1 Experimental Materials

[0020] The slightly acidic hypochlorous acid disinfectant, containing 600 mg / L of available chlorine (Cl) and with a pH of 6.4, was provided by Shanghai Yubozhi Biotechnology Co., Ltd. The control sodium hypochlorite solution, containing 5% of available chlorine (Cl), was purchased from Guangdong Wangxingda Biotechnology Co., Ltd. The African swine fever virus strain, ASFV / China / GZ201801, was provided by the National African Swine Fever Regional Laboratory (Guangzhou). Fetal bovine serum (FBS) was purchased from Vincent Biotechnology Co., Ltd., cell culture medium (1640) was purchased from Biological Industries (BI), phosphate buffered saline solution (PBS) was purchased from Sevier Biotechnology Co., Ltd., and the real-time fluorescence PCR kit was purchased from Qingdao Lijian Biotechnology Co., Ltd.

[0021] Example 2 Virus Titer HAD 50 Determination

[0022] Resuscitate porcine alveolar macrophages (PAM cells). When the growth condition is good, seed the cells into a 96-well cell culture plate and observe the cell growth state. When the cells grow to 75% - 90%, wash the cells twice with PBS and discard the cell culture medium. After serially diluting the ASFV virus solution 10-fold, inoculate it into the cell plate, 100 μL per well, and then incubate it in an incubator at 37 °C with 5% CO₂ for 10 - 12 h. Use a pipette to aspirate the cell maintenance medium after inoculation, then wash the cells once with 2 mL PBS and discard the liquid. Then add the cell maintenance medium to the cell culture flask; place it in an incubator at 37 °C with 5% CO₂ and culture for 4 - 6 h; take out the 96-well plate, add 20 μL of 1% porcine red blood cells to each well, and continue to incubate overnight; observe and record the number of wells with red blood cell adsorption phenomenon under a microscope for each gradient, and calculate the virus titer HAD 50 。

[0023] Take the cell culture supernatant after the proliferation and culture of ASFV on PAM cells, measure the virus titer to be 10⁴.⁷ HAD 50 / mL, the Ct value detected by qPCR = 17.04, and there is an obvious exponential growth phase. Store it at -80 °C; the pictures of red blood cell adsorption when measuring the titer are as Figure 1 。

[0024] Example 3 Evaluation of the Qualitative Virucidal Effects of Slightly Acidic Hypochlorous Acid Disinfectant and Sodium Hypochlorite Disinfectant

[0025] Select two disinfectants to evaluate the virucidal effects on African swine fever virus. Mix the disinfectants at double the concentration to be tested with an equal volume of virus solution (virus titer ≥ 10 3 TCID 50 ) respectively, and let them act for 5, 10, 15, 30, 60 min; determine the dilution degree for terminating the identification test according to the dilution method (the slightly acidic hypochlorous acid solution and sodium hypochlorite solution are diluted 2000-fold and 3200-fold respectively as the concentrations for terminating dilution in this test) to terminate the reaction by the dilution method; inoculate PAM cells, set 5 replicates, and culture for 10 - 12 h; use a pipette to aspirate the cell maintenance medium after inoculation, then wash the cells once with 2 mL PBS and discard the liquid. Then add the cell maintenance medium to the cell culture flask; place it in an incubator at 37 °C with 5% CO₂ and culture for 4 - 6 h; add 20 μL of 1% porcine red blood cells to each well and continue to incubate overnight; observe the red blood cell adsorption phenomenon under a microscope. When virus replication cannot be observed in all 5 replicates, it means that the disinfectant at this concentration has an inactivating effect under this condition.

[0026] As can be seen from Table 1 and Table 2, the minimum effective disinfection concentration of the slightly acidic hypochlorous acid solution against African swine fever virus is 100 mg / L, and the virus can be completely killed within 60 min at this concentration; the minimum effective disinfection concentration of the sodium hypochlorite solution against African swine fever virus is 200 mg / L, and the fastest effective disinfection time at this concentration is 5 min.

[0027] Table 1 Results of qualitative killing test of slightly acidic hypochlorous acid solution against ASFV

[0028]

[0029] Note: "+" indicates erythrocyte adsorption; "-" indicates no erythrocyte adsorption.

[0030] Table 2 Results of qualitative killing test of sodium hypochlorite solution against ASFV

[0031]

[0032]

[0033] Note: "+" indicates erythrocyte adsorption; "-" indicates no erythrocyte adsorption.

[0034] Example 4 Evaluation of quantitative killing effects of slightly acidic hypochlorous acid disinfectant and sodium hypochlorite disinfectant on virus

[0035] Referring to the "Technical Specification for the Identification of Veterinary Disinfectants" promulgated in 1992 and the "Disinfection Technical Specification" in 2002 edition, suspension quantitative killing tests were respectively carried out on African swine fever virus strains using slightly acidic hypochlorous acid disinfectant and sodium hypochlorite disinfectant to evaluate their killing effects.

[0036] 4.5 mL of disinfectants at 300, 200 and 100 mg / L were respectively mixed with 0.5 mL of virus solution (virus titer HAD 50 = 10 6.5 / mL), and after acting for 5, 10, 15, 30, 60 min respectively, the reaction was terminated by adding culture medium and diluting 1000 times, inoculating cells, and measuring the virus titer. Calculate the average virus titer TCID 50 as N x . In the control group, the diluent was used instead of the disinfectant, and other steps were the same as those in the disinfection group, and the virus titer TCID 50 was calculated as N0. The average inactivation logarithm value = logN0 - logN x , and when the average inactivation logarithm value of the disinfectant against the virus ≥ 2, it was judged as qualified.

[0037] As can be seen from Table 3, there was no virus proliferation after the two disinfectants at 300 and 200 mg / L acted on the virus for 5 min, and there was no virus proliferation after the slightly acidic hypochlorous acid solution at 100 mg / L acted on the virus for 60 min. The average log inactivation value of the disinfectant against the virus was greater than 2, indicating that the slightly acidic hypochlorous acid solution and sodium hypochlorite solution at the above-mentioned action concentrations had qualified inactivation effects on ASFV. However, after the sodium hypochlorite solution at 100 mg / L acted on ASFV for 60 min, the virus proliferation was obvious, and the titer was not much different from that of the control group, showing that it had basically no killing effect on the virus.

[0038] Table 3 Effects of Disinfectants on the Logarithmic Value of ASFV Titer

[0039]

[0040]

[0041] Example 5 Evaluation of the Bactericidal Effects of Slightly Acidic Hypochlorous Acid Disinfectant and Sodium Hypochlorite Disinfectant

[0042] Using the suspension quantitative bactericidal method, Staphylococcus aureus (ATCC29213) and Escherichia coli (ATCC25922) were used as indicator bacteria, and the bacterial suspension used in the experiment contained 10 6 ~10 7 cfu / mL. The neutralizer was TSB broth containing 0.5% sodium thiosulfate and 0.5% Tween 80, which was autoclaved before use, and the concentration of the prepared disinfectant solution was 1.25 times the concentration to be measured. The test results of the neutralizer selection proved that the neutralizer selection was reasonable.

[0043] Experimental procedure: Pipette 0.5 mL of the test bacterial suspension into 0.5 mL of 3% BSA and mix well for 5 min; add 4 mL of the disinfectant solution at 1.25 times the concentration to be measured and start timing immediately; after acting for 5, 10, 15, 30, and 60 min respectively, take 0.5 mL of the bacterial-drug mixture and add it to a test tube containing 4.5 mL of the neutralizing solution and mix well; after neutralizing for 10 min, take 0.1 mL of the final reaction solution and spread it on an agar plate, and incubate at 37°C for counting; take its original solution or the diluted solution after continuous 10-fold dilution and inoculate it on the plate, place it in a 37°C incubator for 24 h, and then perform colony counting; in the control group, the diluent was used instead of the disinfectant solution, and the other steps were the same as those in the disinfection group; the test was repeated 3 times and the average value was taken. Calculate the bactericidal rate of the disinfectant against microorganisms, and if the bactericidal rate ≥ 99%, it is judged as qualified. Bactericidal rate = (number of bacteria in the control group - number of bacteria in the disinfection group) / number of bacteria in the control group × 100%.

[0044] As shown in Tables 4 and 5, the 100 mg / L slightly acidic hypochlorous acid solution can effectively kill Staphylococcus aureus after acting for 60 min, while the same concentration of sodium hypochlorite solution cannot completely kill Staphylococcus aureus within 60 min, and the 200 mg / L sodium hypochlorite solution needs to act for 30 min to achieve the killing effect; the 50 mg / L slightly acidic hypochlorous acid solution can effectively kill Escherichia coli after acting for 30 min, and the 25 mg / L sodium hypochlorite solution needs to act for 60 min to achieve the killing effect.

[0045] Table 4 Quantitative killing test results of disinfectants against Escherichia coli

[0046]

[0047]

[0048] Table 5 Quantitative killing test results of disinfectants against Staphylococcus aureus

[0049]

[0050] Example 5 Carrier disinfection test

[0051] Select white plain cotton cloth pieces and stainless steel round pieces as carriers for immersion tests. Select a suitable concentration of disinfectant according to suspension quantitative sterilization, prepare bacterial tablets so that the recovered bacteria count of each bacterial tablet is 5×10 5 ~5×10 6 cfu / tablet. Draw the corresponding concentration of disinfectant solution into a petri dish in an amount of 5 mL per bacterial tablet; use sterile forceps to pick up 3 pre-prepared bacterial tablets, soak them in the disinfectant, and start timing immediately; when the bacteria and medicine have acted for 5, 10, and 15 min respectively, use sterile forceps to take out the bacterial tablets and put them into a test tube containing 5 mL of neutralizer, shake for 20 s with an electric mixer to elute the bacteria, and then let it stand for 10 min for full neutralization; conduct a positive control test with sterile distilled water instead of the disinfectant solution; take 0.1 mL of the final reaction solution and spread it on an agar plate, inoculate 2 petri dishes per tube, and incubate at 37°C for counting; repeat the test 3 times and take the average value. The recovered bacteria amount of the control group is 5×10 5 ~5×10 6 CFU / tablet, and the killing index reaches 10 3 It is judged that disinfection is qualified.

[0052] As shown in Tables 6 and 7, when using stainless steel round pieces as carriers, both groups of disinfectants can completely kill Escherichia coli when acting at 50 mg / L within 5 min. The 200 mg / L slightly acidic hypochlorous acid solution can effectively kill Staphylococcus aureus after acting for 10 min, while the 100 mg / L solution cannot effectively kill Staphylococcus aureus after acting for 15 min. The 50 mg / L sodium hypochlorite solution can completely kill Staphylococcus aureus after acting for 5 min.

[0053] Table 6 Bactericidal effects of different disinfectant concentrations on Escherichia coli (immersion of stainless steel discs)

[0054]

[0055]

[0056] Note: No bacteria grew in each negative control group.

[0057] Table 7 Bactericidal effects of different disinfectant concentrations on Staphylococcus aureus (immersion of stainless steel discs)

[0058]

[0059]

[0060] Note: No bacteria grew in each negative control group.

[0061] As shown in Tables 8 and 9, when using cloth pieces as carriers for immersion tests, the two groups of disinfectants can effectively kill Escherichia coli when acting at 100 mg / L for 5 min and 50 mg / L for 10 min. When the sodium hypochlorite solution at 200 mg / L acts for 5 min and the weakly acidic hypochlorous acid solution at 400 mg / L acts for 10 min, Staphylococcus aureus can be completely killed. Reducing the concentration of the sodium hypochlorite solution to 100 mg / L and acting for 5 min can effectively kill Staphylococcus aureus. However, when the weakly acidic hypochlorous acid solution at 200 mg / L and the sodium hypochlorite solution at 50 mg / L act on Staphylococcus aureus for 15 min, the logarithmic reduction values are all less than 3, and Staphylococcus aureus cannot be effectively killed.

[0062] Table 8 Bactericidal effects of different disinfectant concentrations on Escherichia coli (immersion of cloth pieces)

[0063]

[0064]

[0065] Note: No bacteria grew in each negative control group.

[0066] Table 9 Bactericidal effects of different disinfectant concentrations on Staphylococcus aureus (immersion of cloth pieces)

[0067]

[0068]

[0069] Note: No bacteria grew in each negative control group.

[0070] Example 6 On-site disinfection (flushing of slatted floor) test of virus

[0071] Take 50 μL of the virus solution (HAD 50 = 104.7 / mL) and drop it onto the slotted floor, then spread it evenly. The slotted floor was not rinsed, rinsed with tap water 5 times, 10 mL each time, and rinsed 5 times, 10 mL each time, with micro-acid hypochlorous acid solution or sodium hypochlorite solution with concentrations of 200, 100, 50, 25, and 12.5 mg / L respectively. Samples were taken after 30 min for nucleic acid extraction and qPCR detection.

[0072] Validity determination: The positive control has a typical amplification curve with a Ct value ≤ 30, and the negative control has no Ct value or no amplification curve, and the line is a straight line or a slight oblique line, without an exponential growth period. Then the test result is judged to be valid; otherwise, this test is regarded as invalid. Positive determination: Ct value ≤ 35 and there is an obvious exponential growth period, judged as positive. Suspected determination: Ct value between 35 - 38, judged as suspected negative. Repeat the detection. If the Ct value of the repeated experiment is still between 35 - 38 and there is an obvious exponential growth period, it is judged as positive; otherwise, it is negative. Negative determination: Ct value > 38 or no Ct value, judged as negative.

[0073] The results are shown in Table 10. The Ct value of the positive non-rinsed group is 29, and there is an obvious exponential growth period, judged as positive; the Ct value of the tap water rinsed group is 32, and there is an obvious exponential growth period, judged as positive; the 200 and 100 mg / L disinfectant rinsed groups have no Ct value, judged as negative; the Ct value after rinsing with 50 mg / L micro-acid hypochlorous acid solution is 35, and there is an obvious exponential growth period, judged as positive. There is no Ct value after rinsing with 50 mg / L sodium hypochlorite solution, judged as negative.

[0074] Table 10 Fluorescent quantitative PCR results (Ct values) of on-site disinfection (slotted floor rinsing) test

[0075]

[0076] Note: " / " indicates no Ct value and no obvious amplification curve.

[0077] Example 7 On-site disinfection test of natural bacteria on the surface

[0078] The livestock and poultry house was rinsed with tap water and dried for later use; on the ground and wall, 2 adjacent areas of 25 cm each were marked with a gauge board 2Blocks, one for sampling before disinfection and one for sampling after disinfection; Moisten a sterile cotton swab in a test tube containing 10 mL of neutralizer solution, squeeze it dry on the tube wall, smear and sample the control block, wipe back and forth vertically and horizontally 8 times each with force, and continuously switch the wiping surface of the swab. After sampling, cut the sampled end of the cotton swab into the original neutralizer solution test tube in a sterile operation manner, shake it with an electric mixer for 20 s, and after appropriate dilution, use it as a sample for the positive control group; Use a 500 mL spray bottle to evenly spray the disinfectant to be tested onto the test area, using 200 mL of disinfectant per square meter, and start timing immediately; When the action reaches 5, 10, and 15 min, moisten a sterile cotton swab in a test tube containing 10 mL of neutralizer solution, squeeze it dry on the tube wall, smear and sample the disinfected block, wipe back and forth vertically and horizontally 8 times each with force, and continuously switch the wiping surface of the swab. After sampling, cut the sampled end of the cotton swab into the original neutralizer solution test tube in a sterile operation manner, shake it with an electric mixer for 20 s, and use it as a sample for the test group; Take 0.1 mL of the final reaction solution and spread it on an agar plate, and incubate it at 37 °C for counting; Set negative controls for the same batch of neutralizer solution, diluent, cotton swabs, culture medium, etc. that were not used up in this test. There should be no bacterial growth in the negative control group, there should be more bacterial growth in the positive control group, and the average killing logarithm value of the disinfected sample ≥ 1 is judged as qualified disinfection.

[0079] Calculate the killing logarithm values before and after disinfection at each detection point on the ground and walls of the test pen. The 400 and 200 mg / L slightly acidic sodium hypochlorite solutions act for 5 min, and the killing logarithm values are both greater than 1. Therefore, the on-site disinfection effect of the 400 and 200 mg / L slightly acidic sodium hypochlorite solutions is qualified. The relevant data are shown in Table 11.

[0080] Table 11 Influence of slightly acidic sodium hypochlorite solution on killing logarithm values before and after disinfection at different positions in the pen

[0081]

[0082] Note: There is no bacterial growth in each negative control group.

[0083] The sodium hypochlorite solution at 400 mg / L acts for 5 min, and the bactericidal rate can reach 100%. The 200 and 100 mg / L act for 5 min, and the killing logarithm value is greater than 1. Therefore, the 400, 200, and 100 mg / L sodium hypochlorite solutions are qualified for on-site disinfection. The relevant data are shown in Table 12.

[0084] Table 12 Influence of sodium hypochlorite solution on killing logarithm values before and after disinfection at different positions in the pen

[0085]

[0086] Note: There is no bacterial growth in each negative control group.

Claims

1. Use of slightly acidic hypochlorous acid in the preparation of a disinfectant for preventing and controlling African swine fever.

2. A disinfectant for preventing and controlling African swine fever, characterized in that, It contains an aqueous solution of slightly acidic hypochlorous acid.

3. The disinfectant for preventing and controlling African swine fever according to claim 2, characterized in that, In the described disinfectant, the available chlorine content is greater than or equal to 600 mg / L and the pH is 6.4.

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