CN110643742A - Method for removing residual disinfectant in virus inactivation test - Google Patents
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Abstract
The invention belongs to the technical field of disinfectometry, and relates to a virus inactivation test, in particular to a method for removing residual disinfectants in the virus inactivation test, which comprises the following steps: 1) the preparation method comprises the following steps: preparing a virus suspension; 2) removing: firstly, absorbing a disinfectant into a test tube, carrying out drug removal treatment after water bath, then carrying out 30-50 times dilution and then ultracentrifugation, then discarding supernatant, finally adding a cell maintenance solution, diluting and uniformly mixing again, and removing residual disinfectant; 3) a detection step: sucking and adding the virus suspension, uniformly mixing, sucking the final sample and performing virus titer determination; the method for removing the residual disinfectant can reduce the test time for removing the disinfectant in the whole process, greatly improve the test efficiency of the virus inactivation test and avoid the influence of disinfectant and neutralizer in a chemical neutralization mode on cells and viruses.
Description
Technical Field
The invention belongs to the technical field of disinfectometry, and relates to a virus inactivation test, in particular to a method for removing residual disinfectant in the virus inactivation test.
Background
With the development of social economy, the change of environment, the change of life style of people and the like, some new and unknown infectious diseases (AIDS, SARS, avian influenza, Zika, hand, foot and mouth, Ebola, Eico virus and the like), nosocomial infection outbreaks and emergent public health events frequently occur, and the health and social stability of people are seriously influenced. The infectious diseases caused by the virus are very easy to cause serious consequences due to quick outbreak and strong infectivity, once the infectious diseases occur, the transmission path and the infection mechanism are difficult to be clear in a short time, the targeted prevention and control measures are difficult to be carried out in time, the research and the production of the specific drugs and the vaccines for treating the viral infectious diseases need to carry out a large amount of early research, and a series of reasons cause that the human measures have considerable hysteresis in emergent public health events and hospital infection events; therefore, in order to solve the above problems, disinfection is generally considered to be the simplest and most effective means for cutting off the viral transmission pathway and preventing and controlling viral transmission.
The disinfection work is realized by a disinfection product, and in article 29 of infectious disease control law of the people's republic of China, the attribute of the disinfection product for infectious disease control is determined, so that the virus inactivation effect of the disinfection product is directly related to the health and social stability of people, the method for evaluating the virus inactivation effect of the disinfection product in China mainly refers to law and regulations such as disinfection technical Specification (2002 edition) and related standards, a poliovirus-I type vaccine strain (PV-I) is generally selected as an index virus, the removal of residual disinfectant in the evaluation method is critical, and if the residual disinfectant is not completely removed, the most accurate and objective result of virus inactivation cannot be obtained, so that the evaluation result is distorted.
In order to accurately detect, identify and evaluate the virus inactivation performance of the disinfectant, the residual disinfectant needs to be removed after a certain inactivation time.
The current methods for removing residual disinfectants mainly comprise a chemical neutralization method and a physical method, wherein the chemical neutralization method utilizes antagonism between chemical substances to remove the residual disinfectants, but different chemical neutralizers and neutralized products in the method can influence cells. The method for removing residual disinfectant in each detection needs to be applicable after qualified identification, but the neutralizing agent method has the following defects: 1. the screening of the components of the compound disinfectant neutralizer is very complicated and the success rate is very low; 2. the concentrations of the effective substances of different disinfectants are inconsistent, and the concentration of the components of the common neutralizer is not necessarily reasonable; 3. the neutralizer component itself must not be harmful to the microorganisms or cells. Therefore, the chemical neutralization method needs to perform a sufficient amount of preliminary tests, consumes a great deal of time and labor, and becomes difficult to find a proper chemical neutralizing agent along with the increasing complexity of the components of the disinfectant, so that the difficulty in virus inactivation tests is achieved; the physical methods include dilution, adsorption column, molecular sieve column and carrier washing, which have special requirements for disinfectants or viruses and cannot be generalized.
Disclosure of Invention
The present invention relates to a virus inactivation test, and more particularly, to a method for removing residual disinfectant in a virus inactivation test.
In order to achieve the purpose, the invention adopts the technical scheme that: a method for removing residual disinfectant in a virus inactivation test is characterized by comprising the following steps:
1) the preparation method comprises the following steps: preparing a virus suspension;
2) removing: firstly, absorbing a disinfectant into a test tube, carrying out drug removal treatment after water bath, then carrying out 30-50 times dilution and then ultracentrifugation, then discarding supernatant, and finally adding a cell maintenance solution for dilution and uniform mixing to remove residual disinfectant;
3) a detection step: the virus suspension is added and mixed evenly for virus titer determination.
Preferably, the temperature of the water bath in the step 2) is 20 ℃ +/-1 ℃, the time is 5min, and the centrifugation conditions are as follows: the rotating speed is 10000rpm to 25000rpm, the centrifugation time is 1h to 2.5h, and the temperature is 4 ℃.
Preferably, the volume of the cell maintenance solution added in the step 2) after dilution and mixing is 1 mL-2 mL.
Preferably, the step of detecting in step 3) is performed by calculating virus infection titer by using an end point dilution method or a plaque method, so as to perform virus titer determination.
Preferably, the preparing of the virus suspension of step 1) comprises the following steps:
(1) taking frozen host cells, quickly melting the frozen host cells in warm water at 37 ℃, transplanting the frozen host cells into a cell tube of a cell maintenance liquid by using a capillary pipette, blowing and sucking for 4-7 times, uniformly mixing the cells and the cell maintenance liquid, then centrifuging at a high speed, and removing supernatant; then adding cell maintenance liquid, blowing and sucking for several times to uniformly mix, centrifuging again, and transferring into a culture bottle with complete culture medium;
(2) taking virus seeds frozen at low temperature, thawing in water bath at 37 ℃, diluting by 10 times with cell maintenance liquid, and inoculating into the culture bottle in the step (1); then placing the mixture in a 37 ℃ incubator for subsequent virus harvesting;
(3) breaking host cells by adopting an ultrasonic method or a repeated freeze-thaw method in an ice bath condition to release viruses in a culture solution containing the viruses and the host cells; then centrifuging and removing the precipitate, and taking supernatant, namely virus liquid; 1.0mL of the solution is respectively filled into a sterile centrifuge tube with the volume of 1.5mL, and is frozen and preserved at minus 80 ℃;
(4) determining the virus titer by adopting 1 virus liquid according to a virus titer determination method;
(5) when in use, the virus liquid and the organic interferent are mixed at a ratio of 1:1 to prepare virus suspension.
Preferably, the centrifugation condition of the step (1) is 3000rpm for 3 min; the centrifugation condition of the step (3) is 6000rpm, and the time is 15 min.
The invention has the beneficial effects that: the method for removing the residual disinfectant can reduce the test time for removing the disinfectant in the whole process, reduces the traditional working time of 21-30 days to 4-7 days, can greatly improve the detection efficiency of a virus inactivation test, and can overcome the influence of a chemical agent due to the reduction of the application of the chemical agent because the side effect influence is generated when the existing chemical neutralization method is applied, so that the adverse effect of the neutralizing agent on viruses and cells can be completely eliminated by avoiding the adoption of the chemical neutralization mode; meanwhile, the disinfectant is suitable for various disinfectants and viruses, and has wide application range. The invention is suitable for disinfectant detection or disinfection effect evaluation monitoring, is a detection technology for live virus and cell infection thereof, and can be used for evaluating the killing effect of disinfection factors with various purposes on test viruses.
Detailed Description
The following detailed description of the embodiments of the invention:
a method for removing residual disinfectant in a virus inactivation test is characterized by comprising the following steps:
1) the preparation method comprises the following steps: preparing a virus suspension; diluting the disinfectant to be tested to 1.25 times of the use concentration by using sterilized hard water;
2) removing: firstly, absorbing a disinfectant into a test tube, carrying out drug removal treatment after water bath, then carrying out 30-50 times dilution and then ultracentrifugation, then discarding supernatant, and finally adding a cell maintenance solution for dilution and uniform mixing to remove residual disinfectant; in the embodiment, the volume of the cell maintenance solution is 1 mL-2 mL after the cell maintenance solution is added, diluted and uniformly mixed, the volume ratio of the virus suspension to the disinfectant is 1:4, and the volume ratio of the virus suspension is 0.2 mL-0.4 mL; wherein the water bath temperature is 20 +/-1 ℃, the time is 5min, and the centrifugation conditions are as follows: the rotating speed is 10000 rpm-25000 rpm, the centrifugation time is 1 h-2.5 h, and the temperature is 4 ℃;
3) a detection step: sucking and adding the virus suspension, uniformly mixing, sucking the final sample and performing virus titer determination; in the detection step, the virus infection titer is calculated by adopting an end point dilution method or a plaque method so as to determine the virus titer;
the preparation of the virus suspension of the step 1) further comprises the following steps:
(1) taking the frozen host cells out of liquid nitrogen, quickly melting the frozen host cells in warm water at 37 ℃, transplanting the frozen host cells into a cell tube of a cell maintenance liquid by using a capillary pipette, blowing and sucking for 4-7 times to uniformly mix the cells and the cell maintenance liquid, then centrifuging at high speed, and removing supernatant; then adding cell maintenance liquid, blowing and sucking for several times to mix uniformly, centrifuging again, transferring into a culture bottle with 10mL of complete culture medium, observing the growth condition of the cells day by day, and when the cells grow to be full of a monolayer, using the cells for disinfection detection; the centrifugation condition is 3000rpm, the time is 3 min;
(2) taking virus seeds frozen at low temperature, thawing in water bath at 37 ℃, diluting by 10 times with cell maintenance liquid, and inoculating into the culture bottle in the step (1), such as the culture bottle full of monolayer cells in the step (1); then placing the cells in a 37 ℃ incubator to adsorb and grow the cells, observing lesions day by day, and harvesting the viruses when 3/4 cells have lesions;
(3) breaking host cells by adopting ultrasonic waves or repeated freezing and thawing in a culture solution containing viruses and the host cells under an ice bath condition to release the viruses; then centrifuging and removing the precipitate, wherein the precipitate is mainly cell debris, and taking supernatant, namely virus liquid; 1.0mL of the solution is respectively filled into a sterile centrifuge tube with the volume of 1.5mL, and is frozen and preserved at minus 80 ℃; the centrifugation condition is 6000rpm, and the time is 15 min;
(4) determining the virus titer by adopting 1 virus liquid according to a virus titer determination method;
(5) when in use, the virus liquid and the organic interferent are mixed at a ratio of 1:1 to prepare virus suspension.
The invention is in the specific embodiment of the disinfection product monitoring:
first, application scope
The embodiment is mainly suitable for disinfection product identification or daily monitoring, and utilizes a certain representative and living virus and cell infection technology thereof to evaluate the killing effect of disinfection factors with various purposes on test viruses and verify the important aspect of the virus inactivation capacity of the disinfection factors.
Second, test equipment
(1) Test virus strains: poliovirus type I (PV-I) vaccine strains, and enterovirus type 71 (EV71) are provided by the Guangdong province disease prevention and control center;
(2) host cell: hep-2 cells as test cells for PV-1; VERO cell line, as test cell for EV 71; both cells are provided by the Guangdong province disease prevention and control center;
(3) cell culture bottles and 96-well culture plates;
(4) test equipment: a constant temperature water bath tank; a carbon dioxide incubator; a laminar flow superclean bench; low temperature refrigerator (-20 ℃, -80 ℃); a liquid nitrogen tank; inverting the microscope; a centrifuge; an adjustable pipettor and a matched disposable plastic sucker;
(5) culture medium: a cell maintenance medium; a cell complete medium;
(6) deionized water;
(7) the disinfectant is a compound disinfectant of ethanol and trichloro-hydroxy-diphenyl ether.
Preparation of virus suspension
(1) Taking out the frozen host cells for test from liquid nitrogen, rapidly thawing in 37 deg.C warm water, transplanting in cell tube containing cell maintenance liquid with capillary pipette, blowing and sucking for several times, mixing, immediately centrifuging (centrifuging at 3000rpm for 3min), and removing supernatant; adding appropriate cell maintenance liquid, blowing and sucking for several times, mixing, centrifuging, and transferring into culture flask containing 10mL complete culture medium; observing the growth condition of the cells day by day, and when the cells grow to be full of a monolayer, using the cells for a disinfection test;
(2) taking out the virus seeds of the test virus frozen at low temperature, thawing in water bath at 37 ℃, diluting by 10 times with cell maintenance liquid, then inoculating into a cell bottle full of monolayer cells, and placing in a 37 ℃ incubator to be adsorbed and grown with the cells; observing lesions day by day, and harvesting the virus when 3/4 cells have lesions;
(3) breaking host cells by using ultrasonic waves or repeated freezing and thawing in an ice bath condition to release viruses in a culture solution containing the viruses and the host cells; then centrifuging as soon as possible (6000rpm, 15min) to remove the precipitate (mainly cell debris), and obtaining the supernatant as the required virus suspension; 1.0mL of each tube is respectively filled into a sterile centrifuge tube (1.5 mL);
(4) taking 1 virus suspension, and determining the virus titer according to a virus titer determination method; the rest materials are frozen and stored at-80 ℃ for later use.
Fourth, virus inactivation titer calculation method
Virus inactivation titer method virus infection titer can be calculated by end point dilution or plaque method to determine virus titer:
(I) calculation of Virus infection Titers by endpoint dilution
At half The Cell Infectious Dose (TCID)50) Representation, TCID50The logarithmic value of (c) is calculated as follows:
TCID50the ratio of logarithm of lesion rate to logarithm of dilution of group of 50% + distance
The group with the lesion rate higher than 50 percent refers to the lowest group with the lesion rate higher than 50 percent, and is referred to as the group higher than 50 percent for short; the group with the lesion rate lower than 50% refers to the highest group with the lesion rate lower than 50%, and is referred to as the group with the lesion rate lower than 50% for short;
the specific calculation method is as follows:
1) calculating the cell morbidity rate: counting the number of holes with and without cytopathic effect of samples with different dilutions on a culture plate, and then respectively calculating the cumulative total value of ' cytopathic effect (-) ' and ' cytopathic effect (+); when the 'cytopathic (-)' accumulation value is calculated, accumulation is carried out from a dilution low sample group to a dilution high sample group; the accumulation value of the cytopathic (+) "is opposite, and the accumulation is carried out from a high-dilution sample group to a low-dilution sample group;
dividing the cumulative total value of 'cytopathy (+)' of each dilution sample group by the sum of the cumulative total value of 'cytopathy (-)' and 'cytopathy (+)' of the dilution sample group to obtain the lesion ratio, and obtaining the lesion rate (%);
2) calculating a distance proportion: the distance ratio can be calculated as follows:
(II) calculation of titer of viral infection by plaque assay
Plaque-associated viral infectious titer, expressed as number of plaque-forming units (pfu), abbreviated as plaque number; the counting method is the same as viable bacteria culture counting technology (see disinfection technical Specification (2002 edition)' 2.1.1.3);
the amount of virus per mL of test sample was calculated (pfu/mL) as the average plaque number per plate x dilution.
(3) Calculation of the mean log inactivation value
The average log inactivation value was calculated as follows: mean virus infection Titer (TCID) of positive (virus) control group50Or pfu) is N0Mean viral infectious Titer (TCID) in test (Disinfection) groups50Or pfu) is Nx；
To obtain: mean log N of inactivationo－log Nx。
Fifth, the appraisal test of the dilution method to remove the residual disinfectant
Test grouping:
group 1: disinfectant + virus suspension → inoculation culture
Observing whether the tested disinfectant has inactivation or inhibition effect on the virus and has no influence on the normal growth of cells;
group 2: (disinfectant + viral suspension) + dilution method → inoculation culture
Observing whether the virus can recover the infection effect on the cells after the residual medicine is removed;
group 3: viral suspension + dilution method → inoculation culture
Observing whether the virus titer is affected by the drug removal treatment;
group 4: (disinfectant + dilution method) + viral suspension → inoculation culture
Group 5: viral suspension → inoculation culture
Observing whether the virus grows normally or not, and taking the result as a positive control value;
group 6: cells not inoculated with virus → culture
Observing whether the cell growth is normal;
(II) quantitative operation procedure of virus suspension
Preparation of virus suspension: virus liquid and organic interferent 1:1, mixing;
the disinfectant is used as a stock solution according to the product specification;
1) group 1: sucking 0.8mL of disinfectant into a test tube, putting the test tube in a water bath with the temperature of 20 +/-1 ℃ for 5min, sucking 0.2mL of virus suspension, and uniformly mixing; sucking the final sample liquid (or serial diluted sample liquid by using proper diluent harmless to virus) according to the specified amount of the test until the test specified inactivation time is reached, and carrying out subsequent virus titer determination;
2) group 2: 0.8mL of disinfectant is absorbed into a test tube, and is put into a water bath with the temperature of 20 +/-1 ℃ for 5min, 0.2mL of virus suspension is absorbed and mixed evenly; after the solution acts for a specified time, the solution is subjected to drug removal treatment, a test sample is diluted by 50 times and then is subjected to ultraspeed (25000rpm, 2.5h and 4 ℃) centrifugation, supernatant is discarded, and the cell maintenance solution is used for diluting and uniformly mixing the solution to 1mL so as to remove residual disinfectant; taking the final sample (or serial diluted sample solution by using appropriate virus-harmless diluent solution) according to the specified amount of the test, and carrying out subsequent virus titer determination;
3) group 3: sucking 0.1mL of virus suspension, adding 0.9mL of cell basic culture solution, and performing drug removal treatment; diluting the test sample by 50 times, then centrifuging at an overspeed (25000rpm, 2.5h, 4 ℃), discarding the supernatant, diluting the supernatant with a cell maintenance solution, and uniformly mixing to 1mL to remove the residual disinfectant; taking the final sample (or serial diluted sample solution by using appropriate virus-harmless diluent solution) according to the specified amount of the test, and carrying out subsequent virus titer determination;
4) group 4: 0.8mL of disinfectant is absorbed in a test tube, the test tube is placed in a water bath with the temperature of 20 +/-1 ℃ for 5min, the solution is subjected to drug removal treatment, a test sample is diluted by 50 times and then centrifuged at an overspeed (25000rpm, 2.5h and 4 ℃), supernatant is discarded, and the test sample is diluted by cell maintenance solution and mixed uniformly to 0.8mL so as to remove residual disinfectant; sucking 0.2mL of virus suspension, mixing uniformly, sucking a final sample (or serial diluted sample solution by using appropriate virus-harmless diluent) according to a specified amount of a test, and carrying out subsequent virus titer determination;
5) group 5: sucking 0.2mL of virus suspension, adding 0.8mL of cell maintenance liquid, and not adding a disinfectant or performing any drug removal treatment; according to the specified amount of the test, sucking the virus suspension (or serial dilution of the virus-harmless dilution solution) and carrying out subsequent virus titer determination;
6) group 6: adding complete cell culture medium into the cell tubes which are not inoculated with the virus for culture.
(III) provision of evaluation
The test result meets all the following conditions, and the tested physical drug-removing method can be judged to be qualified:
1) group 1 had no test virus, or only a small growth;
2) group 2 had far more test virus growth than group 1, but significantly less test virus growth than groups 3-5;
3) the growth amounts of the test viruses of the 3 rd, 4 th and 5 th groups are similar;
4) obtaining qualified evaluation by continuous 3 times of tests;
5) the same principle and procedures can be used for testing using viral vectors, and the qualification criteria are the same.
Sixth, virus inactivation test
1. Test grouping
1) Test groups: setting proper concentration and action time groups (no less than 1 concentration and 3 action times) according to the estimation of the killing or inactivation dose of the measured disinfectant on other microorganisms, wherein the action time is designed to be no less than 30 s;
2) positive control group: deionized water is used for replacing a disinfectant, virus suspension is added according to the specified steps of a test group for testing and culturing, and whether the virus grows well is observed;
3) negative control group: using a complete culture medium without virus as a negative control, and observing whether the used culture medium is polluted or not and whether the cells grow well or not;
2. operating procedure of virus suspension quantitative inactivation test
1) Taking out the frozen test host cells from the liquid nitrogen, quickly melting the test host cells in warm water at 37 ℃, washing the test host cells twice by using a cell maintenance solution, and then transferring the test host cells into a culture bottle with 10mL of complete culture medium; observing the growth condition of the cells day by day, and when the cells grow full of a monolayer, using the cells for an inactivation test;
2) taking out the virus frozen at low temperature, thawing in water bath at 37 ℃, diluting by 10 times with cell maintenance liquid, then inoculating into a cell bottle full of monolayer cells, and placing in a 37 ℃ incubator to adsorb and grow with the cells; observing lesions day by day, and harvesting the virus when 3/4 cells have lesions; when the virus is harvested, the culture solution is taken out, the host cells are crushed by ultrasonic waves or repeated freeze thawing, the centrifugation is carried out as soon as possible, the supernatant containing the virus is subpackaged into a sterile centrifuge tube (1.5mL) according to 1.0mL per tube, and the supernatant is frozen and stored at minus 80 ℃ for later use;
3) taking a disinfectant to be detected, and placing the disinfectant in a water bath at 20 +/-1 ℃ for later use;
4) mixing 100 μ l of organic interfering substance with 100 μ l of virus stock solution, acting in water bath at 20 + -1 deg.C for 5min, adding 0.8mL of disinfectant to be detected, mixing immediately and timing; acting for a specified time, and treating by using a qualified medicine removing method;
5) in the positive (virus) control group test, the disinfectant is replaced by sterile deionized water;
6) the virus titer of each group is respectively determined by adopting an end point dilution method or a plaque method;
7) the test was repeated 3 times;
8) the operation steps of the end point dilution method are as follows: firstly, carrying out 10-fold serial dilution on a sample to be titrated by using a cell maintenance culture solution, then titrating the amount of viruses remained in each dilution sample on a 96-hole culture plate, wherein each dilution is 4 holes (each hole should be filled with a monolayer of host cells), and placing for 1-2 h at 37 ℃ to ensure that the residual viruses are completely adsorbed on the cells; taking out the culture plate, and replacing the cell maintenance culture solution; continuously placing into carbon dioxide incubator (37 deg.C, 5% CO)2) Culturing, observing cytopathic effect under microscope day by day, continuously observing for 3d, and observing and recording cytopathic effect;
calculation of virus infection titer by end point dilution: at half The Cell Infectious Dose (TCID)50) And (4) showing.
3. Provisions for evaluation
1) The virus inactivation test can be used for evaluating the virus inactivation effect of the chemical disinfectant for medical instruments, tableware, object surfaces and skin; the inactivation titer of the virus should reach 4 log values;
2) under normal conditions, the average inactivation log value of 3 times of tests is more than or equal to 4.00, and the laboratory test for disinfecting virus pollutants is judged to be qualified; meanwhile, the titer of the positive control group virus is between 5 and 7.
Seventh, experimental results
PV-I inactivation dilution method:
1. identification test for removing residual disinfectant by PV-I inactivation dilution method
Group (1) mean viral Titer (TCID)50) Has a log value of 0.82, mean viral Titer (TCID) of group (2)50) The logarithmic value of (1) is 1.58, and the mean viral Titers (TCID) of the groups (3), (4) and (5)50) Log values of (a) were similar, the error rate between three groups was 3.86%, and the cells in group (6) grew well (table 1):
TABLE 1 results of the residual disinfectant neutralization dilution test
2. PV-I inactivation Effect
3 repeated tests show that: the disinfectant is as such, and the average inactivation log values of 0.5min, 1min, 1.5min on PV-I are all >4.00 at the test temperature of 20 ℃. + -. 1 ℃ (Table 2):
TABLE 2 inactivation of PV-I by disinfectant test results
Cell control: the cells grew well.
EV71 virus inactivation assay:
1. neutralizer identification test in EV71 virus inactivation test
Group (1) mean viral Titer (TCID)50) Has a logarithmic value of<0.5, group (2) mean viral Titer (TCID)50) Has a logarithmic value of 1.00, and the mean viral Titers (TCID) of the groups (3), (4) and (5)50) Have similar log values, three groupsThe inter-error rate was 0.73%; (6) cell growth was good (table 3):
TABLE 3 results of the residual disinfectant neutralization dilution test
2. EV71 virus inactivation effect
3 repeated tests show that: at test temperatures of 19 ℃ to 21 ℃, the disinfectant is intact, 1min, 2min and 3min, and the average inactivation log values of EV71 virus are all >4.00 (Table 4):
TABLE 4 test results of the effect of disinfectant on inactivation of EV71 virus
Cell control: the cells grew well.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the technical scope of the present invention, and those skilled in the art may make modifications and variations within the spirit of the present invention, and all modifications, equivalents and modifications of the above embodiments according to the technical spirit of the present invention are within the scope of the present invention.
Claims (6)
1. A method for removing residual disinfectant in a virus inactivation test is characterized by comprising the following steps:
1) the preparation method comprises the following steps: preparing a virus suspension;
2) removing: firstly, absorbing a disinfectant into a test tube, carrying out drug removal treatment after water bath, then carrying out 30-50 times dilution and then ultracentrifugation, then discarding supernatant, and finally adding a cell maintenance solution for dilution and uniform mixing to remove residual disinfectant;
3) a detection step: the virus suspension is added and mixed evenly for virus titer determination.
2. The method for removing residual disinfectant in virus inactivation test according to claim 1, wherein the water bath temperature in step 2) is 20 ℃ ± 1 ℃ and the time is 5min, and the centrifugation conditions are as follows: the rotating speed is 10000rpm to 25000rpm, the centrifugation time is 1h to 2.5h, and the temperature is 4 ℃.
3. The method for removing residual disinfectant in virus inactivation test according to claim 1, wherein the volume of the cell maintenance solution added in step 2) after dilution and mixing is 1mL to 2 mL.
4. The method of claim 1, wherein the detection step of step 3) is performed by calculating virus infection titer by end point dilution or plaque assay to determine virus titer.
5. The method for removing residual disinfectant in virus inactivation test according to claim 1, wherein the preparing the virus suspension of step 1) comprises the following steps:
(1) taking frozen host cells, quickly melting the frozen host cells in warm water at 37 ℃, transplanting the frozen host cells into a cell tube of a cell maintenance liquid by using a capillary pipette, blowing and sucking for 4-7 times, uniformly mixing the cells and the cell maintenance liquid, then centrifuging at a high speed, and removing supernatant; then adding cell maintenance liquid, blowing and sucking for several times to uniformly mix, centrifuging again, and transferring into a culture bottle with complete culture medium;
(2) taking virus seeds frozen at low temperature, thawing in water bath at 37 ℃, diluting by 10 times with cell maintenance liquid, and inoculating into the culture bottle in the step (1); then placing the mixture in a 37 ℃ incubator for subsequent virus harvesting;
(3) breaking host cells by adopting an ultrasonic method or a repeated freeze-thaw method in an ice bath condition to release viruses in a culture solution containing the viruses and the host cells; then centrifuging and removing the precipitate, and taking supernatant, namely virus liquid; 1.0mL of the solution is respectively filled into a sterile centrifuge tube with the volume of 1.5mL, and is frozen and preserved at minus 80 ℃;
(4) determining the virus titer by adopting 1 virus liquid according to a virus titer determination method;
(5) when in use, the virus liquid and the organic interferent are mixed at a ratio of 1:1 to prepare virus suspension.
6. The method for removing residual disinfectant in virus inactivation test as claimed in claim 5, wherein the centrifugation condition of step (1) is 3000rpm for 3 min; the centrifugation condition of the step (3) is 6000rpm, and the time is 15 min.
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| CN114480310A (en) * | 2022-01-28 | 2022-05-13 | 内蒙金属材料研究所 | Packaging process and inactivation method for infectious virus liquid |
| CN114480310B (en) * | 2022-01-28 | 2023-07-14 | 内蒙金属材料研究所 | Packaging process and inactivating method for infectious virus liquid |
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