CN113528705A

CN113528705A - Method for detecting inactivation efficiency of surface virus and application thereof

Info

Publication number: CN113528705A
Application number: CN202110714437.1A
Authority: CN
Inventors: 林弘毅
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-06-25
Filing date: 2021-06-25
Publication date: 2021-10-22
Also published as: CN115521994A

Abstract

The invention discloses a method for detecting inactivation efficiency of surface viruses and application thereof, comprising the following steps: (1) determining the type of the carrier; (2) judging the effectiveness of the experiment; (3) detecting inactivation efficiency; (4) and judging the effectiveness and inactivation efficiency of the disinfection product or the disinfection operation to be detected. The detection method can simulate the multi-step virus disinfection operation flow or the use condition of virus inactivation products in the form of combined medication, and simultaneously considers the virus residual proportion of the vector, thereby realizing the quantification of the disinfection capability of the products. Meanwhile, the test result of the detection method is closer to the reality, especially for the multi-step disinfection steps, the accuracy is higher, a quantitative index for measuring the material disinfection difficulty is provided, and the detection method has extremely important significance for the multi-step disinfection operation scheme and the development of antiviral products.

Description

Method for detecting inactivation efficiency of surface virus and application thereof

Technical Field

The invention belongs to the field of public health, and particularly relates to a method for detecting inactivation efficiency of surface viruses and application thereof.

Background

Along with the continuous improvement of the economy and the living standard of people, the consciousness of people on medical treatment and health is gradually improved, and various disinfection and sterilization products gradually enter every family. Meanwhile, the national importance on medical treatment and health also enables enterprises to consciously improve the self sanitary standards, and various environment disinfection operation flows and product disinfection operation flows are established so as to ensure the cleanliness of production environments and products. At present, most disinfection procedures are mainly aimed at pursuing better inactivation effect, so virus inactivation and disinfection operations are often carried out by using medicaments with high cytotoxicity and large dose, but the use of medicaments with large dose can bring about serious disinfectant residues. In particular, residues of acidic or toxic disinfectants can cause corrosion of products, instruments and equipment, shorten the service life of the equipment, or threaten the health of workers, and increase the risk of occupational diseases. Therefore, it is important to develop a multi-step disinfection protocol with high inactivation efficiency, low concentration of disinfectant used, and low toxicity.

The multi-step virus inactivation method is mainly applied to various scenes such as food packaging disinfection, commodity integral disinfection, work and production environment disinfection and the like. For example, in the food packaging process, the manufacturing enterprise may use a low concentration drug to soak the surface of the package multiple times to achieve a corresponding inactivation efficacy, while reducing drug residue in the product to meet food production safety standards. In the process of environmental disinfection, disinfection personnel can spray low-concentration medicines for many times, so that the harm of the medicines to the health of the personnel is reduced. Therefore, the development of a multi-step sterilization process is particularly important to the manufacturing operation.

However, in the related art, an effective method and a corresponding algorithm capable of accurately determining the inactivation efficiency and the overall inactivation efficiency of each step in the multi-step disinfection scheme are not available, and effective data cannot be obtained, so that the development of the multi-step disinfection scheme is greatly limited. Therefore, developing a set of method for detecting the inactivation efficiency of surface viruses, which is complete in algorithm and applicable to single-step and multi-step disinfection schemes, is of great significance in promoting the perfection and development of disinfection operation systems.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a method for detecting the virus inactivation efficiency of a disinfection product or disinfection operation, which can simulate the use condition of a multi-step virus disinfection operation flow or an antiviral product in a combined drug mode, simultaneously considers the virus residual ratio of the vector, and quickly calculates the step-by-step (single step) inactivation efficiency and the total inactivation efficiency of the flow by using the residual ratio, thereby realizing the quantification of the product disinfection capacity and having extremely important significance for the development of a multi-step disinfection operation scheme and the antiviral product.

In a first aspect of the invention, there is provided a method of detecting the virus-inactivating effect of a sterilized product or sterilization work, comprising the steps of:

(1) determining the type of the carrier;

(2) judging the effectiveness of the experiment;

(3) detecting inactivation efficiency;

(4) and judging the effectiveness and inactivation efficiency of the disinfection product or the disinfection operation to be detected.

The inventor finds that the existing detection means is difficult to simulate a multi-step disinfection operation flow or a disinfection product needing multiple times of medication, particularly the inactivation efficiency of each step in the multi-step disinfection operation is difficult to determine, and if the detection means is applied with a strong algorithm, the problems that the error of a simulation test result from the actual situation is large, the inactivation efficiency of multiple times of medication is difficult to compare with the inactivation efficiency of single time of medication and the like can be caused. In addition, in the detection process, carrier containers made of different materials have different virus residual ratios, which affect the actual inactivation efficiency of the drugs and the corresponding drug application methods, and currently, there is no effective means for detecting the virus residual ratio on the surfaces of the materials made of different materials. Based on the above problems, the inventors developed a method for detecting the virus inactivation efficiency of a disinfection product or disinfection operation, which can simulate the use of a multi-step virus disinfection operation process or an antiviral product in a combined drug form, and simultaneously can obtain the virus residual ratio of the vector, and rapidly calculate the step-by-step inactivation efficiency and the total inactivation efficiency of the process by using the residual ratio. In addition, the method can also know the virus residual ratio among each step, so that the method can be used as an important index to judge the difficulty of cleaning and sterilizing materials, and can further effectively know the disinfection operation mode suitable for the method.

According to a first aspect of the invention, in some embodiments of the invention, the method comprises the steps of:

(1) and (3) preparation test:

the preliminary test includes: the method comprises three parts of titer identification of the virus to be detected, cytotoxicity identification of the product and effective neutralization test of the product, and is provided with a positive control and a negative control.

Before all tests are started, carriers with proper sizes are selected according to the to-be-detected disinfection products or disinfection process usage (the selection standard is shown in the specification table 1), and the number of disinfection steps is determined. The number of test wells to be tested and the dilution procedure are shown in Table 2.

The titer of the virus to be detected is identified:

the virus suspension of the test virus was diluted 10-fold as shown in Table 2, and 100. mu.L of each dilution was inoculated on a cell plate, and 4 wells were inoculated in each dilution. After inoculation, the cell plates were placed in a carbon dioxide incubator (37 ℃, 5% CO)₂) Culturing for 3-5 days. Half of the tissue culture infectious dose (TCID50) of the test virus after culture was tested using methods routine in the art to ensure that its TCID50 log/ml was greater than 7.

② identifying cytotoxicity:

according to the table 1, a carrier with a suitable size was selected, a corresponding volume of cell maintenance solution was dropped on the carrier, and the carrier was allowed to stand and air-dried. After air drying, applying the disinfection product to be tested with the corresponding volume on the carrier by the corresponding action method, uniformly mixing, diluting according to the corresponding method, inoculating 100 mu L of each dilution on a cell plate, and inoculating 4 holes in each dilution. Wherein, if the disinfection product to be detected can not be diluted in the cell maintenance liquid to remove the cytotoxicity, the disinfection product can be uniformly mixed, filtered in Sephadex G25 gel and then diluted according to the method shown in Table 2. After inoculation, the cell plate is placed into a carbon dioxide incubator (37 ℃, 5% CO2) to be cultured for 3-5 days, and the influence of the disinfection product to be detected on cell growth is observed.

Efficient neutralization test

According to the table 1, a carrier with a suitable size was selected, a corresponding volume of cell maintenance solution was dropped on the carrier, and the carrier was allowed to stand and air-dried. After air drying, the corresponding volume of the disinfection product to be tested was applied to the carrier by the corresponding action method, after mixing well, 1200 μ L of the mixed solution (cell maintenance solution + disinfection product to be tested) was taken, after gel filtration by Sephadex G25, diluted according to the method shown in Table 2. After dilution, 100. mu.L of each dilution was seeded onto cell plates4 wells were inoculated for each dilution. Wherein, if the disinfection product to be tested is known to be non-cytotoxic or can be detoxified by dilution in a cell maintenance solution, the product can be directly diluted and seeded on a cell plate as shown in Table 2 without using Sephadex G25 gel filtration. Then, 100. mu.L of a viral suspension having a logarithmic value of TCID50 of 2 to 3 was added to each well, and the mixture was placed in a carbon dioxide incubator (37 ℃ C., 5% CO)₂) And culturing for 3-5 days, observing whether the to-be-detected disinfection product has influence on cell growth, and if each cell is infected by virus, indicating that the to-be-detected disinfection product is effectively neutralized.

Positive control and negative control:

positive control: according to the table 1, a vector with a suitable size is selected, a corresponding volume of virus suspension is added dropwise to the vector, and the vector is allowed to stand and air-dry. Air drying, applying cell maintenance solution with corresponding volume on the carrier by corresponding action method, mixing, inoculating 100 μ L onto cell plate, inoculating 4 holes, placing the cell plate into carbon dioxide incubator (37 deg.C, 5% CO)₂) And (4) culturing for 3-5 days, if each hole of cells is infected by the virus, determining that the positive control passes, collecting experimental data, and if the cells are not infected by the virus, restarting the experiment.

Negative control: according to the table 1, a carrier with a suitable size was selected, a corresponding volume of cell maintenance solution was dropped on the carrier, and the carrier was allowed to stand and air-dried. Air drying, applying cell maintenance solution with corresponding volume on the carrier by corresponding action method, mixing, inoculating 100 μ L onto cell plate, inoculating 4 holes, placing the cell plate into carbon dioxide incubator (37 deg.C, 5% CO)₂) And (3) culturing for 3-5 days, if the cells in each hole have no abnormality and grow normally, determining that the negative control passes, collecting experimental data, and if any abnormal condition or virus infection occurs, restarting the experiment.

(2) And (3) inactivation efficacy detection:

the inactivation efficacy assay comprises: the method comprises three parts of titer identification of the virus to be detected, inactivation efficiency test and vector virus amount detection, and is provided with a positive control and a negative control.

Identification of titer (Cv) of virus to be detected:

the test method is the same as that of the step (1).

② inactivation efficiency test:

according to the table 1, a vector with a suitable size is selected, a corresponding volume of virus suspension is added dropwise to the vector, and the vector is allowed to stand and air-dry. After air drying, applying the disinfection product to be tested with the corresponding volume on the carrier by a corresponding action method, uniformly mixing, and standing for full reaction. Then, 1200. mu.L of the mixed solution (virus suspension + disinfection product to be tested) was gel-filtered in Sephadex G25, and after dilution according to Table 1 using an appropriate dilution method, 100. mu.L of each dilution was inoculated on a cell plate, and each dilution was performed, wherein, if the disinfection process flow is a multi-step disinfection method, after the first inactivation efficacy test, the carrier was taken out with tweezers, and placed in a sterile petri dish prepared in advance, and the above-described inactivation efficacy test operation was repeated to perform the second disinfection inactivation efficacy test. The number of repetitions is determined according to the number of sterilizations of the sterilization process, but the number of applications is not more than three in principle. If the sterilization product to be tested is known to be non-cytotoxic or can be rendered cytotoxic by dilution in a cell maintenance solution, it is directly diluted and seeded on a cell plate as described above without gel filtration using Sephadex G25, placed in a carbon dioxide incubator (37 ℃, 5% CO)₂) And culturing for 3-5 days, observing the test result, and recording the disinfection result of the nth time as Ct1-Ctn according to the disinfection times in the disinfection operation flow.

Thirdly, detecting the amount of the vector viruses:

according to the table 1, a vector with a suitable size is selected, a corresponding volume of virus suspension is added dropwise to the vector, and the vector is allowed to stand and air-dry. After air drying, the corresponding volume of cell maintenance solution was applied to the carrier by the corresponding method of action, mixed and diluted according to the method of table 1. After dilution, 100 μ L of the mixture (virus suspension + cell maintenance) was plated onto the cell plate, 4 wells for each dilution.

Wherein, if the sterilization process is a multi-step sterilization method, after the first time of the vector virus amount detection, the vector is taken out by using tweezers and put into a prepared sterile culture dish, and the operation of the vector virus amount detection is repeated to carry out the second time of the vector virus amount detection after the sterilization. The number of repetitions is determined according to the number of sterilizations of the sterilization process, but the number of applications is not more than three in principle. The results of the nth sterilization are recorded as Cc1-Ccn based on the number of sterilizations in the sterilization workflow.

Positive control and negative control:

the positive control and the negative control in the step (1) are the same.

The tests are repeated three times at different time points, and the result of each test is calculated independently to ensure the accuracy of the test.

(3) And (3) statistics and analysis:

after the experimental results are obtained, the surface inactivation efficiency and unit area inactivation efficiency of the disinfection product to be tested or the disinfection process are calculated by using the following formula, and the final result is obtained.

The calculation of the inactivation potency comprises three steps: calculating the stepwise inactivation efficiency, calculating the total inactivation efficiency and converting the unit area inactivation efficiency, and through the three steps, the titer value of the inactivated virus per ml, the reduction value of the effective virus infection unit per square centimeter and the residual ratio of the tested virus of the disinfection product or the disinfection process to be tested can be obtained. The algorithm is divided into a one-step test calculation formula and a multi-step (n steps) calculation formula according to the number of steps (disinfection times) of the disinfection process.

Wherein all valid data for inactivation potency test (Ct) and vector virus amount test (Cc), including TCID50 titer values for each step (per sterilization) should be accurately recorded and formatted as 10 before being substituted into the algorithmⁿAnd (4) format.

The specific algorithm is as follows:

calculating stepwise inactivation efficiency:

the formula for titer per ml of inactivated virus is:

R_logn＝log(10Ct(n-1)×R_ratio)-log(10Ctn)；

wherein in the above formula, R_ratioCalculating the surface virus residual ratio of the nth step by the following formula:

ccn is the test result of step n for vector virus amount;

cc (n-1) is the test result of the virus quantity of the vector in the step (n-1);

ctn is the result of the inactivation efficacy test in the nth step;

ct (n-1) is the inactivation efficiency test result of the step (n-1);

R_lognthe titer value of the inactivated virus per ml in the nth step is shown.

Calculating the total inactivation efficiency:

the test result (Ctn) obtained in the last step (the last sterilization in the sterilization process) of the inactivation efficacy test and the test result (Cc1) obtained in the first step (the first sterilization in the sterilization process) of the vector virus amount test are substituted into the following algorithm, so that the overall inactivation efficacy can be obtained.

For ease of understanding, n in the present step formula represents the number of steps (number of sterilizations) of the last step.

The general titer value formula of the inactivated virus per ml of the disinfection product or the disinfection process to be tested is as follows:

R_logt＝log(10Cc1)-log(10Ctn)；

wherein, in the formula, the first and second groups,

cc1 is the result of the test for the amount of the vector virus in step 1;

ctn is the result of the final inactivation efficiency test;

R_logtthe titer value of the total inactivated virus per ml is shown.

③ conversion of inactivation efficiency per unit area:

in this step, the inactivation efficiency per unit area was calculated by substituting the following formula according to the specific method of action (see Table 1). Wherein n in the formula of this step represents the number of steps (number of times of sterilization) at the nth step (nth sterilization).

(a) When the action method is a single-side soaking method or an integral soaking action method:

the formula for the overall average reduction in effective infectious units of virus per square centimeter is:

the formula of the average reduction value of effective infection units of the virus per square centimeter in the nth step is as follows:

wherein, in the formula, the first and second groups,

cc1 is the result of the test for the amount of the vector virus in step 1;

R_ratiocalculating the surface virus residual ratio of the nth step by the formula of the first step;

ct (n-1) is the inactivation efficiency test result of the step (n-1);

ctn is the inactivation efficiency test result of the last step/nth step;

Rt/cm²reduction of the overall mean effective infectious units per square centimeter of virus

Rn/cm²The reduction in effective viral infection units per square centimeter is averaged for step n.

(b) When the action method is a spraying action method:

judging the standard:

after the calculation is carried out by using the algorithm, the actual efficacy of the disinfection product to be detected or the disinfection process is judged according to the calculation result.

Wherein the content of the first and second substances,residual ratio (R) to surface virus_ratio) If the virus residual ratio is less than 3%, the virus residual ratio is extremely low residual, which indicates that the surface viscosity is extremely low and the inactivation efficiency is extremely high; if the ratio of virus residues is 3-10%, the virus residues are low-degree residues, which indicates that the surface viscosity is low and the inactivation efficiency is high; if the ratio of virus residues is 10-20%, the virus residues are moderate residues, which indicates that the surface viscosity is in the inactivation effect; if the ratio of virus residues is more than 20%, the virus residues are severe residues, which indicates that the surface viscosity is high and the inactivation efficiency is low. The residual ratio of the virus obtained in the above examples can be used as an index of the virus elution efficiency of the material under the sterilization method, and a larger residual ratio indicates that the virus is more likely to adhere to the carrier and the material is less likely to be washed, or the sterilization method has a weaker elution efficiency against the virus on the carrier. In actual operation, the residual ratio should be less than 0.43, and will be influenced by the carrier material, virus suspension titer, temperature, sterilization procedure, etc., and the operation should be performed as much as practical during the test. Applying the inactivation log (R) in calculating the actual disinfection efficacy log_logt、R_logn) The log of the test's effective neutralization, or the log of the minimum dilution (effective neutralization is preferred and the minimum dilution is used without this data) is subtracted.

The flow chart of the detection method in the invention is shown in the attached figure 1 in the specification.

In the invention, 4 residual proportion levels are set, virus inactivation efficiencies of different materials can be quantitatively and qualitatively embodied, so that a person skilled in the art can quantitatively judge the influence of physical properties of different materials on the inactivation efficiencies, and a new dimension is given to compare whether the disinfection step is suitable for the materials. In addition, the method provided by the invention has the advantages that the test result is closer to the actual result, especially for multi-step disinfection steps, the accuracy is higher, and the virus inactivation amount of each step can be quantitatively measured, so that the comprehensive evaluation of the virus inactivation efficiency in a novel multi-step disinfection operation flow or the development process of a disinfection product is facilitated. Furthermore, the present invention also provides a comparative method of testing the virus inactivation efficacy in steps (single step) or in total in a multi-step sterilization process, thereby allowing the multi-step sterilization process using low concentrations, low efficacy or low cytotoxic agents to be compared to a single step sterilization process using high concentrations, high efficacy or high cytotoxic agents.

According to the first aspect of the present invention, in some embodiments of the present invention, the method for determining the validity of the experiment in step (2) is: performing virus titer identification, cytotoxicity identification and effective neutralization test, and if the logarithm value of each milliliter of TCID50 of the virus titer identification is more than 7 and the positive control and the negative control are not abnormal, judging that the experimental result is effective; if not, the method is invalid.

According to the first aspect of the present invention, in some embodiments of the present invention, the inactivation potency assay in step (3) comprises viral titer identification, an inactivation potency test and a vector viral load assay.

According to the first aspect of the present invention, in some embodiments of the present invention, the determination method of the effectiveness of the disinfection product or the disinfection operation to be tested in the step (4) is:

calculating the total inactivation efficiency and the single-step inactivation efficiency according to the inactivation efficiency detection result;

if the log value of the total inactivation efficiency is more than or equal to 4, the log value of the single-step inactivation efficiency is more than or equal to 1, and after repeated tests, when the difference value between the log values of the total inactivation efficiency and the difference value between the log values of the single-step inactivation efficiency are both less than 0.5, the disinfection product to be tested or the disinfection operation is effective;

if not, the method is invalid.

According to a first aspect of the invention, in some embodiments of the invention, the algorithmic formula of the single-step inactivation efficacy is:

R_logn＝log(10Ct(n-1)×R_ratio)-log(10Ctn)；

wherein R is_ratioCalculating the surface virus residual ratio of the nth step by the following formula:

ccn is the test result of step n for vector virus amount; cc (n-1) is the n-1 th vector virus measurementTesting results; ctn is the result of the inactivation efficacy test in the nth step; ct (n-1) is the inactivation efficiency test result of the step (n-1); r_lognThe titer value of the inactivated virus per ml in the nth step is;

the algorithm formula for the overall inactivation efficiency is:

R_logt＝log(10Cc1)-log(10Ctn)；

wherein Cc1 is the result of the test on the viral load of the vector in the step 1; ctn is the result of the final inactivation efficiency test; r_logtThe titer value of the total inactivated virus per ml is shown.

According to a first aspect of the present invention, in some embodiments of the present invention, the method of inactivating the sterilization product or sterilization job to be tested in step (4) is:

calculating the virus residual ratio according to the inactivation efficiency detection result, wherein the calculation formula is as follows:

wherein Ccn is the test result of the vector virus amount in the step n; cc (n-1) is the test result of the virus quantity of the vector in the step (n-1);

if the virus residual ratio is less than 3%, the virus residual ratio is extremely low residual, the surface viscosity is extremely low, and the inactivation efficiency is extremely high;

if the ratio of virus residues is 3-10%, the virus residues are low-degree residues, the surface viscosity is low, and the inactivation efficiency is high;

if the ratio of virus residues is 10-20%, the virus residues are moderate residues, the surface viscosity is moderate, and the inactivation efficiency is moderate;

if the ratio of virus residues is more than 20%, the virus residues are severe residues, the surface viscosity is high, and the inactivation efficiency is low.

According to a first aspect of the present invention, in some embodiments of the present invention, the surface of the carrier in step (1) is provided with L-shaped snaps. The buckle is perpendicular to the surface of the carrier, and the preparation material of the carrier comprises glass, stainless steel and plastic.

Of course, those skilled in the art can reasonably select an appropriate material as the carrier material according to the actual use requirement.

In some preferred embodiments of the present invention, the carrier in the present invention is any one of (1) or (2):

(1) circular 20cm as shown in FIG. 2²And (3) a carrier. The method specifically comprises the following steps: the carrier is a circular structure with a diameter of 50.5mm and a thickness of 0.5mm, and the area is 20cm²The outer side of the carrier is provided with an L-shaped clamp position with the length of 2mm, the width of 2mm and the protrusion of 2mm to one side, the surface of the carrier is provided with marking lines, the surface of the carrier is equally divided into 20 grids with each grid being 1cm²。

(2) Square 25cm as shown in figure 3²And (3) a carrier. The method specifically comprises the following steps: the carrier is a square structure with side length of 5cm and thickness of 0.5mm, and area of 25cm²The outer side of the carrier is provided with an L-shaped clamp position with the length of 20mm and the width of 2mm and the protrusion of 2mm to one side, the surface of the carrier is provided with marking lines, the surface of the carrier is equally divided into 25 grids with 1cm of each grid²。

In the invention, the process of dripping the carrier and the reagent can ensure that the virus to be tested is equally and uniformly distributed on the carrier, thereby avoiding false positive caused by overhigh concentration due to uneven distribution and ensuring that the test scheme is more accurate and reliable.

According to a first aspect of the invention, in some embodiments of the invention, the type of carrier in step (1) is determined according to a disinfection mode of action, wherein the disinfection mode of action comprises single-sided immersion, whole immersion and spraying.

In some preferred embodiments of the invention, the carrier type corresponds to the mode of disinfection action in the following manner:

if the disinfection action mode is single-side immersion or integral immersion, the carrier type is a round carrier;

if the disinfection mode is spraying, the carrier is a square carrier.

In a second aspect of the invention, there is provided the use of a method according to the first aspect of the invention in antiviral product screening.

In a third aspect of the invention, there is provided the use of a method according to the first aspect of the invention for the quantitative determination of the ease of cleaning of a material.

The invention has the beneficial effects that:

1. the detection method can simulate the multi-step virus disinfection operation flow or the use condition of virus inactivation products in the form of combined medication, simultaneously considers the virus residual proportion of the vector, and quickly calculates the step-by-step (single step) inactivation efficiency and the total inactivation efficiency of the flow by utilizing the residual proportion, thereby realizing the quantification of the product disinfection capacity and having very important significance for the development of multi-step disinfection operation schemes and antiviral products.

2. The detection method of the invention can quantitatively and qualitatively reflect the virus inactivation efficiency among different materials, so that a person skilled in the art can quantitatively judge the influence of physical properties of different materials on the inactivation efficiency, and a new dimension is provided for comparing whether the disinfection step is suitable for the materials. In addition, the method provided by the invention has the advantages that the test result is closer to the actual result, especially for a plurality of disinfection steps, the accuracy is higher, the virus inactivation amount of each step can be quantitatively measured, and the minimum sensitivity is 10¹A 1.0/mL or per milliliter titer value facilitates a comprehensive assessment of its virus inactivation efficacy during a new multi-step sterilization workflow or development of a sterilization product.

3. The test method of the present invention can also be used as a comparative method to test the virus inactivation efficiency in steps (single step) or in total in a multi-step sterilization process, thereby allowing the multi-step sterilization process using low concentrations, low efficiency or low cytotoxic agents to be compared to the single step sterilization process using high concentrations, high efficiency or high cytotoxic agents.

Drawings

FIG. 1 is a flow chart of a detection method in an embodiment of the invention;

FIG. 2 shows a 20cm circle in an embodiment of the present invention²The carrier is shown in three views, wherein 1 represents a mark line, 2 represents the outer side of the carrier, and 3 represents a buckle;

FIG. 3 shows a square 25cm in an embodiment of the present invention²And a carrier three-view, wherein 1 represents the outer side of the carrier, 2 represents a mark line, and 3 represents a buckle.

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.

Construction of method for detecting inactivation efficiency of surface virus

The method for detecting the inactivation efficiency of the surface virus in the embodiment can be carried out by three parts:

(1) and (3) preparation test:

Before all tests are started, carriers with proper sizes are selected according to the to-be-detected disinfection products or disinfection process usage (the selection standard is shown in table 1), the number of disinfection steps is determined, and equipment (such as a water bath, a sterile culture dish, tweezers and the like) required by experiments is prepared. Meanwhile, corresponding cells are cultured according to virus strains used for testing, and subculture cells are cultured to monolayer cells for later use according to a conventional operation method in the field.

TABLE 1 Carrier types, methods of action and specific operating parameters thereof

Wherein, the circle is 20cm²The vector is shown in FIG. 2. The method specifically comprises the following steps: the carrier is a circular structure with a diameter of 50.5mm and a thickness of 0.5mm, and the area is 20cm²The outer side of the carrier is provided with a carrier with the length of 2mm and the width of 2mm and facing to one sideProtruding 2mm 'L' shape screens, the carrier surface is equipped with the mark line, equally divide the carrier surface into 20 check, 1cm per check²。

Square 25cm²The vector is shown in FIG. 3. The method specifically comprises the following steps: the carrier is a square structure with side length of 5cm and thickness of 0.5mm, and area of 25cm²The outer side of the carrier is provided with an L-shaped clamp position with the length of 20mm and the width of 2mm and the protrusion of 2mm to one side, the surface of the carrier is provided with marking lines, the surface of the carrier is equally divided into 25 grids with 1cm of each grid²。

The carrier material is not limited, and includes, but is not limited to, glass, stainless steel, plastic, and the like.

When the carrier is used, the carrier is placed in an aseptic culture dish, and the L-shaped structure arranged outside the carrier solves the problem that the carrier is difficult to take out due to the difference of liquid level pressure.

The number of test wells and their dilution procedures for the preliminary and inactivation efficacy tests are shown in table 2.

Table 2 number of test wells for preliminary test and inactivation efficacy test

The titer of the virus to be detected is identified:

② identifying cytotoxicity:

according to the table 1, a carrier with a suitable size was selected, a corresponding volume of cell maintenance solution was dropped on the carrier, and the carrier was allowed to stand and air-dried. After air drying, applying the disinfection product to be tested with corresponding volume on a carrier by corresponding action method, uniformly mixing, diluting according to corresponding method, and taking 100 mu for each dilutionL were seeded on cell plates, 4 wells for each dilution. Wherein, if the disinfection product to be detected can not be diluted in the cell maintenance liquid to remove the cytotoxicity, the disinfection product can be uniformly mixed, filtered in Sephadex G25 gel and then diluted according to the method shown in Table 2. After inoculation was complete, the cell plates were placed in a carbon dioxide incubator (37 ℃, 5% CO)₂) And (5) culturing for 3-5 days, and observing the influence of the disinfection product to be detected on the cell growth.

Efficient neutralization test

According to the table 1, a carrier with a suitable size was selected, a corresponding volume of cell maintenance solution was dropped on the carrier, and the carrier was allowed to stand and air-dried. After air drying, the corresponding volume of the disinfection product to be tested was applied to the carrier by the corresponding action method, after mixing well, 1200 μ L of the mixed solution (cell maintenance solution + disinfection product to be tested) was taken, after gel filtration by Sephadex G25, diluted according to the method shown in Table 2. After dilution, 100. mu.L of each dilution was plated on a cell plate, and 4 wells were plated for each dilution. Wherein, if the disinfection product to be tested is known to be non-cytotoxic or can be detoxified by dilution in a cell maintenance solution, the product can be directly diluted and seeded on a cell plate as shown in Table 2 without using Sephadex G25 gel filtration. Then, 100. mu.L of a viral suspension having a logarithmic value of TCID50 of 2 to 3 was added to each well, and the mixture was placed in a carbon dioxide incubator (37 ℃ C., 5% CO)₂) And culturing for 3-5 days, observing whether the to-be-detected disinfection product has influence on cell growth, and if each cell is infected by virus, indicating that the to-be-detected disinfection product is effectively neutralized.

Positive control and negative control:

(2) And (3) inactivation efficacy detection:

As with step (1), before all tests are started, carriers with appropriate sizes are selected according to the to-be-detected disinfection products or disinfection process usage (the selection standard is shown in Table 1), the number of disinfection steps is determined at the same time, and equipment (such as bathtub, sterile culture dish, tweezers and the like) required by experiments is prepared. Meanwhile, corresponding cells are cultured according to virus strains used for testing, and subculture cells are cultured to monolayer cells for later use according to a conventional operation method in the field. In addition, the virus suspension and the reagent need to be placed in an ultra-clean bench or a water bath to be at an appropriate temperature for testing.

Identification of titer (Cv) of virus to be detected:

the test method is the same as that of the step (1).

② inactivation efficiency test:

according to the table 1, a vector with a suitable size is selected, a corresponding volume of virus suspension is added dropwise to the vector, and the vector is allowed to stand and air-dry. After air drying, applying the disinfection product to be tested with the corresponding volume on the carrier by a corresponding action method, uniformly mixing, and standing for full reaction. 1200. mu.L of the mixture (viral suspension + sterile product to be tested) was then gel-filtered on Sephadex G25 and diluted according to the dilution method specified in Table 1. After dilution, 100. mu.L of each dilution was plated on a cell plate, and 10 wells were plated for each dilution.

And if the sterilization operation flow is a multi-step sterilization method, after the first inactivation efficacy test, taking out the carrier by using tweezers, putting the carrier into a prepared sterile culture dish, and repeating the operation of the inactivation efficacy test to perform the second inactivation efficacy test after sterilization. The number of repetitions is determined according to the number of sterilizations of the sterilization process, but the number of applications is not more than three in principle. If the sterilization product to be tested is known to be non-cytotoxic or can be rendered cytotoxic by dilution in a cell maintenance solution, it is directly diluted and seeded on a cell plate as described above without gel filtration using Sephadex G25, placed in a carbon dioxide incubator (37 ℃, 5% CO)₂) And culturing for 3-5 days, observing the test result, and recording the disinfection result of the nth time as Ct1-Ctn according to the disinfection times in the disinfection operation flow.

Third, detecting the virus amount of the vector (Cc):

according to the table 1, a vector with a suitable size is selected, a corresponding volume of virus suspension is added dropwise to the vector, and the vector is allowed to stand and air-dry. After air-drying, the corresponding volume of cell maintenance solution was applied to the carrier by the corresponding method of action, mixed well and diluted according to the dilution method specified in table 1. After dilution, 100 μ L of the mixture (virus suspension + cell maintenance) was plated onto the cell plate, 4 wells for each dilution.

Positive control and negative control:

the positive control and the negative control in the step (1) are the same.

(3) And (3) statistics and analysis:

The specific algorithm is as follows:

calculating stepwise inactivation efficiency:

the formula for titer per ml of inactivated virus is:

R_logn＝log(10Ct(n-1)×R_ratio)-log(10Ctn)；

ccn is the test result of step n for vector virus amount;

ctn is the result of the inactivation efficacy test in the nth step;

ct (n-1) is the inactivation efficiency test result of the step (n-1);

R_lognthe titer value of the inactivated virus per ml in the nth step is shown.

Calculating the total inactivation efficiency:

R_logt＝log(10Cc1)-log(10Ctn)；

wherein, in the formula, the first and second groups,

cc1 is the result of the test for the amount of the vector virus in step 1;

ctn is the result of the final inactivation efficiency test;

R_logtthe titer value of the total inactivated virus per ml is shown.

③ conversion of inactivation efficiency per unit area:

wherein, in the formula, the first and second groups,

cc1 is the result of the test for the amount of the vector virus in step 1;

ct (n-1) is the inactivation efficiency test result of the step (n-1);

ctn is the inactivation efficiency test result of the last step/nth step;

(b) When the action method is a spraying action method:

judging the standard:

Wherein, the ratio of virus residues (R) to surface_ratio) If the virus residual ratio is less than 3%, the virus residual ratio is extremely low residual, which indicates that the surface viscosity is extremely low and the inactivation efficiency is extremely high; if the ratio of virus residues is 3-10%, the virus residues are low-degree residues, which indicates that the surface viscosity is low and the inactivation efficiency is high; if the ratio of virus residues is 10-20%, the virus residues are moderate residues, which indicates that the surface viscosity is in the inactivation effect; if the ratio of virus residues is more than 20%, the virus residues are severe residues, which indicates that the surface viscosity is high and the inactivation efficiency is low. The ratio of virus residues obtained in the above examples can be used as the material for virus disinfectionAs an index of the elution efficiency, a larger residual ratio indicates that the virus is more likely to adhere to the carrier or that the sterilization method has a weaker elution efficiency against the virus on the carrier. In actual operation, the residual ratio should be less than 0.43, and will be influenced by the carrier material, virus suspension titer, temperature, sterilization procedure, etc., and the operation should be performed as much as practical during the test. Applying the inactivation log (R) in calculating the actual disinfection efficacy log_logt、R_logn) The log of the test's effective neutralization, or the log of the minimum dilution (effective neutralization is preferred and the minimum dilution is used without this data) is subtracted. The theoretical minimum dilution of the detection method in the above example was: the single-side immersion method is-2 (10)^-2) The spraying method is-1.3 (10)^-1.3)。

The disinfection operation flow of the whole soaking method or the inactivation efficiency of the disinfection product can be judged according to the following conditions: the log of the total inactivation potency should be equal to or greater than 4, the log of the inactivation potency at each step of the multi-step sterilization test should be equal to or greater than 1, and the log difference between the inactivation potencies from each repeat of the test is less than 0.5, only under these conditions can the sterilization process or sterilization product be judged to be effective in inactivating the virus.

Wherein, according to practical operation, the detection method in the above embodiment has a minimum sensitivity of 10¹titer/mL or titer per mL value was 1.0. The result validity judgment needs to meet the following requirements: the viral titer identification (Cv) should have a log value of TCID50 of greater than 7 per ml, and no abnormalities in both positive and negative controls.

Effect testing experiment

In this example, the detection method in the above example was compared with the detection method in the current conventional protocol (using glass carrier and wood carrier, respectively) by using the current conventional protocol (obtained by modifying the disinfection specification ws/t367-2012 and the new coronavirus evaluation standard ws/t 775-2021) as a control.

It should be noted that in order to make the two detection methods comparable, the following adjustment is made:

1. in the above embodiment, the areas are the sameThe detection method adopts a 20cm²Or 25cm²Carriers, and 20 or 25 1cm are used in the currently common protocols²Vehicle, test 20 or 25 averages.

2. The currently used protocol tests a multi-step disinfection protocol by first testing step 1 separately and then testing steps 1+2 separately twice because it does not have the ability to obtain the disinfection efficacy results of each step separately, and therefore, this method can only be used to obtain the disinfection efficacy results of step 2 (step 2 efficacy is the efficacy of step 1+2 minus the efficacy of step 1), but this method does not consider that part of the virus is washed away in step 1, and that the time between steps is long enough to inactivate part of the virus (the actual disinfection operation is carried out at intervals of 10 minutes to several hours), and therefore, the actual disinfection efficacy should be lower than the experimental value.

Effect comparison experiment 1:

the carrier material of this example is glass material, the disinfection product to be tested is hydrogen peroxide, the disinfection operation steps are 2 steps (disinfection times is 2), the detected virus is H1N1 virus (virus strain purchased from ATCC, No. VR-95) matched MDCK cell strain (used for generating H1N1 virus, cell strain purchased from ATCC, No. CCL-34), and there is no organic interference.

(1) The detection method in the above embodiment:

the detection is carried out according to the method in the embodiment, and the specific steps are as follows:

identification of virus titer: 100. mu. L H1N1 virus suspension was added to 900. mu.L of cell maintenance medium and diluted 10-fold in gradient according to Table 1 (10^-1～10^-9Nine gradients), 100 μ L of each dilution was plated on the cell plate, and 4 wells of each dilution were plated. After inoculation, the cells were incubated in a carbon dioxide incubator (37 ℃ C., 5% CO)₂) Culturing for 3-5 days.

② identifying cytotoxicity: dropping the cell maintenance solution at 20cm²On the surface of the round glass material carrier, 10 mu L of each lattice is dripped into 20 lattices, and the glass material carrier is air-dried in a super clean bench. After air drying, 2mL of 0.5% hydrogen peroxide solution was dropped on the surface of the carrier, 50 seconds later, 1.2mL of the mixed solution was sucked from the surface of the carrier and gel-filtered by Sephadex G25, and the dilution of the filtrate after filtration was regarded as 10^-1. Taking 200 mu L of filtrate, adding 1.8mL of cell maintenance solution for 10-fold gradient dilution, and diluting to 10^-4(10^-1～10^-4Four gradients), 4 wells of 100 μ L per well were seeded at each dilution. After inoculation, the cells were incubated in a carbon dioxide incubator (37 ℃ C., 5% CO)₂) Culturing for 3-5 days.

And thirdly, effective neutralization test: taking a new 20cm²And (3) a round glass carrier, dripping the cell maintenance liquid on the surface of the carrier, dripping the cell maintenance liquid into each cell with the volume of 10 mu L, dripping the cell maintenance liquid into each cell with the volume of 20 cells, and air-drying. After air drying, 2mL of 0.5% hydrogen peroxide solution was dropped on the surface of the carrier, 50 seconds later, 1.2mL of the mixed solution was sucked from the surface of the carrier and gel-filtered with Sephadex G25, and the dilution of the filtrate after filtration was regarded as 10^-1. Taking 200 mu L of filtrate, adding 1.8mL of cell maintenance solution for 10-fold gradient dilution, and diluting to 10^-4(10^-1～10^-4Four gradients), 4 wells of 100 μ L per well were seeded at each dilution. Then 100 mul of TCID50 logarithmic value between 2 and 3H 1N1 virus suspension is added into each well, and after inoculation, the mixture is placed in a carbon dioxide incubator (37 ℃, 5% CO)₂) Culturing for 3-5 days.

And fourthly, setting a positive control and a negative control according to the method in the embodiment.

Fifthly, after the steps of the first step to the fourth step meet the requirement of effective interpretation, the virus titer is identified again, and the steps are the same as the first step.

Sixthly, inactivation efficiency detection: the undiluted virus suspension was added dropwise at 20cm²On the surface of the round glass material carrier, 10 mu L of each lattice is dripped into 20 lattices, and the glass material carrier is air-dried in a super clean bench. After air-drying, 2mL of 0.5% hydrogen peroxide solution was dropped on the surface of the carrier, and after 50 seconds, 1.2mL of the mixture was taken up from the surface of the carrier and gel-filtered with Sephadex G25 (this step is described as Ct 1). The carrier was tilted to the side, the residual liquid on the surface of the carrier was poured out, the carrier was placed in another sterile petri dish, 2mL of 0.3% hydrogen peroxide solution was added dropwise to the surface of the carrier, and after 1 minute, 1.2mL of the mixture was aspirated from the surface of the carrier and gel-filtered with Sephadex G25 (this step is denoted as Ct 2). The dilutions of the filtrates obtained after filtration in steps Ct1 and Ct2 were both considered as 10^-1. Respectively taking 200 mu L of filtrate, adding 1.8mL of cell maintenance solution for 10-fold gradient dilution, and diluting to 10%^-4(10^-1～10^-9Nine gradients), 10 wells per dilution were inoculated, 100 μ L per well. After inoculation, the cells were incubated in a carbon dioxide incubator (37 ℃ C., 5% CO)₂) Culturing for 3-5 days.

Testing the amount of the vector viruses: the undiluted virus suspension was added dropwise at 20cm²On the surface of the round glass material carrier, 10 mu L of each lattice is dripped into 20 lattices, and the glass material carrier is air-dried in a super clean bench. After air-drying, 2mL of the cell-maintaining solution was dropped on the surface of the carrier, and after 50 seconds, 100. mu.L of the mixture was aspirated from the surface of the carrier and seeded on a cell plate, and 200. mu.L of the mixture was used for serial dilution (this step is referred to as Cc 1). The carrier was tilted to remove the residual liquid on the surface of the carrier, the carrier was placed in another sterile petri dish, 2mL of the cell maintenance solution was dropped on the surface of the carrier, and after 1 minute, 100 μ L of the mixture was aspirated from the surface of the carrier and inoculated on a cell plate, and at the same time, 200 μ L of the mixture was used for serial dilution (this step is denoted as Cc 2). The dilutions of the mixtures (virus suspension and cell maintenance solution) in steps Cc1 and Cc2 were all considered to be 10^-1. Respectively taking 200 mu L of filtrate, adding 1.8mL of cell maintenance solution for 10-fold gradient dilution, and diluting to 10%^-4(10^-1～10^-9Nine gradients), 4 wells per dilution were inoculated, 100 μ Ι _ per well. After inoculation, the cells were incubated in a carbon dioxide incubator (37 ℃ C., 5% CO)₂) Culturing for 3-5 days.

And setting positive control and negative control according to the method in the embodiment.

The detection results of the first to fourth steps are shown in tables 3 to 6.

TABLE 3 identification of the virus titration

TABLE 4 results of cytotoxicity identification

Degree of dilution	10^-1	10^-2	10^-3	10^-4
					Diseased cells	0/4	0/4	0/4	0/4

Table 5 effective neutralization test results

Degree of dilution	10^-1	10^-2	10^-3	10^-4
					Number of infection	4/4	4/4	4/4	4/4
Percent by weight%	100	100	100	100

TABLE 6 Positive control and negative control results

Positive control	1	2	3	4
					Whether or not to infect	+	+	+	+
Negative control	1	2	3	4
					Whether or not to infect	-	-	-	-

As shown in the above results, in this embodiment, the titer of the virus suspension is greater than 7, there is no cytotoxicity and no inhibition effect on virus infection after filtration, the procedure can effectively neutralize the sterilized product, the positive controls are all positive, the negative controls are all negative, and the test results all pass, which indicates that the test result is effective, and the inactivation efficacy test can be further performed.

The detection results of the fifth step-the sixth step are shown in tables 7 to 12.

TABLE 7 viral titration identification (Cv) results

TABLE 8 step one inactivation potency (Ct1)

TABLE 9 step two inactivation potency (Ct2)

TABLE 10 step one vector viral load control (Cc1)

TABLE 11 step two vector viral load control (Cc2)

TABLE 12 Positive and negative controls

The data in the above table are shown as 10ⁿSubstituting the format into the algorithm in the above embodiment can obtain: TCID50 inactivation efficiency of step one was 10^5.30The titer per mL for either/mL or TCID50 was 5.30. The inactivation efficiency of TCID50 in step two is 10^2.03The titer per mL for the/mL or TCID50 was 2.03. The overall TCID50 inactivation efficiency of the sterilization job after the substitution of the formula is 10^7.67Conversion of 100. mu.L to 10 per ml^8.67A titer of 8.67 per mL or TCID50, a practical effective titer of 7.67, and an overall unit area inactivation efficiency of 4.68X 10 reduction per square centimeter on average⁶And (4) infectious units. The virus residual ratio in this sterilization operation was 4.57% by substituting Cc1 and Cc2 into the formula, and the data indicated that the virus concentration remained in step two was 4.57% of the virus concentration in step one and was low residual, indicating that this material was easy to clean. Based on the data, the titer logarithmic value of the virus suspension is greater than 7, the virus positive control is all positive, and the virus negative control is all negative, so that the test result can be judged to be a valid result.

(2) The current common scheme is as follows:

20 pieces of glass sheets of 1cm multiplied by 1cm are used, and an effective neutralization test and a cytotoxicity test are carried out before formal test to ensure that data are effective.

Wherein, the effective neutralization test comprises the following steps: 3 glass carriers were taken, 10. mu.L of cell maintenance solution was dropped into each glass carrier, and air-dried. After air drying, 100. mu.L of 0.5% hydrogen peroxide solution was added dropwise to each glass carrier, and after the action time, the glass carrier was immersed in 1mL of a neutralizing agent and allowed to act for 10 minutes. After the cells were taken out, the cells were diluted 10-fold (10 times) with a cell maintenance medium as a diluent^-1、10^-2、10^-3、10^-4Total 4 groups), 100 μ L of each dilution was plated on a cell plate, and 4 wells of each dilution were plated. Subsequently, 100. mu.L of TCID50 log-2-3H 1N1 virus suspension was added to each well. After inoculation, the cells were incubated in a carbon dioxide incubator (37 ℃ C., 5% CO)₂) Culturing for 3-5 days.

The cytotoxicity test procedure was: 3 glass carriers were taken, 10. mu.L of cell maintenance solution was dropped into each glass carrier, and air-dried. After air drying, 100. mu.L of 0.5% hydrogen peroxide solution was dropped on each glass carrier,after the action time, the cell maintenance solution is used as a diluent to carry out 10-fold dilution (10 times)^-1、10^-2、10^-3、10^-4Total 4 groups), 100 μ L of each dilution was plated on a cell plate, and 4 wells of each dilution were plated. After inoculation, the cells were incubated in a carbon dioxide incubator (37 ℃ C., 5% CO)₂) Culturing for 3-5 days.

② setting positive control and negative control. The positive control is virus suspension without adding 0.5% hydrogen peroxide solution, the negative control is cell maintenance liquid without adding virus suspension, and the method is the same as the first step.

Testing the effectiveness:

first efficacy test: 20 glass carriers were taken, 10. mu.L of cell maintenance solution was dropped into each glass carrier, and air-dried. After air drying, 100. mu.L of 0.5% hydrogen peroxide solution was added dropwise to each glass carrier, and after the action time, the glass carrier was immersed in 1mL of a neutralizing agent and allowed to act for 10 minutes. After taking out, the cells were diluted 10-fold with the cell maintenance medium as a diluent, and 100. mu.L of each dilution was inoculated on a cell plate, and 4 wells were inoculated for each dilution. After inoculation, the cells were incubated in a carbon dioxide incubator (37 ℃ C., 5% CO)₂) Culturing for 3-5 days.

Second efficacy test: 20 glass carriers were added dropwise to each glass carrier, and 10. mu.L of the virus suspension was added dropwise to each glass carrier, followed by air-drying. After air drying, 100. mu.L of 0.5% hydrogen peroxide solution was added dropwise to each glass carrier, and after the action time, the glass carrier was placed in a sterile petri dish. Then 100. mu.L of 0.3% hydrogen peroxide solution was dropped on each glass carrier, and after the action time, the glass carrier was immersed in 1mL of a neutralizer and acted for 10 minutes. After taking out, the cells were diluted 10-fold with the cell maintenance medium as a diluent, and 100. mu.L of each dilution was inoculated on a cell plate, and 4 wells were inoculated for each dilution. After inoculation, the cells were incubated in a carbon dioxide incubator (37 ℃ C., 5% CO)₂) Culturing for 3-5 days.

The test results are shown in tables 13 to 17:

TABLE 13 effective neutralization test results

TABLE 14 cytotoxicity test results

TABLE 15 viral suspension titers in Positive controls

TABLE 16 results of the first inactivation potency test

TABLE 17 second inactivation potency test results

Neither the positive control nor the negative control showed abnormalities.

The results of the first inactivation efficacy test are: 10^5.23The titer value per mL or TCID50 was 5.23, and the second inactivation potency test results were: 10^3.27The titer per mL for either/mL or TCID50 was 3.27. The overall TCID50 inactivation efficacy of the disinfection job was 10^8.50The titer value per mL of/mL or TCID50 was 8.50, and the actual titer value was 7.50.

Effect comparison experiment 2:

the carrier material of this embodiment is wood material (waterproof wood chip), and the disinfection product that awaits measuring is hydrogen peroxide, and the disinfection operation step is 2 steps (disinfection number of times is 2), and it is H1N1 virus (virus strain purchases from ATCC, serial number VR-95) supporting MDCK cell strain (is used for producing H1N1 virus, and the cell strain purchases from ATCC, serial number CCL-34), does not have organic interference to detect the virus.

All testing procedures were as in effect control experiment 1.

Wherein, the detection is carried out according to the method in the embodiment, and the detection results of the steps I-IV are shown in tables 18-21.

TABLE 18 identification of Virus titration

TABLE 19 results of cytotoxicity identification

Degree of dilution	10^-1	10^-2	10^-3	10^-4
					Diseased cells	0/4	0/4	0/4	0/4

TABLE 20 effective neutralization test results

TABLE 21 Positive control and negative control results

The results of the detection of the fifth step ((C) - (C)) are shown in tables 22-27.

TABLE 22 viral titration identification (Cv) results

TABLE 23 procedure-inactivation efficiency (Ct1)

TABLE 24 step two inactivation efficiency (Ct2)

TABLE 25 Steps one vector viral load control (Cc1)

TABLE 26 step two vector viral load control (Cc2)

TABLE 27 Positive and negative controls

The data in the above table are shown as 10ⁿSubstituting the format into the algorithm in the above embodiment can obtain: TCID50 inactivation efficiency of step one was 10^5.62The titer per mL for either/mL or TCID50 was 5.62. The inactivation efficiency of TCID50 in step two is 10^2.71The titer per mL for the/mL or TCID50 was 2.71. The overall TCID50 inactivation efficiency of the sterilization job after the substitution of the formula is 10^8.33Conversion of 100. mu.L to 10 per ml^9.33A titer of 9.33/mL or TCID 50/mL, a practical effective titer of 8.33, and an overall unit area inactivation efficiency of 2.14X 10 reduction per square centimeter on average⁷And (4) infectious units. The ratio of virus remaining in the sterilization process was 10.00% by substituting Cc1 and Cc2 into the formula, and the data indicated that the virus concentration remaining in step two was 10.00% of the virus concentration in step one, and was moderate, indicating that the material was difficult to clean. Based on the data, the titer logarithmic value of the virus suspension is greater than 7, the virus positive control is all positive, and the virus negative control is all negative, so that the test result can be judged to be a valid result.

The results of the current conventional scheme are shown in tables 28 to 32.

TABLE 28 effective neutralization test results

TABLE 29 results of cytotoxicity test

TABLE 30 viral suspension titers in Positive controls

TABLE 31 results of the first inactivation potency test

TABLE 32 results of the second inactivation potency test

Neither the positive control nor the negative control showed abnormalities.

The results of the first inactivation efficacy test are: 10^5.71The titer value per mL or TCID50 was 5.71, and the second inactivation potency test results were: 10^3.79The titer per mL for the/mL or TCID50 was 3.79. The overall TCID50 inactivation efficacy of the disinfection job was 10^9.50The titer value per mL of/mL or TCID50 was 9.50, and the actual titer value was 8.50.

Results of the experiment

According to the data (table 33) of the two methods (the detection method in the above embodiment and the current common scheme), it can be seen that the total inactivation efficiency result of the detection method in the above embodiment is very close to the current common scheme, and the detection method in the above embodiment is close to the mean value of the 20 groups of data in the current common scheme in the first detection step, and the second detection step is different from the current common scheme, but considering that the operation in the environment and the current common scheme may cause partial virus loss, the experimental value is falsely high, while the detection method in the above embodiment completely avoids the above problem, and only calculates the calculated inactivation efficiency of the virus actually carried on the vector, and the result is closer to the real value than the current common scheme.

TABLE 33 comparison of the test methods in the above examples with the current protocols in common use

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A method of detecting the virus inactivation efficiency of a sterilized product or sterilization procedure, comprising the steps of:

(1) determining the type of the carrier;

(2) judging the effectiveness of the experiment;

(3) detecting inactivation efficiency;

2. The method of claim 1, wherein the method for determining the validity of the experiment in step (2) comprises: performing virus titer identification, cytotoxicity identification and effective neutralization test, and if the logarithm value of each milliliter of TCID50 of the virus titer identification is more than 7 and the positive control and the negative control are not abnormal, judging that the experimental result is effective; if not, the method is invalid.

3. The method of claim 1, wherein the inactivation potency assay of step (3) comprises viral titer identification, inactivation potency assay, and vector viral load assay.

4. The method according to claim 1, wherein the determination method of the effectiveness of the sterilization product or sterilization operation to be tested in the step (4) is:

if the log value of the total inactivation efficiency is more than or equal to 4, and the log value of the single-step inactivation efficiency is more than or equal to 1, the to-be-tested disinfection product or the disinfection operation is effective;

preferably, if the log value of the total inactivation efficiency is greater than or equal to 4, the log value of the single-step inactivation efficiency is greater than or equal to 1, and after repeated tests, the difference between the log values of the total inactivation efficiency and the difference between the log values of the single-step inactivation efficiency are both less than 0.5, the disinfection product or the disinfection operation to be tested is effective;

if not, the method is invalid.

5. The method of claim 4,

the algorithm formula of the single-step inactivation efficiency is as follows:

R_logn＝log(10Ct(n-1)×R_ratio)-log(10Ctn)；

ccn is the test result of step n for vector virus amount; cc (n-1) is the test result of the virus quantity of the vector in the step (n-1); ctn is the result of the inactivation efficacy test in the nth step; ct (n-1) is the inactivation efficiency test result of the step (n-1); r_lognThe titer value of the inactivated virus per ml in the nth step is;

the algorithm formula for the overall inactivation efficiency is:

R_logt＝log(10Cc1)-log(10Ctn)；

6. The method according to claim 1, wherein the inactivation efficiency of the sterilization product or sterilization work to be tested in the step (4) is judged by:

7. The method of claim 1, wherein the surface of the carrier in step (1) is provided with L-shaped snaps, the snaps being perpendicular to the surface of the carrier, and the carrier is made of materials including glass, stainless steel, and plastic.

8. The method according to claim 1, wherein the type of carrier in step (1) is determined according to a disinfection mode of action, wherein the disinfection mode of action comprises single-sided immersion, whole immersion and spraying;

preferred correspondence of carrier type to mode of disinfection action is:

if the disinfection mode is spraying, the carrier is a square carrier.

9. Use of the method of claims 1-8 in antiviral product screening.

10. Use of the method of claims 1-8 for quantitative determination of ease of cleaning materials.