GB 15981-1995 Evaluation methods and standards for disinfection and sterilization effects
Some standard content:
National Standard of the People's Republic of China
Evaluating method and standard for the efficacy of disinfection and sterilization Part I Evaluation method and standard for the efficacy of pressure steam sterilization 1 Subject content and scope of application
This method specifies the technical standard for pressure steam sterilization and the detection method for evaluating the sterilization efficacy. This method is applicable to the evaluation of the sterilization efficacy of pressure steam sterilization equipment. 2 Reagents
All reagents used in this standard are of analytical grade (AR) if the specifications are not specified, and the water is distilled water. 2.1 Protein Chen.
2.2 Glucose.
2.3 Bromocresol purple alcohol solution: Take 2.0g of bromocresol purple and dissolve it in 100mL 95% ethanol. GB15981-1995
2.4 Bromocresol purple protein water culture medium preparation: protein 10.0g, glucose 5.0g, dissolved in 1000mL distilled water, adjust pH to 7.0~7.2, then add 2% bromocresol purple alcohol solution 0.6mL, shake well, according to 5mL/tube, pack the mouth, put in the pressure steam sterilizer, sterilize at 115C for 40min and set aside.
3 Indicator bacteria
Thermomyces stearophilus spores (ATCC7953 or SSIK31) bacterial tablets, bacterial content is 5×105～5×10°cfu/tablet, at 121C, the time required to kill 90% of microorganisms Di2i value is 1.3～1.9min, the killing time (KT value) is ≤19min, and the survival time (ST value) is ≥3.9min.
4 Chemical indicators
Chemical indicators approved by the Ministry of Health are required. 5 Technical requirements
Pressure steam sterilizer
Downward exhaust type
Prevacuum type
Pressure, MPa/cm2
Approved by the State Administration of Technical Supervision on December 15, 1995 256
Temperature, C
Sterilization time, min
Implemented on July 1, 1996
6 Detection methods
GB15981-1995
6.1 Biological indicators (used as the basis for the sterilization effect of pressure steam sterilization equipment). 6.1.1 Place two pieces of Bacillus stearothermophilus spores in sterilized small paper bags and place them in the center of the standard test package. 6.1.2 In the sterilization cabinet, place a standard test pack (consisting of 3 plain long-sleeved surgical gowns, 4 small surgical towels, 2 medium surgical towels, 1 large surgical towel, 30 10cm×10cm, and 8 layers of gauze dressings wrapped in a size of 25cm×30cm×30cm) in the center of the upper and middle layers and the exhaust port. For portable pressure steam sterilizers, use a ventilated storage box (22cm×13cm×6cm) instead of a standard test pack. Fill the box with medium test tubes, and place the indicator bacteria sheet in the two sterilized test tubes in the center (the test tube mouths are wrapped with sterilized kraft paper). Place the box flat on the bottom of the portable pressure steam sterilizer. 6.1.3 After a sterilization cycle, take out the indicator bacteria sheet from the standard test pack or ventilated storage box under sterile conditions, put it into the bromocresol purple glucose protein aged water culture medium, and culture it at 56℃ for 48h to observe the color change of the culture medium. 6-2 Chemical indicators
Chemical indicator tape is used on the outside of the article package to mark whether the article has been sterilized. Chemical indicators are used in the center of the article package to mark whether the article has been sterilized. 7 Result determination and evaluation
7.1 In the same test, if the bromocresol purple protein water medium inoculated with each indicator bacteria piece in the standard test package or ventilation storage box does not change color, it is determined to be sterilized. When the bromocresol purple protein water medium inoculated with one of the indicator bacteria pieces changes from purple to yellow, it is determined to be sterilized unqualified.
7.2 When the color of the chemical indicator changes to the same color as the sterilization qualified standard color, or when it melts, it is used as a reference standard for sterilization qualified. Part II Methods and standards for evaluating the effect of ultraviolet surface disinfection 8 Subject content and scope of application
This method specifies the wavelength and intensity of ultraviolet rays used for surface disinfection of objects, as well as the physical indicators and biological detection methods for evaluating its disinfection effect.
This method is applicable to the evaluation of the disinfection effect of objects directly irradiated by ultraviolet rays. 9 Indicator bacteria
9.1 Escherichia coli (8099 or ATCC25922). 9.2 Bacillus subtilis var. niger spores (ATCC9372). 10 Physical indicators
10.1 When the voltage is 220V, the ordinary 30W straight tube UV lamp, when the room temperature is 20~25℃, the 253.7nm UV radiation intensity (vertical 1m) should be ≥70μW/cm2. 10.2 When the voltage is 220V, the high-intensity UV lamp, when the room temperature is 20~25℃, the 253.7nm UV radiation intensity (vertical 1m) should be ≥200μW/cm.
10.3 The irradiation dose is calculated according to formula (1):
Dose (μW·s/cm\) - Intensity (μW/cm2) × Time (s)… 11 Detection method
11.1 Physical detection method
11.1.1 The ultraviolet intensity (uW/cm2) of the lamp tube is measured at a vertical position of 1m with an ultraviolet intensity meter with a central wavelength of 253.7nm (within the calibration validity period).
GB15981—1995
11.1.2 In actual application, the irradiation intensity of the disinfected surface should be measured based on the actual distance between the lamp tube and the disinfected object. 11.1.3 The irradiation dose received by the surface disinfection should reach the required amount to kill the target microorganisms. For Escherichia coli, the irradiation dose should reach 20,000μW·s/cm2, and for Bacillus subtilis var. niger spores, it should reach 100,000μW·s/cm. 11.2 Biological detection method
11.2.1 Use carrier quantitative disinfection test. Carrier preparation shall be carried out according to Appendix C of this standard. 11.2.2 After turning on the ultraviolet lamp for 5 minutes, place 8 stained glass slides flat in the sterilization container, place them horizontally at an appropriate distance for irradiation, take out 2 stained glass slides at 4 different intervals, put them into 2 test tubes containing 5mL eluent (1% Tween 80 1% protein-aged saline), and shake them 80 times.
11.2.3 After appropriate dilution, take 0.5mL of eluent, pour it into a plate, inoculate two on each stained glass slide, and incubate at 37℃ for 48h for viable bacteria count.
11.2.4 For the positive control, except for not irradiating, take 2 stained glass slides, put them into 2 test tubes containing 5mL eluent, and shake them 80 times, and the rest shall be carried out according to 4.2.3.
11.2.5 Calculation of killing rate
Killing rate (%) = Number of recovered bacteria in the positive control group x 10 Number of recovered bacteria in the positive control group
12 Judgment standard
12.1 The killing rate of indicator bacteria ≥ 99.9% is judged as qualified disinfection. 12.2 When the physical test standard is reached, it is used as a reference standard for qualified disinfection. Part III Evaluation Methods and Standards for Disinfection Effect of Liquid Disinfectants 13 Subject Content and Scope of Application
This method specifically stipulates the biological detection method and evaluation standard for the disinfection effect of disinfectants. This method is applicable to the evaluation of the disinfection effect of disinfectants on various objects. 14 Physical and chemical indicators
Put the disinfectant in a 20±2℃ water bath and measure the shortest time (min) required to kill the indicator microorganisms to achieve disinfection or sterilization at the use concentration.
15 Indicator Microorganisms
15.1 Bacteria
15.1.1 Bacterial propagules: Staphylococcus aureus (ATCC6538), Escherichia coli (8099 or ATCC25922). 15.1.2 Bacterial spores: Bacillus subtilis var. niger spores (ATCC9732). 15.2 Fungi: Candida albicans (ATCC10231). 15.3 Hepatitis B surface antigen: purified antigen (1.0 mg/mL). 16 Detection Methods
16.1 Neutralization test (see Appendix A).
16.2 Qualitative disinfection test of disinfectants (see Appendix B). 16.3 Quantitative disinfection test of disinfectants (see Appendix C). 16.4 Disinfectant bactericidal energy test (see Appendix D). 16.5 Hepatitis B surface antigen (HBsAg) antigenic destruction test (see Appendix E). 258
Evaluation Standards for Mold Removal Effect
GB15981-1995
17.1 The killing rate of bacteria and fungi is ≥99.9%. For HBsAg, if the HBsAg antigenicity is destroyed by 10* times or 5×101 times (carrier test) of the detection method sensitivity, it can be judged as qualified disinfection. 17.2 If all spores of Bacillus subtilis var. niger are killed, it can be judged as qualified sterilization. 17.3 In practical application, the lowest concentration and shortest time of the organic matter protection test are used as the concentration and time required for the disinfectant to achieve practical disinfection.
A1 Summary
GB 15981—1995
Appendix A
Neutralizing effect test of neutralizer
(Supplement)
In order to accurately evaluate the killing effect of disinfectants on microorganisms, it is required to select appropriate neutralizers in the disinfection test. The selected neutralizer should not only be able to stop the microbial killing effect of the disinfectant in time, but also the neutralizer itself and its reaction product with the disinfectant (hereinafter referred to as the neutralization product) should have no inhibitory or killing effect on microorganisms and no adverse effect on the culture medium. A2 culture medium and reagents
A2.1 Nutrient agar culture medium
Ingredients: egg white,
beef extract
sodium chloride
distilled water
1000.00mL
Preparation method: Except for agar, other ingredients are dissolved in distilled water, pH is adjusted to 7.2-7.4, agar is added and heated to dissolve, filtered and packaged, sterilized at 121C and pressure steam for 30 minutes, and then used for later use. A2.2 0.03mol/L phosphate buffer (pH7.2-7.6, hereinafter referred to as PBS). Ingredients: disodium hydrogen phosphate
potassium dihydrogen phosphate
distilled water
1000.00ml
Preparation method: dissolve disodium hydrogen phosphate and potassium dihydrogen phosphate in distilled water, pH 7.2-7.4, divide into packages, sterilize by pressure steam at 121℃ for 30min and set aside.
A3 Equipment
A3.1 Conical flask.
A3.2 Flat III (diameter 9cm).
A3.3 Measuring cylinder.
A3.4 Precision pH test paper.
A3.5 Sterile test tube.
Sterile graduated pipette (1.0, 5.0, 10.0mL). A3.7 Constant temperature incubator.
A3.8 Refrigerator.
A3.9 Colony counter.
A3.10 Alcohol lamp.
Neutralizer (specify manufacturer and batch number)
A5 Operation method
A5.1Use PBS to make indicator bacteria into 5×105~5×10°cfu/mL suspension. 260
GB 15981—1995
A5.2Use sterile distilled water to prepare disinfectant into 3 different concentrations. Without adding neutralizer, measure the minimum effective concentration of the disinfectant that can inhibit and kill indicator bacteria by more than 99.9% in 10 minutes. A5.3Test the minimum effective concentration of the disinfectant that can inhibit and kill indicator bacteria in 10 minutes with the neutralizer to be selected, select the type of neutralizer and adjust the concentration of the neutralizer according to the principle of equivalent neutralization, and select the concentration of the neutralizer used for the disinfectant of the test concentration. A5.4 In the neutralizer selection test, first mix 1.0mL of disinfectant with 9.0ml of neutralizer solution to make a neutralization product solution, and then perform the test in groups according to Table A1.
Table A1 Neutralizer Selection Test
0.5ml of bacterial solution is added to:
4.5ml of disinfectant.
4.5ml of disinfectant
4.5ml of neutralization product
4.5ml of neutralizer
Take 0.5ml of the mixed solution and add it to:
(the total amount after addition is 5mL)
4.5ml of PRS
4.5ml of neutralizer
4.5ml of PBS.
4.5ml of PBS 5. Oml
After 10 minutes of action, take 0.5ml of the original solution or diluted solution. Inoculate plates (2/sample): original solution, ×10
original solution, ×10
X100, ×1000
X100, ×1000
X100, X1000
Then, pour the plate and culture at 37C for 48 hours, count the number of colonies, and calculate the number of recovered bacteria (cfu/ml) according to the dilution multiple. A6 Reporting method of neutralization test results (as shown in Table A2) Table A2 Neutralization test results example
Neutralizing agent
1% lecithin
1% lecithin + 0.1% Tween 80
1% Tween 80
0.5% sodium thiosulfate
Number of colonies recovered in each group, cfu/ml
4.67X1064.83X1064.11X106
5.81×106 5.89×106 5.78×1063.31×1055.31×1065.21×106
3.20×1065.03×10%5.18×106
3, 4.5Inter-group
Error rate, %
3, 4.5Inter-group error rate calculation formula
Error rate (%)=（[mean of three groups - number of bacteria in group 3 ± mean of three groups number of bacteria in group 4 ± mean of three groups - number of bacteria in group 5 1) ± 3 ×100mean of blue group
（Al）
A7Judgment criteria
A7.13, 4, 5 groups have similar bacterial counts, and the error rate is ≤10%. A7.26 group has no sterile growth.
A7.32 group has significantly fewer bacteria than 3, 45 groups. A7.4 Group 1 has no bacteria or is significantly less than Group 2. Neutralizers that meet the above standards indicate that they can eliminate the effect of disinfectants on indicator bacteria. Neutralizers and their neutralization products with disinfectants are non-toxic to indicator bacteria and are determined to be neutralizers for the disinfectant. A8 Selection of neutralizer concentration for disinfection test
Follow the steps in A5.4 and determine according to the standards of A7.1 to 7.4. 261
B1 Summary
GB159811995
Appendix B
Qualitative disinfection test for disinfectants
(Supplement)
Qualitative disinfection test is a test method to determine whether there is bacterial growth in samples after being acted on by disinfection factors. It is used to identify the sterilization effect of disinfection factors and preliminarily evaluate the bactericidal effect of disinfectants. B2 Culture medium and reagents
B2.1 Ordinary broth culture medium
B2.1.1 Ingredients: protein chen
sodium chloride
meat extract
1000.00mL
B2.7.2 Preparation method: Take protein chen and sodium fluoride and add them to the meat extract, dissolve them at a low temperature, adjust the pH to weak alkalinity, boil and filter, adjust the pH to 7.2-7.4 after sterilization, and sterilize by pressure steam for use. B2.2 Reagents
B2.2.1 Diluent: 0.03 mol/L PBS (pH 7.2-7.4) containing 1% protein chen. B2.2.2 Sterile distilled water.
B2.2.3 Neutralizer: Select according to Appendix A of this standard. B3 Equipment
B3.1 Sterile graduated pipette (1.0, 5.0, 10.0mL). B3.2
Sterile test tube.
Sterile conical flask.
B3.4Alcohol lamp.
Constant temperature water bath.
Constant temperature incubator.
B4Test method
B4.1 Count the live bacteria in the bacterial solution and use diluent to prepare a bacterial suspension with a bacterial content of 5×105～5×10°cfu/mL. B4.2 Arrange 10 sterile test tubes on a test tube rack and mark the tube numbers. B4.3 Add 2.5mL of sterile distilled water to each test tube and place it in a 20±2℃ water bath. B4.4 Add 2.5mL of disinfectant of appropriate concentration to the first tube, mix well and transfer 2.5mL to the second tube, mix well again, transfer 2.5mL from the second tube to the third tube, and so on to the ninth tube. Discard 2.5mL after mixing. No disinfectant is added to the tenth tube as a control. B4.5 Add 2.5mL of bacterial suspension to each tube, mix well and record the time of adding bacteria to each tube, so that the bacterial content in the bacterial and drug mixture is 105~10cfu/mL.
B4.6 Take out 0.5mL from each tube at 4 different intervals after adding bacteria, add it to 4.5mL neutralizer, neutralize it for 10 minutes, take out 0.5mL and add it to 4.5mL nutrient broth tube. B4.7 Place the broth tube inoculated with bacteria at 37℃ for 48h, observe the preliminary results, and continue to culture the sterile growth tube until the 7th day. B4.8 Repeat the test 5 times.
B5 Result determination
GB15981-1995
B5.1 If the broth tube is turbid, it means bacterial growth, which is recorded as positive and indicated by (+). B5.2 If the broth tube is clear on the 7th day of culture, it means sterile growth, which is recorded as negative and indicated by (-). B5.3 For broth tubes that are difficult to determine, take 0.1mL and inoculate it on the nutrient agar plate, spread it evenly with a sterile 1. stick, and culture it at 37℃ for 48h to observe the colony morphology; and perform smear staining and microscopic examination to determine whether there is growth of indicator bacteria. Growth of indicator bacteria is recorded as positive. B5.45 tests, no indicator bacteria growth, indicating sterilization. Appendix
Quantitative disinfection test of fungicides
(Supplement)
C1 Summary
Quantitative disinfection test is a test method to determine the number of microorganisms remaining in a sample after being acted upon by disinfection factors, and the result is expressed as a killing rate. It is used to evaluate the killing effect of disinfectants.
C2 Culture medium and reagents
C2.1 Ordinary nutrient agar culture medium: prepared according to A2.1 of this standard. C2.2 Reagents
C2.2.1 Diluent: 0.03 mol/L PBS (pH 7.2~7.4) containing 1% protein. C2.2.2 Sterile distilled water.
C2.2.3 Neutralizer: selected according to Appendix A of this standard. C2. 2. 40. 03mol/L PBS (pH 7. 2~~7. 4). C2.2.5 Eluent: PBS containing neutralizer, 1% protein Chen, 0.1% Tween 80. C3 Equipment
C3.1 Sterile graduated pipette (1.0, 5.0, 10.0mL). C3.2 Sterile test tube.
C3.3 Sterile conical flask
C3.4 Sterile flat III (9cm in diameter).
Constant temperature water bath.
Constant temperature incubator.
C3.7 Alcohol lamp.
C3.8 Colony counter.
C3.9 Microinjector.
0 Carrier: Select degreased 0.5cmX1.0cm cloth, paper, glass, rubber, plastic, stainless steel or aluminum sheets according to needs and test purposes.
C4 Test method
C4.1 Quantitative suspension test
C4.1.1 Count the live bacteria in the bacterial solution and dilute it with diluent to a bacterial suspension with a bacterial content of 5×105~～~5×10°cfu/mL. C4.1.2 Dilute the disinfectant with sterile distilled water to 3 different concentrations, take 4.5mL of each and add them to three test tubes, and place them in a 20±263
2℃ water bath.
GB15981--1995
C4.1.3 After the temperature of the liquid in the test tube is balanced with the temperature of the water bath, add 0.5mL of bacterial suspension (bacterial content of 5×105～5×10°cfu/mL) to each of the three test tubes, mix well and start timing. C4.1.4 Take 0.5 mL of the bacterial solution at 4 different intervals and transfer it to 4.5 mL of neutralizer to mix. C4.1.5 Neutralize for 10 minutes, and then make appropriate dilution to count the live bacteria. C4.1.6 Replace the disinfectant with the eluent for the positive control, and perform the test according to C4.1.2 to C4.1.5. C4.1.7 Calculate the number of surviving bacteria (cfu/mL) of each sample according to different dilutions, and calculate the killing rate according to formula (C1): Killing rate (%) = Number of surviving bacteria in the control group × 100 Number of surviving bacteria in the control group
C4.1.8 Repeat the test 5 times.
C4.2 Carrier quantitative test
C4.2.1 Place the sterilized carrier flat in sterilization plane III, drop a predetermined amount of bacterial solution on each carrier (the number of bacteria recovered by the carrier reaches 5×105～5×106cfu/piece), spread it evenly, and place it in a 37℃ incubator to dry. When using commercially available bacterial carriers, the amount of recovered bacteria should also reach 5×105~5×10°cfu/piece). C4.2.2 Use sterile distilled water to dilute the disinfectant into 3 different concentrations, take 5mL of each and add them to three test tubes, and place them in a 20±2℃ water bath.
C4.2.3 After the temperature of the liquid in the test tube is balanced with the temperature of the water bath, add the bacterial carrier and act for the specified time. Move the bacterial carrier into a 5mL eluent test tube containing a neutralizer, neutralize for 10 minutes, shake 80 times, dilute appropriately, and inoculate two plates. Incubate at 37℃ for 24~48h, count the viable bacteria,
C4.2.4 Positive control, use the eluent instead of the disinfectant and proceed according to C4.2.2~C4.2.3. C5 Result judgment
C5.1 The killing rate of 5 tests is ≥99.9% and the disinfection is qualified. C5.2 All the spores of Bacillus subtilis var. niger were killed in 5 tests and the disinfection was qualified. Appendix D
Organic protection test
(Supplement)
D1 Summary
Organic protection test is to determine the killing effect of disinfectants on microorganisms under organic protection conditions, expressed as a killing rate. The results are compared with the quantitative disinfection test of the disinfectant to evaluate the effect of organic matter on the bactericidal ability of the disinfectant. D2 Culture medium and reagents
D2.1 Ordinary nutrient agar culture medium, prepared according to A2.1 of this standard. D2.2 Reagents:
D2.2, 1 diluent: the same as C2.2.1.
D2.2.2 Sterile distilled water.
D2.2.3 Neutralizer: selected according to Appendix A of this standard. D2.2.4 0.03 mol/L phosphate buffer (pH7.2~7.4) (referred to as PBS). D2.2.5 Eluent: physiological saline containing 1% protein Chen and 0.1% Tween 80. D2.2.6 Calf serum is added to the bacterial suspension to make the final concentration 10%. 264
D3 Equipment
Same as C3 of this standard.
D4 Test method
GB15981-1995
D4.1 Before the experiment, the bacterial suspension was counted for live bacteria, diluted with diluent, and calf serum was added to make the final serum content 10%, and the bacterial count was 5×105～5×10℃cfu/mL, which was used as the test bacterial suspension. D4.2 The following steps are the same as C4.1.2～C4.1.8 of this standard. D5 Result determination
D5.1 The killing rate of the 5 tests was greater than the minimum concentration and shortest time required for 99.9%, and it was judged that the disinfectant could achieve the effective concentration and time for disinfection in the presence of organic matter. D5.2 This effective concentration and time are the same or similar to the effective concentration and time of the quantitative disinfection test, and it is judged that the organic matter has no significant effect on the bactericidal effect of the disinfectant. The minimum effective concentration for disinfection is increased by more than one time or the shortest action time is extended by more than one time, which can be regarded as having a significant effect.
Appendix E
Hepatitis B surface antigen destruction test
(Supplement)
E1 Summary
Hepatitis B surface antigen (HBsAg) destruction test is a test method that uses the antigen activity of HBsAg as an indirect marker to evaluate the inactivation ability of disinfection factors on hepatitis B virus (HBV). It is suitable for evaluating the disinfection effect of chemical disinfectants and ultraviolet rays on HBV. E2 Reagents
E2.1 Calf serum (56C, 30min inactivation). E2.2 0.01mol/L phosphate buffer (PBS, pH7.2~7.4). E2.3 Purified HBsAg (1.0mg/mL).
E2.4 Solid phase radioimmunoassay reagents, requiring specificity 100%, precision CV≤15%, sensitivity ≤1.0ng/mL, linearity r>0.95.
Enzyme-linked immunosorbent assay reagents, requiring specificity 100%, precision CV≤15%, sensitivity ≤3.2ng/mL, linearity r>0.95. E3 Equipment
Pipette: including test tubes, pipettes (4 types: 0.1, 1.0, 5.0 and 10.0mL) and micro-injectors (100.0μL). E3.11
E3.2 Carrier (stainless steel sheet with a diameter of 1.5cm). E3.3r Immunocounter.
E3.4 Enzyme-linked immunosorbent assay instrument.
E3.5 Low temperature refrigerator (-30℃～-70C). E4 Preparation of HBsAg suspension
E4.1 Take 9.4mL of 0.01mol/LPBS (pH7.2~7.4) and add 0.6mL of calf serum to prepare a suspension containing 6% calf serum. Then 26.5
GB15981--1995
Take 9.0mL of the PBS and add 1.0mL of purified HBsAg suspension with a concentration of 1.0mg/mL to prepare a test HBsAg suspension containing 5.4% calf serum and 100μg/mL HBsAg concentration. E4.2 Put 1.0mL of the test HBsAg suspension into a 1.5mL glass bottle, seal it, and store it in a refrigerator at 30℃~～~70℃ for later use. The shelf life is three months. E5 HBsAg detection method
E5.1 Solid phase radioimmunoassay (SPRIA). E5.2 Enzyme-linked immunosorbent assay (ELISA). E6 Selection of neutralizer
When determining the destructive effect of disinfectants on HBsAg, the appropriate neutralizer and its concentration should be selected before conducting the destructive test. The neutralizer used: a. Should be able to effectively and promptly terminate the residual effect of the disinfectant; b.The neutralizer and its neutralization product with the disinfectant have no effect on the antigenic activity of HBsAg, nor do they affect the sensitivity of the detection method for HBsAg detection. E6.1 The disinfectant is prepared into different concentrations with sterile distilled water, 1.2mL of disinfectant is mixed with 0.3mL of HBsAg suspension, and placed in a 20±2°C water bath for 10 minutes. The minimum effective concentration of the disinfectant that inhibits or destroys the antigenicity of HBsAg for 10 minutes is determined. E6.2 The minimum effective concentration of the disinfectant that inhibits or destroys the antigenicity of HBsAg for 10 minutes is tested with the selected neutralizer. The test can be carried out according to the following groups: a. 0.9 mL of disinfectant + 0.1 mL of HBsAg suspension; b. 0.4 mL of disinfectant + 0.1 mL of HBsAg suspension. After 10 minutes of action, add 0.5 mL of neutralizer containing 20% calf serum; c. First take 1 mL of neutralizer containing 20% calf serum and 1 mL of disinfectant, mix them, act for 10~~30 minutes, make a neutralization product solution, and then test it. Neutralization product solution 0.9 mL + 0.1 mL of HBsAg suspension; d. 0.9 mL of PBS containing 10% calf serum + 0.1 mL of HBsAg suspension; e. 0.9 mL of neutralizer containing 10% calf serum + 0.1 mL of HBsAg suspension; f. 0.9 mL of neutralization product solution + 0.1 mL of PBS. Only when the measured values of HBsAg activity in groups c, d, and e are similar (difference less than 10%) and are significantly higher than group b, the HBsAg activity in group a cannot be detected or the detected activity is significantly lower than that in group b, and the negative control in group f is normal, can it be proved that the selected neutralizer and its dosage are appropriate. E6.3 When conducting disinfection tests, the dosage of neutralizers should be appropriately adjusted according to the principle of equal neutralization of disinfectants and neutralizers. After the neutralization effect is determined again according to the steps of E6.2, the antigenic destruction test can be carried out. E7
Destruction test method
E7.1 Suspension method
The suspension method refers to the test method of interacting HBsAg with disinfectants in suspension and observing the antigenic destruction effect. E7.1.1 The concentration of HBsAg suspension is 104 times the sensitivity of the detection reagent. If the sensitivity of the detection reagent is 1ng/mL, the concentration of HBsAg should be 10ug/mL.
E7.1.2 Take 2.7mL of PBS containing 5% calf serum and add it to 0.3mL of HBsAg suspension with a concentration of 100μg/mL, and mix well. E7.1.3 Take 20mL of calf serum and add it to 80mL of sterilized neutralizer solution, and mix well. E7.1.4 Destruction test: Mix the disinfectant with a concentration of 1.25 times the predetermined disinfectant concentration with the HBsAg suspension in a ratio of 4:1, and the volume of the mixed solution shall not be less than 1.5mL. Then place it in a water bath at 20±2℃, and act for a specified time. Immediately take 0.3mL of the mixed solution and mix it with an equal volume of neutralizer containing 20% calf serum, and act for 10 to 30 minutes. Take a sample to determine the activity of the residual HBsAg. Each sample is measured in parallel for 2 copies, each copy is 0.1mL, and the average value is taken to determine the destruction effect. Observe 3 concentrations of each disinfectant and observe 4 action times for each concentration. Repeat the test 5 times.
E7.1.5 Positive control: Take 1mL of neutralizer containing 20% calf serum, add it to a test tube containing 1mL of test disinfectant, mix well, and let it act for 10-30min to make a neutralization product solution. Take 0.9mL of the neutralization product solution and add 0.1mL of HBsAg suspension of the test concentration to sample and test. Each sample is tested in triplicate, 0.1mL per portion, and the average value is taken as the positive control value. E7.1.6 Negative control: Take 1mL of neutralizer containing 20% calf serum and add it to a test tube containing 1mL of test disinfectant, mix well, let it act for 10-3min, sample and test. Each sample is tested in triplicate, 0.1mL per portion, and the average value is taken as the negative control value. 266
GB15981-1995
Note that the negative control sample of the kit cannot be used for the negative control. E7.2 Carrier method
The carrier method refers to the test method of making the HBsAg on the carrier surface interact with the disinfection factor and observing the antigenic destruction effect. E7.2.1 Preparation of HBsAg carrier
E7.2.1.1 The carrier is a stainless steel sheet with a diameter of 1.5 cm. It is degreased by washing with detergent and sterilized by pressure steam. E7.2.1.2 The concentration of HBsAg suspension is 5×10* times the sensitivity of the detection method. If the sensitivity is 1ng/mL, the concentration of HBsAg should be 50μg/ml.
E7.2.1.3 The contamination method of HBsAg is the dripping method. When dripping, spread the sterilized carrier flat in a sterile plate, use a microinjector to absorb the HBsAg suspension, and drip it in the center of the carrier, 20μL per carrier. Then use L-shaped platinum wire to evenly spread the suspension, place it in a 37C incubator for 40-60 minutes, and conduct an antigenic destruction test after the suspension dries. E7.2.2 Antigenic destruction test
Take the carrier contaminated with HBsAg, place it flat in a sterile flat plate, use a pipette to absorb 50uL of the predetermined concentration of disinfectant, drip it in the center of the carrier, and quickly spread it evenly so that the entire carrier is evenly treated. Each group of 2 pieces is placed in a 20±2C water bath. After the prescribed time, use sterile tweezers to move the carrier into a neutralizer tube containing 1.0mL10% calf serum. After acting for 10-30 minutes, knock and shake 200 times, take samples to detect the activity of residual HBsAg, take 2 samples of each sample, each 0.1mL, take the average value, and determine the antigenic destruction effect. Observe 3 concentrations of each disinfectant and 4 action times for each concentration. Repeat the test 5 times. When observing the effect of ultraviolet rays on HBsAg destruction, place the contaminated carrier directly under the action factor. After the prescribed dose is reached, move the carrier into a test tube containing 1.0 ml of 10% calf serum PBS, tap and shake 200 times, take samples to detect the residual HBsAg activity, take 2 samples of each sample, each 0.1 ml, take the average, and determine the destruction effect. Repeat the test 5 times. E7.2.3 Positive control
When observing the effect of disinfectants on HBsAg destruction, take 50 μL of the test disinfectant and add it to a test tube containing 1.0 ml of 10% calf serum neutralizer. Act for 10 to 30 minutes to prepare a neutralization product solution. Take 1.0 ml of the neutralization product solution and add it to a large test tube, then move the carrier stained with HBsAg into it, tap and shake 200 times, test 3 samples of each sample, each 0.1 ml, and take the average as the positive control value. When observing the effect of ultraviolet rays destroying HBsAg, directly transfer the carrier contaminated with HBsAg into a test tube containing 1.0mL 10% calf serum in PBS (pH7.2-7.4), tap and shake 200 times, test 3 copies of each sample, 0.1mL each, and take the average value as the positive control value. bzxZ.net
E7.2.4 Negative control
When observing the effect of disinfectant destroying HBsAg, take 50μL disinfectant and add it to a test tube containing 1mL.10% calf serum neutralizer, act for 10~~30min, take samples for testing, and test 3 copies of each sample, 0.1mL each. Take the average value as the negative control value. When observing the effect of ultraviolet rays destroying HBsAg, the negative control is PBS (pH7.2-7.4) containing 10% calf serum. Test 3 copies of each sample, 0.1mL each, and take the average value as the negative control value. It should be noted that the negative control sample of the kit cannot be used for the negative control. E8 Destruction effect determination
S/N<2.1 is used as the qualified standard for HBsAg antigenic destruction. Where S is the average pulse count per minute (cpm) or optical density (OD) of the sample under test or the positive control sample after the disinfection factor acts. N is the average cpm or OD value of the negative control sample in the test. Additional notes:
This standard is proposed by the Ministry of Health of the People's Republic of China. This standard is drafted by the Institute of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine. The main drafters of this standard are Yuan Qia, Wang Taixing and Gu Jian. This standard is interpreted by the Office of Supervision and Administration of Communicable Disease Prevention and Control of the Ministry of Health, the technical coordination unit entrusted by the Ministry of Health. 267First, take 1mL of neutralizer containing 20% calf serum and 1mL of disinfectant, mix them, and let them react for 10~~30min to make a neutralization product solution, and then test it. Neutralization product solution 0.9mL + HBsAg suspension 0.1mL; d. PBS containing 10% calf serum 0.9mL + HBsAg suspension 0.1mL; e. Neutralizer containing 10% calf serum 0.9mL + HBsAg suspension 0.1mL; f. Neutralization product solution 0.9mL + PBS 0.1mL. After testing, only when the measured values of HBsAg activity in groups c, d, and e are similar (difference less than 10%) and are significantly higher than group b, the HBsAg activity in group a cannot be detected or the detected activity is significantly less than that in group b, and the negative control in group f is normal, can it be proved that the selected neutralizer and its dosage are appropriate. E6.3 When conducting disinfection tests, according to the principle of equal neutralization of disinfectants and neutralizers, the dosage of neutralizers should be appropriately adjusted. After the neutralization effect is determined again according to step E6.2, the antigenic destruction test can be carried out. E7
Destruction test method
E7.1 Suspension method
The suspension method refers to a test method in which HBsAg interacts with the disinfectant in a suspension and observes the antigenic destruction effect. E7.1.1 The concentration of the HBsAg suspension is 104 times the sensitivity of the detection reagent. If the sensitivity of the detection reagent is 1ng/mL, the concentration of HBsAg should be 10ug/mL.
E7.1.2 Take 2.7mL of PBS containing 5% calf serum and add it to 0.3mL of HBsAg suspension with a concentration of 100μg/mL, and mix well. E7.1.3 Take 20mL of calf serum and add it to 80mL of sterilized neutralizer solution and mix well. E7.1.4 Destruction test: Mix a disinfectant with a concentration of 1.25 times the predetermined disinfectant concentration with the HBsAg suspension in a ratio of 4:1, and the volume of the mixed solution should be no less than 1.5 mL. Then place it in a water bath at 20 ± 2 ° C, and act for a specified time. Immediately take 0.3 mL of the mixed solution and mix it with an equal volume of a neutralizer containing 20% calf serum. Act for 10 to 30 minutes, take a sample to determine the activity of the residual HBsAg, and measure 2 copies of each sample in parallel, each copy is 0.1 mL, and take the average value to determine the destruction effect. Observe 3 concentrations of each disinfectant, and observe 4 action times for each concentration. Repeat the test 5 times.
E7.1.5 Positive control: Take 1 mL of a neutralizer containing 20% calf serum, add it to a test tube containing 1 mL of the test disinfectant, mix it, and act for 10 to 30 minutes to make a neutralization product solution. Take 0.9mL of the neutralization product solution and add 0.1mL of HBsAg suspension of the test concentration for sampling and testing. Test three copies of each sample, 0.1mL each, and take the average value as the positive control value. E7.1.6 Negative control: Take 1mL of the neutralizer containing 20% calf serum and add it to a test tube containing 1mL of the test disinfectant, mix well, act for 10-3min, sample and test. Test three copies of each sample, 0.1mL each, and take the average value as the negative control value. 266
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It should be noted that the negative control sample of the kit cannot be used for the negative control. E7.2 Carrier method
The carrier method refers to the test method in which the HBsAg on the carrier surface interacts with the disinfection factor and the antigenic destruction effect is observed. E7.2.1 Preparation of HBsAg carrier
E7.2.1.1 The carrier is a stainless steel sheet with a diameter of 1.5 cm. It is washed with detergent and degreased, and sterilized with pressure steam for use. E7.2.1.2 The concentration of HBsAg suspension is 5×10* times the sensitivity of the detection method. If the sensitivity is 1ng/mL, the concentration of HBsAg should be 50μg/ml.
E7.2.1.3 The contamination method of HBsAg is the dripping method. When dripping, spread the sterilized carrier flat in a sterile plate, use a microinjector to absorb the HBsAg suspension, and drip it in the center of the carrier, 20μL per carrier. Then use L-shaped platinum wire to evenly spread the suspension, put it in a 37C incubator for 40-60min, and perform the antigenic destruction test after the suspension is dry. E7.2.2 Antigenicity destruction test
Take the carrier contaminated with HBsAg, place it flat in a sterile blood container, use a pipette to absorb 50uL of the disinfectant of the predetermined concentration, drip it in the center of the carrier, and spread it evenly so that the entire carrier is evenly treated. Place each group of 2 tablets in a 20±2C water bath. After the prescribed time, use sterile tweezers to move the carrier into a neutralizer tube containing 1.0mL10% calf serum. After 10-30min of action, knock and shake 200 times, take samples to detect the activity of residual HBsAg, take 2 samples of each sample, each 0.1mL, take the average value, and determine the antigenicity destruction effect. Observe 3 concentrations of each disinfectant and 4 action times for each concentration. Repeat the test 5 times. When observing the effect of ultraviolet rays on HBsAg destruction, place the contaminated carrier directly under the action factor. After the prescribed dose is reached, move the carrier into a test tube containing 1.0 ml of 10% calf serum PBS, tap and shake 200 times, take samples to detect the residual HBsAg activity, take 2 samples of each sample, each 0.1 ml, take the average, and determine the destruction effect. Repeat the test 5 times. E7.2.3 Positive control
When observing the effect of disinfectants on HBsAg destruction, take 50 μL of the test disinfectant and add it to a test tube containing 1.0 ml of 10% calf serum neutralizer. Act for 10 to 30 minutes to prepare a neutralization product solution. Take 1.0 ml of the neutralization product solution and add it to a large test tube, then move the carrier stained with HBsAg into it, tap and shake 200 times, test 3 samples of each sample, each 0.1 ml, and take the average as the positive control value. When observing the effect of ultraviolet rays destroying HBsAg, directly transfer the carrier contaminated with HBsAg into a test tube containing 1.0mL 10% calf serum in PBS (pH7.2-7.4), tap and shake 200 times, test 3 copies of each sample, 0.1mL each, and take the average value as the positive control value.
E7.2.4 Negative control
When observing the effect of disinfectant destroying HBsAg, take 50μL disinfectant and add it to a test tube containing 1mL.10% calf serum neutralizer, act for 10~~30min, take samples for testing, and test 3 copies of each sample, 0.1mL each. Take the average value as the negative control value. When observing the effect of ultraviolet rays destroying HBsAg, the negative control is PBS (pH7.2-7.4) containing 10% calf serum. Test 3 copies of each sample, 0.1mL each, and take the average value as the negative control value. It should be noted that the negative control sample of the kit cannot be used for the negative control. E8 Destruction effect determination
S/N<2.1 is used as the qualified standard for HBsAg antigenic destruction. Where S is the average pulse count per minute (cpm) or optical density (OD) of the sample under test or the positive control sample after the disinfection factor acts. N is the average cpm or OD value of the negative control sample in the test. Additional notes:
This standard is proposed by the Ministry of Health of the People's Republic of China. This standard is drafted by the Institute of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine. The main drafters of this standard are Yuan Qia, Wang Taixing and Gu Jian. This standard is interpreted by the Office of Supervision and Administration of Communicable Disease Prevention and Control of the Ministry of Health, the technical coordination unit entrusted by the Ministry of Health. 267First, take 1mL of neutralizer containing 20% calf serum and 1mL of disinfectant, mix them, and let them react for 10~~30min to make a neutralization product solution, and then test it. Neutralization product solution 0.9mL + HBsAg suspension 0.1mL; d. PBS containing 10% calf serum 0.9mL + HBsAg suspension 0.1mL; e. Neutralizer containing 10% calf serum 0.9mL + HBsAg suspension 0.1mL; f. Neutralization product solution 0.9mL + PBS 0.1mL. After testing, only when the measured values of HBsAg activity in groups c, d, and e are similar (difference less than 10%) and are significantly higher than group b, the HBsAg activity in group a cannot be detected or the detected activity is significantly less than that in group b, and the negative control in group f is normal, can it be proved that the selected neutralizer and its dosage are appropriate. E6.3 When conducting disinfection tests, according to the principle of equal neutralization of disinfectants and neutralizers, the dosage of neutralizers should be appropriately adjusted. After the neutralization effect is determined again according to step E6.2, the antigenic destruction test can be carried out. E7
Destruction test method
E7.1 Suspension method
The suspension method refers to a test method in which HBsAg interacts with the disinfectant in a suspension and observes the antigenic destruction effect. E7.1.1 The concentration of the HBsAg suspension is 104 times the sensitivity of the detection reagent. If the sensitivity of the detection reagent is 1ng/mL, the concentration of HBsAg should be 10ug/mL.
E7.1.2 Take 2.7mL of PBS containing 5% calf serum and add it to 0.3mL of HBsAg suspension with a concentration of 100μg/mL, and mix well. E7.1.3 Take 20mL of calf serum and add it to 80mL of sterilized neutralizer solution and mix well. E7.1.4 Destruction test: Mix a disinfectant with a concentration of 1.25 times the predetermined disinfectant concentration with the HBsAg suspension in a ratio of 4:1, and the volume of the mixed solution should be no less than 1.5 mL. Then place it in a water bath at 20 ± 2 ° C, and act for a specified time. Immediately take 0.3 mL of the mixed solution and mix it with an equal volume of a neutralizer containing 20% calf serum. Act for 10 to 30 minutes, take a sample to determine the activity of the residual HBsAg, and measure 2 copies of each sample in parallel, each copy is 0.1 mL, and take the average value to determine the destruction effect. Observe 3 concentrations of each disinfectant, and observe 4 action times for each concentration. Repeat the test 5 times.
E7.1.5 Positive control: Take 1 mL of a neutralizer containing 20% calf serum, add it to a test tube containing 1 mL of the test disinfectant, mix it, and act for 10 to 30 minutes to make a neutralization product solution. Take 0.9mL of the neutralization product solution and add 0.1mL of HBsAg suspension of the test concentration for sampling and testing. Test three copies of each sample, 0.1mL each, and take the average value as the positive control value. E7.1.6 Negative control: Take 1mL of the neutralizer containing 20% calf serum and add it to a test tube containing 1mL of the test disinfectant, mix well, act for 10-3min, sample and test. Test three copies of each sample, 0.1mL each, and take the average value as the negative control value. 266
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It should be noted that the negative control sample of the kit cannot be used for the negative control. E7.2 Carrier method
The carrier method refers to the test method in which the HBsAg on the carrier surface interacts with the disinfection factor and the antigenic destruction effect is observed. E7.2.1 Preparation of HBsAg carrier
E7.2.1.1 The carrier is a stainless steel sheet with a diameter of 1.5 cm. It is washed with detergent and degreased, and sterilized with pressure steam for use. E7.2.1.2 The concentration of HBsAg suspension is 5×10* times the sensitivity of the detection method. If the sensitivity is 1ng/mL, the concentration of HBsAg should be 50μg/ml.
E7.2.1.3 The contamination method of HBsAg is the dripping method. When dripping, spread the sterilized carrier flat in a sterile plate, use a microinjector to absorb the HBsAg suspension, and drip it in the center of the carrier, 20μL per carrier. Then use L-shaped platinum wire to evenly spread the suspension, put it in a 37C incubator for 40-60min, and perform the antigenic destruction test after the suspension is dry. E7.2.2 Antigenicity destruction test
Take the carrier contaminated with HBsAg, place it flat in a sterile blood container, use a pipette to absorb 50uL of the disinfectant of the predetermined concentration, drip it in the center of the carrier, and spread it evenly so that the entire carrier is evenly treated. Place each group of 2 tablets in a 20±2C water bath. After the prescribed time, use sterile tweezers to move the carrier into a neutralizer tube containing 1.0mL10% calf serum. After 10-30min of action, knock and shake 200 times, take samples to detect the activity of residual HBsAg, take 2 samples of each sample, each 0.1mL, take the average value, and determine the antigenicity destruction effect. Observe 3 concentrations of each disinfectant and 4 action times for each concentration. Repeat the test 5 times. When observing the effect of ultraviolet rays on HBsAg destruction, place the contaminated carrier directly under the action factor. After the prescribed dose is reached, move the carrier into a test tube containing 1.0 ml of 10% calf serum PBS, tap and shake 200 times, take samples to detect the residual HBsAg activity, take 2 samples of each sample, each 0.1 ml, take the average, and determine the destruction effect. Repeat the test 5 times. E7.2.3 Positive control
When observing the effect of disinfectants on HBsAg destruction, take 50 μL of the test disinfectant and add it to a test tube containing 1.0 ml of 10% calf serum neutralizer. Act for 10 to 30 minutes to prepare a neutralization product solution. Take 1.0 ml of the neutralization product solution and add it to a large test tube, then move the carrier stained with HBsAg into it, tap and shake 200 times, test 3 samples of each sample, each 0.1 ml, and take the average as the positive control value. When observing the effect of ultraviolet rays destroying HBsAg, directly transfer the carrier contaminated with HBsAg into a test tube containing 1.0mL 10% calf serum in PBS (pH7.2-7.4), tap and shake 200 times, test 3 copies of each sample, 0.1mL each, and take the average value as the positive control value.
E7.2.4 Negative control
When observing the effect of disinfectant destroying HBsAg, take 50μL disinfectant and add it to a test tube containing 1mL.10% calf serum neutralizer, act for 10~~30min, take samples for testing, and test 3 copies of each sample, 0.1mL each. Take the average value as the negative control value. When observing the effect of ultraviolet rays destroying HBsAg, the negative control is PBS (pH7.2-7.4) containing 10% calf serum. Test 3 copies of each sample, 0.1mL each, and take the average value as the negative control value. It should be noted that the negative control sample of the kit cannot be used for the negative control. E8 Destruction effect determination
S/N<2.1 is used as the qualified standard for HBsAg antigenic destruction. Where S is the average pulse count per minute (cpm) or optical density (OD) of the sample under test or the positive control sample after the disinfection factor acts. N is the average cpm or OD value of the negative control sample in the test. Additional notes:
This standard is proposed by the Ministry of Health of the People's Republic of China. This standard is drafted by the Institute of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine. The main drafters of this standard are Yuan Qia, Wang Taixing and Gu Jian. This standard is interpreted by the Office of Supervision and Administration of Communicable Disease Prevention and Control of the Ministry of Health, the technical coordination unit entrusted by the Ministry of Health. 2674 Destruction test: Mix the disinfectant with a concentration of 1.25 times the predetermined disinfectant concentration with the HBsAg suspension in a ratio of 4:1, and the volume of the mixed solution shall not be less than 1.5mL. Then place it in a water bath at 20±2℃, and act for a specified time. Immediately take 0.3mL of the mixed solution and mix it with an equal volume of a neutralizer containing 20% calf serum. Act for 10 to 30 minutes, take a sample to determine the activity of the residual HBsAg, and measure 2 copies of each sample in parallel, each copy is 0.1mL, and take the average value to determine the destruction effect. Observe 3 concentrations of each disinfectant, and observe 4 action times for each concentration. Repeat the test 5 times.
E7.1.5 Positive control: Take 1mL of the neutralizer containing 20% calf serum, add it to a test tube containing 1mL of the test disinfectant, mix it, and act for 10 to 30 minutes to make a neutralization product solution. Take 0.9mL of the neutralization product solution and add 0.1mL of HBsAg suspension of the test concentration for sampling and testing. Test three copies of each sample, 0.1mL each, and take the average value as the positive control value. E7.1.6 Negative control: Take 1mL of the neutralizer containing 20% calf serum and add it to a test tube containing 1mL of the test disinfectant, mix well, act for 10-3min, sample and test. Test three copies of each sample, 0.1mL each, and take the average value as the negative control value. 266
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It should be noted that the negative control sample of the kit cannot be used for the negative control. E7.2 Carrier method
The carrier method refers to the test method in which the HBsAg on the carrier surface interacts with the disinfection factor and the antigenic destruction effect is observed. E7.2.1 Preparation of HBsAg carrier
E7.2.1.1 The carrier is a stainless steel sheet with a diameter of 1.5 cm. It is washed with detergent and degreased, and sterilized with pressure steam for use. E7.2.1.2 The concentration of HBsAg suspension is 5×10* times the sensitivity of the detection method. If the sensitivity is 1ng/mL, the concentration of HBsAg should be 50μg/ml.
E7.2.1.3 The contamination method of HBsAg is the dripping method. When dripping, spread the sterilized carrier flat in a sterile plate, use a microinjector to absorb the HBsAg suspension, and drip it in the center of the carrier, 20μL per carrier. Then use L-shaped platinum wire to evenly spread the suspension, put it in a 37C incubator for 40-60min, and perform the antigenic destruction test after the suspension is dry. E7.2.2 Antigenicity destruction test
Take the carrier contaminated with HBsAg, place it flat in a sterile blood container, use a pipette to absorb 50uL of the disinfectant of the predetermined concentration, drip it in the center of the carrier, and spread it evenly so that the entire carrier is evenly treated. Place each group of 2 tablets in a 20±2C water bath. After the prescribed time, use sterile tweezers to move the carrier into a neutralizer tube containing 1.0mL10% calf serum. After 10-30min of action, knock and shake 200 times, take samples to detect the activity of residual HBsAg, take 2 samples of each sample, each 0.1mL, take the average value, and determine the antigenicity destruction effect. Observe 3 concentrations of each disinfectant and 4 action times for each concentration. Repeat the test 5 times. When observing the effect of ultraviolet rays on HBsAg destruction, place the contaminated carrier directly under the action factor. After the prescribed dose is reached, move the carrier into a test tube containing 1.0 ml of 10% calf serum PBS, tap and shake 200 times, take samples to detect the residual HBsAg activity, take 2 samples of each sample, each 0.1 ml, take the average, and determine the destruction effect. Repeat the test 5 times. E7.2.3 Positive control
When observing the effect of disinfectants on HBsAg destruction, take 50 μL of the test disinfectant and add it to a test tube containing 1.0 ml of 10% calf serum neutralizer. Act for 10 to 30 minutes to prepare a neutralization product solution. Take 1.0 ml of the neutralization product solution and add it to a large test tube, then move the carrier stained with HBsAg into it, tap and shake 200 times, test 3 samples of each sample, each 0.1 ml, and take the average as the positive control value. When observing the effect of ultraviolet rays destroying HBsAg, directly transfer the carrier contaminated with HBsAg into a test tube containing 1.0mL 10% calf serum in PBS (pH7.2-7.4), tap and shake 200 times, test 3 copies of each sample, 0.1mL each, and take the average value as the positive control value.
E7.2.4 Negative control
When observing the effect of disinfectant destroying HBsAg, take 50μL disinfectant and add it to a test tube containing 1mL.10% calf serum neutralizer, act for 10~~30min, take samples for testing, and test 3 copies of each sample, 0.1mL each. Take the average value as the negative control value. When observing the effect of ultraviolet rays destroying HBsAg, the negative control is PBS (pH7.2-7.4) containing 10% calf serum. Test 3 copies of each sample, 0.1mL each, and take the average value as the negative control value. It should be noted that the negative control sample of the kit cannot be used for the negative control. E8 Destruction effect determination
S/N<2.1 is used as the qualified standard for HBsAg antigenic destruction. Where S is the average pulse count per minute (cpm) or optical density (OD) of the sample under test or the positive control sample after the disinfection factor acts. N is the average cpm or OD value of the negative control sample in the test. Additional notes:
This standard is proposed by the Ministry of Health of the People's Republic of China. This standard is drafted by the Institute of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine. The main drafters of this standard are Yuan Qia, Wang Taixing and Gu Jian. This standard is interpreted by the Office of Supervision and Administration of Communicable Disease Prevention and Control of the Ministry of Health, the technical coordination unit entrusted by the Ministry of Health. 2674 Destruction test: Mix the disinfectant with a concentration of 1.25 times the predetermined disinfectant concentration with the HBsAg suspension in a ratio of 4:1, and the volume of the mixed solution shall not be less than 1.5mL. Then place it in a water bath at 20±2℃, and act for a specified time. Immediately take 0.3mL of the mixed solution and mix it with an equal volume of a neutralizer containing 20% calf serum. Act for 10 to 30 minutes, take a sample to determine the activity of the residual HBsAg, and measure 2 copies of each sample in parallel, each copy is 0.1mL, and take the average value to determine the destruction effect. Observe 3 concentrations of each disinfectant, and observe 4 action times for each concentration. Repeat the test 5 times.
E7.1.5 Positive control: Take 1mL of the neutralizer containing 20% calf serum, add it to a test tube containing 1mL of the test disinfectant, mix it, and act for 10 to 30 minutes to make a neutralization product solution. Take 0.9mL of the neutralization product solution and add 0.1mL of HBsAg suspension of the test concentration for sampling and testing. Test three copies of each sample, 0.1mL each, and take the average value as the positive control value. E7.1.6 Negative control: Take 1mL of the neutralizer containing 20% calf serum and add it to a test tube containing 1mL of the test disinfectant, mix well, act for 10-3min, sample and test. Test three copies of each sample, 0.1mL each, and take the average value as the negative control value. 266
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It should be noted that the negative control sample of the kit cannot be used for the negative control. E7.2 Carrier method
The carrier method refers to the test method in which the HBsAg on the carrier surface interacts with the disinfection factor and the antigenic destruction effect is observed. E7.2.1 Preparation of HBsAg carrier
E7.2.1.1 The carrier is a stainless steel sheet with a diameter of 1.5 cm. It is washed with detergent and degreased, and sterilized with pressure steam for use. E7.2.1.2 The concentration of HBsAg suspension is 5×10* times the sensitivity of the detection method. If the sensitivity is 1ng/mL, the concentration of HBsAg should be 50μg/ml.
E7.2.1.3 The contamination method of HBsAg is the dripping method. When dripping, spread the sterilized carrier flat in a sterile plate, use a microinjector to absorb the HBsAg suspension, and drip it in the center of the carrier, 20μL per carrier. Then use L-shaped platinum wire to evenly spread the suspension, put it in a 37C incubator for 40-60min, and perform the antigenic destruction test after the suspension is dry. E7.2.2 Antigenicity destruction test
Take the carrier contaminated with HBsAg, place it flat in a sterile blood container, use a pipette to absorb 50uL of the disinfectant of the predetermined concentration, drip it in the center of the carrier, and spread it evenly so that the entire carrier is evenly treated. Place each group of 2 tablets in a 20±2C water bath. After the prescribed time, use sterile tweezers to move the carrier into a neutralizer tube containing 1.0mL10% calf serum. After 10-30min of action, knock and shake 200 times, take samples to detect the activity of residual HBsAg, take 2 samples of each sample, each 0.1mL, take the average value, and determine the antigenicity destruction effect. Observe 3 concentrations of each disinfectant and 4 action times for each concentration. Repeat the test 5 times. When observing the effect of ultraviolet rays on HBsAg destruction, place the contaminated carrier directly under the action factor. After the prescribed dose is reached, move the carrier into a test tube containing 1.0 ml of 10% calf serum PBS, tap and shake 200 times, take samples to detect the residual HBsAg activity, take 2 samples of each sample, each 0.1 ml, take the average, and determine the destruction effect. Repeat the test 5 times. E7.2.3 Positive control
When observing the effect of disinfectants on HBsAg destruction, take 50 μL of the test disinfectant and add it to a test tube containing 1.0 ml of 10% calf serum neutralizer. Act for 10 to 30 minutes to prepare a neutralization product solution. Take 1.0 ml of the neutralization product solution and add it to a large test tube, then move the carrier stained with HBsAg into it, tap and shake 200 times, test 3 samples of each sample, each 0.1 ml, and take the average as the positive control value. When observing the effect of ultraviolet rays destroying HBsAg, directly transfer the carrier contaminated with HBsAg into a test tube containing 1.0mL 10% calf serum in PBS (pH7.2-7.4), tap and shake 200 times, test 3 copies of each sample, 0.1mL each, and take the average value as the positive control value.
E7.2.4 Negative control
When observing the effect of disinfectant destroying HBsAg, take 50μL disinfectant and add it to a test tube containing 1mL.10% calf serum neutralizer, act for 10~~30min, take samples for testing, and test 3 copies of each sample, 0.1mL each. Take the average value as the negative control value. When observing the effect of ultraviolet rays destroying HBsAg, the negative control is PBS (pH7.2-7.4) containing 10% calf serum. Test 3 copies of each sample, 0.1mL each, and take the average value as the negative control value. It should be noted that the negative control sample of the kit cannot be used for the negative control. E8 Destruction effect determination
S/N<2.1 is used as the qualified standard for HBsAg antigenic destruction. Where S is the average pulse count per minute (cpm) or optical density (OD) of the sample under test or the positive control sample after the disinfection factor acts. N is the average cpm or OD value of the negative control sample in the test. Additional notes:
This standard is proposed by the Ministry of Health of the People's Republic of China. This standard is drafted by the Institute of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine. The main drafters of this standard are Yuan Qia, Wang Taixing and Gu Jian. This standard is interpreted by the Office of Supervision and Administration of Communicable Disease Prevention and Control of the Ministry of Health, the technical coordination unit entrusted by the Ministry of Health. 2671mL, take the average value, and determine the destruction effect. Repeat the test 5 times. E7.2.3 Positive control
When observing the effect of disinfectant in destroying HBsAg, take 50μL of the test disinfectant and add it to a test tube containing 1.0mL10% calf serum neutralizer. Act for 10 to 30 minutes to make a neutralization product solution. Take 1.0mL of the neutralization product solution and add it to a large test tube, then move the carrier stained with HBsAg into it, knock and shake it 200 times, and test 3 copies of each sample, each with 0.1mL, and take the average value as the positive control value. When observing the effect of ultraviolet rays in destroying HBsAg, directly move the carrier contaminated with HBsAg into a test tube containing 1.0mL10% calf serum PBS (pH7.2-7.4), knock and shake it 200 times, and test 3 copies of each sample, each with 0.1mL, and take the average value as the positive control value.
E7.2.4 Negative control
When observing the effect of disin
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