This standard specifies the diagnostic criteria and treatment principles for influenza. This standard is applicable to the diagnosis, reporting and treatment of influenza by medical, health and healthcare institutions and personnel at all levels and types. GB 15994-1995 Diagnostic criteria and treatment principles for influenza GB15994-1995 standard download decompression password: www.bzxz.net

GB15994—1995
Influenza (abbreviated as influenza) is an acute respiratory infectious disease caused by influenza virus, which is mainly transmitted through air droplets. There are three types of influenza virus: A, B and C. Among them, influenza caused by type A is the most widespread and serious, type B often causes outbreaks, and type C often causes sporadic diseases in children. Although this disease is classified as a statutory Class C infectious disease in my country, once it becomes popular, it spreads quickly and affects a wide range of areas, which has a great impact on people's health and labor productivity, and poses a greater threat to the elderly, weak and sick people and infants. In order to implement the "Law of the People's Republic of China on the Prevention and Control of Infectious Diseases" and the "Implementation Measures for the Law of the People's Republic of China on the Prevention and Control of Infectious Diseases", this standard is specially formulated. Appendix A of this standard is the appendix of the standard;
Appendix B and Appendix C of this standard are both indicative appendices. This standard was proposed by the Ministry of Health of the People's Republic of China. The drafting unit of this standard: Institute of Virology, Chinese Academy of Preventive Medicine. The main drafter of this standard: Tao Sanju.
This standard is interpreted by the Ministry of Health's technical coordination unit, the Ministry of Health's Office of Supervision and Administration of Infectious Disease Prevention and Control. 3.59
1 Scope
National Standard of the People's Republic of China
Diagnostic criteria and principles of management of influenza This standard specifies the diagnostic criteria and management principles of influenza. GB15994--1995
This standard applies to the diagnosis, reporting and management of influenza by medical, health and health care institutions and personnel at all levels and of all types. 2 Diagnostic principles
When influenza is prevalent, a preliminary diagnosis can be made for the patient based on clinical symptoms and epidemiology. To confirm the diagnosis, it is necessary to isolate virus-positive or double serum antibodies of the patient, and to determine whether the antibody in the recovery period is 4 times or more higher than that in the acute period. 3 Diagnostic criteria
3.1 Epidemiological history
A large number of patients with upper respiratory tract infection appear in a unit or region at the same time during the epidemic season; or the number of patients with upper respiratory tract infection in the region or neighboring regions has increased significantly in the near future, or the number of patients with upper respiratory tract infection in the hospital outpatient department has increased significantly. 3.2 Clinical symptoms
3.2.1 Symptoms of poisoning such as acute chills, high fever, headache, dizziness, body aches, and fatigue may occur. 3.2.2 May be accompanied by respiratory symptoms such as sore throat, dry cough, runny nose, and tearing. 3.2.3 A few cases have loss of appetite, accompanied by digestive symptoms such as abdominal pain, abdominal distension, vomiting, and diarrhea. 3.3 Laboratory diagnosis
3.3.1 Blood test shows that the total white blood cell count is not high or is low. 3.3.2 Influenza virus was isolated from the patient's nasopharyngeal secretions (see Appendix A). 3.3.3 The anti-influenza virus antibody titer in the serum of patients in the recovery period is 4 times or more higher than that in the acute period (see Appendix B). 3.3.4 Direct examination of respiratory epithelial cells for influenza virus antigen is positive (see C1 in Appendix C). 3.3.5 The specimen is antigen positive after one generation of sensitive cell proliferation (see C2 in Appendix C). 3.4 Case classification
3.4.1 Suspected cases
Meet 3.1 plus 3.2 or 3.1 plus 3.2 plus 3.3.1. 3.4.2 Confirmed cases
Suspected cases plus 3.3.2 or 3.3.3 or 3.3.4 or 3.3.5. 4 Treatment principles
4.1 Early isolation of patients, symptomatic treatment of patients and prevention and treatment of complications Early detection of patients and early separation of patients are the most important measures. Symptomatic treatment is generally used for patient treatment. Prevention and treatment of complications should mainly prevent influenza virus pneumonia and bacterial pneumonia, especially for the elderly and weak or those with cardiovascular and chronic respiratory diseases or infants and young children. Approved by the State Administration of Technical Supervision on December 15, 1995 360
Implementation on July 1, 1996
GB15994-1995
Influenza itself or its complications may cause death, and special attention should be paid to strengthening treatment and care. Preventive measures
Generally, comprehensive preventive measures are adopted, such as paying attention to hygiene, physical exercise and nutrition, and maintaining indoor air circulation; relative isolation measures should be taken for susceptible people, such as avoiding contact with patients and not going to public places; for the elderly and weak, inactivated vaccination or taking adamantane can be used for prevention when necessary (but it should be noted that adamantane is only effective for the prevention and treatment of influenza A). 361
A1 Virus isolation
GB15994-1995
Appendix A
(Standard Appendix)
Etiological diagnosis method
Collect specimens from the respiratory tract of suspected influenza patients to isolate pathogens. A2 Collection and processing of specimens
A2.1 The specimen collection time should be as early as possible in the early stage of the disease (within 1 to 3 days). A2.2 The specimen collection method is usually cotton swab wiping or throat gargling. The cotton swab method is used to collect specimens from children. When collecting, slightly dip the sterile cotton swab in the specimen collection solution (pH 7.2-7.6 containing 20%-40% sterile broth in saline or Hanks solution or saline), repeatedly wipe the patient's throat several times, and then put the cotton swab into a test tube containing the above collection solution and plug it with a cotton plug. The throat wash method is used to collect specimens from adults. When collecting, let the patient cough first, then use about 10mL of collection solution to repeatedly wash the throat for about 1 minute and spit it into the tube. If conditions permit, use negative pressure suction to extract nasopharyngeal secretions for children's specimens and nasopharyngeal wash solution for adult specimens to increase the positive rate of separation. After the specimen is collected, place it in an ice pot (4C) and send it to the laboratory as soon as possible.
A2.3 Specimen processing: Use a capillary pipette to repeatedly blow the specimen liquid (if it is a throat swab specimen, first squeeze the throat swab repeatedly and then discard it) to break up the mucus, let it stand at 4'C for 5-10 minutes, take 3mL of supernatant after natural precipitation, add 1000 units of penicillin and 1000ug of streptomycin per milliliter, mix well, treat at 4℃ for 2-4 hours, and then inoculate. If the specimen is heavily contaminated, it can be placed at 4℃ overnight. If it is expected that the specimen cannot be inoculated within 48 hours, the specimen should be stored at a low temperature (preferably -70℃). A3 Inoculation and culture method
The most commonly used chicken embryo culture method and tissue culture method, if conditions permit, can also be used for virus isolation by both methods. A3.1 Chicken embryo culture method: Take 9~11 day old chicken embryos, inoculate 0.2mL of the treated specimen fluid through the amniotic cavity and the allantoic cavity, inoculate 3~4 embryos for each specimen, and culture in a 33~35℃ incubator for 3 days, then place the chicken embryo in a 4℃ refrigerator overnight (or place in a 20℃ refrigerator for 1 hour and then place in 4℃ for several hours), then harvest urine and amniotic fluid respectively, and make preliminary hemagglutination (the method is to use a capillary pipette to take 1~2 drops of urine or amniotic fluid, place in a large-pore plastic plate, then add 1~2 drops of 1% chicken red blood cells, shake well, place at room temperature or 4℃ for 30~45 minutes to observe the results, according to the different degrees of hemagglutination, it can be divided into 10+10+, 10+10+, 10+10+, 10+10+, ±, and -). If the hemagglutination is 10+10+-10+10+, then determine the hemagglutination titer according to the serial multiple dilution, if it has a certain hemagglutination titer, it can be identified. If the hemagglutination is negative, it can be blindly propagated for 2 generations, if it is still negative, it will be discarded. A3.2 Tissue culture method: 0.2 mL of the treated specimen solution is inoculated into primary human embryonic kidney (or monkey kidney or hamster kidney) or dog kidney passage cells (Madin Darby Canine Kidney, MDCK) grown into a monolayer. Each specimen is inoculated into 4 tubes. After adsorption at 37°C for 1~2 hours, the specimen solution is discarded. Then 1 mL of 199 maintenance solution containing 2 μg of pancreatic enzyme per liter is added. Culture at 33°C for 7~10 days and observe the lesions every day. From the third day on, 0.4% chicken or guinea pig red blood cells are used to perform a red blood cell adsorption test on the culture tube every other day (the cell culture supernatant is harvested aseptically before the test). If negative, the harvested supernatant is used for incubation and passage. If positive, the hemagglutination titer of the supernatant is measured. If the specimen is negative in the first generation of red blood cell adsorption test, all the harvested supernatant can be mixed and blindly passaged for 2 generations. If it is still negative, it is discarded. A4 Identification of Newly Isolated Strains
Newly isolated influenza viruses can be identified by hemagglutination inhibition, complement fixation, neutralization, and hemagglutination inhibition tests. The most commonly used and simplest method is the hemagglutination inhibition test, and complement fixation test can be used when necessary. A4.1 Hemagglutination inhibition test: Use immune sera of currently popular influenza A3, A1, and B virus representative strains to perform hemagglutination inhibition tests on newly isolated influenza viruses to observe whether the newly isolated strains can be inhibited by the immune sera. See Appendix B for the hemagglutination inhibition test method. A4.2 Hemagglutination inhibition test: Take tissue culture tubes with positive hemagglutination test results and normal cell control tubes, wash the cells twice with Hanks solution, add 0.2 mL of immune serum treated with cholera filtrate (RDE) (1:10), then add 0.6 mL of Hanks, and let it stand at room temperature for 30 minutes, then add 0.4% chicken (or guinea pig) red blood cells.2mL, place at room temperature for 30 minutes, and observe under an ordinary optical microscope for blood cell adsorption. If blood cell adsorption is inhibited by the immune serum used, it indicates that the isolated virus is consistent with or close to the type of the immune serum used. A4.3 Complement fixation test: If the hemagglutination of the newly isolated virus cannot be inhibited by the known influenza A (A3, A1) or B or C virus immune serum, it should be considered that it may be a new subtype of influenza A virus. The immune serum against the nucleoprotein of influenza A and B viruses can be used for complement fixation test to determine the type.
Appendix B
(Suggested Appendix)
Serological Diagnosis Method
B1 Hemagglutination Inhibition Test Method
B1.1 Principle
Some animal red blood cells (such as chickens, guinea pigs, etc.) and human "O" type red blood cells have influenza virus receptors, which can produce red blood cell agglutination when encountering influenza viruses, referred to as hemagglutination. If specific antibodies are reacted with influenza virus (hemagglutinin) before adding red blood cells, no agglutination will occur, which is called hemagglutination inhibition. After using quantitative hemagglutinin to react with serum antibodies of different dilutions, the highest dilution that can completely inhibit hemagglutination is the titer of hemagglutination inhibition antibody.
B1.2 Main materials
Macroporous plastic plate, 1mL pipette or 1mL pipette. B1.3 Collection and processing of double serum
Acute serum is collected within 3 days of onset, and convalescent serum is collected 2~~4 weeks after onset. Take 0.1mL of separated serum and add 0.9 (or 0.4)ml. RDE (i.e. 1:10 or 1:5 dilution) and shake well, and place in a 37℃ water bath overnight to remove nonspecific inhibitors in the serum. The next day, place in a 56℃ water bath for 50min to inactivate excess RDE. B1.4 Preparation of influenza virus hemagglutinin
The allantoic fluid harvested 48 hours after the influenza virus was inoculated into the chicken embryo was preserved by adding 1/10000 thimerosal. In order to delay the precipitation of urine salts, it can be diluted with sterile saline in equal amounts and stored at 4°C. B1.5 Preparation of chicken red blood cell suspension
Normal healthy chicken blood is extracted from the vein or heart, stored in Alsevers solution, and stored at 4°C. Wash with saline three times before use, and centrifuge at 2000r/min for 10 minutes at the last time. The red blood cells are diluted with saline to a concentration of 1%. B1.6 Hemagglutinin titration
Dilute influenza virus hemagglutinin in a large-pore plastic plate with physiological saline starting from 1:5 in series, add an equal amount of 1% chicken red blood cells to each well with 0.25mL, shake well, place at room temperature or 4℃ for 30-45 minutes and observe the results. The highest dilution of hemagglutinin that shows ++ hemagglutination is its hemagglutination titer, that is, 1 hemagglutination unit. Four hemagglutinin units are used in the experiment. For example, the hemagglutinin titer is 1:320, which is 1 unit, and 4 units are 1:80 dilution. Before the formal experiment, 4 hemagglutination units are taken for the experiment after proofreading. B1.7 Hemagglutination inhibition test steps
B1.7.1Dilute the above-mentioned treated serum to be tested (1:10 or 1:5) in series with physiological saline, and add 0.25ml to each well. B1.7.2Add an equal amount of 4 units of prepared hemagglutinin. B1.7.3 Add 0.25mL 1% chicken red blood cells to each well, shake well and place at room temperature or 4C for 30-45 minutes. B1.8 Observation of results
When observing the results, tilt the blood coagulation plate for tens of seconds, and read the results when the red blood cells in the negative well slide freely and form teardrops. The reciprocal of the highest dilution of the serum that shows complete inhibition is the antibody titer. When testing two serum samples from patients, they must be tested in the same test, and the antibody titer of the convalescent serum is 4 times or more higher than the acute phase antibody titer. 363
B2 Type-specific complement fixation test
B2.1 Principle
GB15994-1995
Each of the three influenza viruses A, B, and C has a specific soluble antigen (nucleoprotein antigen). Each subtype of influenza A virus has the same soluble antigen, which is completely different from the soluble antigen of type B or C. Therefore, the complement fixation test can be used to distinguish them. When influenza viruses undergo great mutations or new subtypes emerge and cause a pandemic, it is often necessary to use complement fixation tests and hemagglutination inhibition tests simultaneously to perform serological diagnosis of new strains because there is no time to fully grasp the characteristics of the newly isolated strains. B2.2 Preparation of type-specific immune serum
B2.2.1 Antigens for immunization
Type A is the PR8 strain and type B is the Lee strain, both of which are mouse-adapted strains. Under ether anesthesia, 12-16g mice are infected nasally with 0.05mL per mouse. When most mice become ill and some die 3-5 days after infection, the mice are dissected. The lungs are ground and made into a 10% suspension with saline. The coarse pieces are removed by low-speed centrifugation. The supernatant mouse lung suspension is the antigen for immunization. B2.2.2 Preparation of immune serum
Select healthy guinea pigs weighing 300-500g, collect 3-5mL of blood before immunization, separate the serum, and freeze it for use as a control. The guinea pigs were lightly anesthetized with ether, and 0.5 mL of the above mouse lung suspension antigen was dripped into the nasal cavity. The immunization was performed 3 to 4 times, with an interval of one week between each immunization. At the same time as the last immunization, 2 mL of mouse lung suspension was injected into the abdominal cavity. A week later, blood was tested. If the serum titer to the same type of urine membrane antigen exceeded 1:80, and there was no cross reaction with the heterotypic antigen, blood was immediately collected and the serum was separated and aliquoted and frozen at low temperature (below -20°C). The serum was inactivated in a 56°C water bath for 30 minutes before the test. B2.3 Preparation of soluble antigens
Use influenza A and B viruses or newly isolated viruses to inoculate and harvest allantoic fluid as usual. If the urine hemagglutination is above 1:40, harvest the urine membrane, wash the allantoic membrane with saline once, cut the membrane into several small pieces with sterile scissors and put them into a test tube. Add 1ml of sterile physiological saline to each chicken embryo, freeze and thaw three times, centrifuge at 3000r/min for 15min after the last thaw, aspirate the supernatant, which is the soluble antigen, add 1:10000 thimerosal for preservation, and place in a 4℃ refrigerator for at least one month. B2.4 Collection of double serum
Collect serum from patients in the acute phase (within 3 days of onset) and the recovery phase (10 to 30 days after onset). The serum was inactivated at 56℃ for 30min before the test. B2.51% sheep red blood cell suspension preparation, hemolysin, complement preparation and titration according to conventional methods. B2.6 Complement fixation test steps
Perform as usual.
B2.7 Result determination
B2.7.1 If the urine membrane antigen of the tested virus reacts positively with the immune serum of influenza A (or B), it can be diagnosed as influenza A (or B). If both A and B are negative, influenza C or other viruses should be considered. B2.7.2 If the double serum has a 4-fold or more increase in the antigen of type A (or type B), it can be diagnosed as influenza A (or type B) virus infection.
B2.7.3 The complement fixation antibody titer of the normal population is generally below 1:16. If the antibody of a single convalescent serum is above 1:32, it can be used as a reference for diagnosis in combination with clinical symptoms.
B2.7.4 In the survey of serum antibody levels in the population, complement fixation antibodies above 1:32 can be used as evidence of recent influenza infection. B3 Neuraminidase inhibition test
B3.1 Principle
After the fetuin substrate is acted upon by the influenza virus neuraminidase, N-acetylneuraminic acid is released, and N-acetylneuraminic acid is converted into β-formaldehydepyruvate by oxidation with iodate, which forms a chromophore by the action of thiobarbituric acid. The chromophore is extracted with an organic solvent and the color is compared on a spectrophotometer. The above reaction can be used to measure the neuraminidase activity of the influenza virus. The darker the color of the reaction, the stronger the enzyme activity. A standard dose can also be selected for the neuraminidase inhibition test. The neuraminidase inhibition test is to incubate a standardized dose of neuraminidase (i.e., influenza virus allantoic fluid at a certain dilution) with a serially diluted test serum and normal serum, measure the inhibitory titer of the serum on the neuraminidase activity, and calculate the neuraminidase inhibition titer of the serum. B3.2 Equipment and reagents
B3.2.1 Small test tubes, 10mL pipettes and spectrophotometer, etc. B3.2.2 Phosphate buffer (PBS pH 5.9) and physiological saline. B3.2.3 Fetuin substrate solution: 450~500mg of freeze-dried fetuin is added to 10mL of distilled water, dissolved and stored at 4℃, and freshly prepared every week.
B3.2.4 Sodium periodate solution: Weigh 4.8g of sodium periodate (NaIO.) and dissolve it in 38mL of distilled water, add 62mL of orthophosphoric acid (concentrated), mix thoroughly, pour into a brown ground-mouth bottle, and store at room temperature. B3.2.5 Arsenic reagent: weigh 10g sodium arsenate (VaAsO,) and 7.1g anhydrous sodium sulfate and dissolve them in 100mL distilled water, add 0.3mL concentrated sulfuric acid, place in a bottle with a glass stopper, and add 4% thiobarbituric acid. B3.2.6 Thiobarbituric acid reagent: weigh 1.2g thiobarbituric acid and 14.Add 2g of anhydrous sodium sulfate to 200mL of distilled water, heat in a boiling water bath to dissolve, and prepare freshly every week. B3.2.7 Acidified butanol reagent: Add 5ml of concentrated hydrochloric acid to 100mL of n-butanol, place in a brown bottle with a glass stopper, and store at room temperature. B3.3 Determination of neuraminidase activity
B3.3.1 Use physiological saline to make serial dilutions of the virus (1:2 to 1:32) and divide into small test tubes, 0.1mL per tube. Neuraminidase of standard virus and unknown virus should be determined in the same test. B3.3.2 Add 0.1mL of physiological saline to each tube, and then add 0.1mL of fetal protein substrate prepared with pH 5.9 PBS. Replace virus with saline in the control tube, mix thoroughly, and place in a 37C water bath for 18h. B3.3.3 Take out the test tube from the water bath and cool at room temperature. Add 0.1mL of periodate reagent to each tube, mix thoroughly, and let stand at room temperature for 20min (time must be accurate).
B3.3.4 Add 1mL of arsenic reagent to each tube. Brown color appears due to the release of iodine. Shake the test tube until the brown color disappears. B3.3.5 Add 2.5mL of thiobarbital reagent to each tube and mix thoroughly (this reagent needs to be added after boiling and dissolving). Use cotton plugs to seal the tube mouth.
B3.3.6 Immediately place the test tube in a boiling water bath and boil for 10 minutes. Red color will appear, which is the manifestation of neuraminidase activity. The darker the color, the higher the enzyme activity.
B3.3.7 Cool the test tube in ice water, remove the cotton plug, add 4ml of butanol reagent, replace with a clean rubber plug, shake the test tube vigorously by hand, extract the color and transfer it to the organic (butanol) phase. B3.3.8 Centrifuge at 1500r/min for 5~~10min to make the butanol layer clear and transparent. Carefully aspirate the butanol on the upper layer and put it into a clean test tube for inspection.
B3.3.9 Pour the above-mentioned samples into the light-transmitting pools from high dilution to low dilution, and add the virus-free control tube liquid to the first light-transmitting pool. Adjust the wavelength of the spectrophotometer to 549nm, adjust the control tube to zero, and then read the optical density of each dilution sample in turn. B3.3.10 Calculation of enzyme units: The virus dilution with an optical density of 0.45 to 0.85 during the enzyme activity determination is regarded as an enzyme unit. B3.4 Neuraminidase inhibition test
B3.4.1 Determine the enzyme activity according to the above method, select a virus dilution of 1 enzyme unit (i.e., a virus dilution with an optical density of 0.45 to 0.85 during the enzyme activity determination), and add 0.1ml of Lc to each tube. B3.4.2 Make a series of multiple dilutions of the tested serum, and add 0.1mL of serum of different dilutions to each virus tube. The serum control tube is the lowest dilution serum with physiological saline instead of virus, and the reaction is carried out in a 37C water bath for 1h. The normal serum control group is made in the same way. B3.4.3 Add 0.1 mL of fetuin prepared in pH 5.9 PBS to each tube, shake well, and incubate in a 37°C water bath for 18 hours. B3.4.4 Remove the test tube from the water bath and gradually add the aforementioned reagents according to B3.3.3.B3.3.7. B3.4.5 When measuring the optical density, start from the low dilution serum tube to the high dilution serum tube, and the detection method is the same as B3.3.9. B3.5 Calculation method of neuraminidase inhibition antibody titer B3.5.1 Based on the experimental results of the two groups of tests on antiserum and normal serum, the quotient of the optical density of each group of serum at the same dilution is obtained, which is the percentage of enzyme activity remaining after the action of the serum. The higher the serum dilution, the higher the residual activity. Find the two serum dilutions before and after 50% enzyme activity and their corresponding enzyme activity percentages, and calculate the serum neuraminidase inhibitory antibody titer according to the formula B3.5.2 Calculation formula
C, -C2
A=(Bl-B2)+ 50-C)
Wherein: A-the reciprocal of serum neuraminidase inhibitory antibody titer, B,——the reciprocal of serum dilution with enzyme activity higher than 50%; B,——the reciprocal of serum dilution with enzyme activity lower than 50%; C,—the percentage of enzyme activity higher than 50%; C2——the percentage of enzyme activity lower than 50%; 50—constant.
B3.5.3 Calculation example
First calculate the enzyme activity after the serum is acted on:
Serum dilution (0.5log10)
Immune serum + 1 unit virus
Normal serum + 1 unit virus
Neuraminidase activity, %
1) Optical density.
(B1)
Then convert the logarithmic dilution into geometric dilution: for example, 10-3 is converted to 1/1000, and 10-3.5 is converted to 1/3200. Finally, substitute into the formula:
(3200 - 1000) -
70—45
+10002200→5+1000=1440
50—45
The neuraminidase inhibition titer of this antiserum is 1:1440. Appendix C
(Suggested Appendix)
Rapid diagnosis method for influenza
C1 Direct examination of antigens in respiratory epithelial cells C1.1 Principle
Since the main infection site of influenza virus is respiratory epithelial cells, the exfoliated cells of the patient's respiratory specimen contain influenza virus antigens, and diagnosis can be made by directly examining the antigens in the exfoliated epithelial cells. The examination method can be immunofluorescence, which can generally be completed within 3 to 4 hours and can be directly typed. C1.2 Collection and preparation of specimensWww.bzxZ.net
For children, it is best to use negative pressure suction to collect respiratory secretions, and for adults, it is best to use nasopharyngeal wash. Each specimen can be divided into two parts, one part is frozen at low temperature for virus isolation, and the other part is used with pH7.2 phosphate buffer (PBS). The mucus is blown and dispersed, and the supernatant is removed by centrifugation at 1500r/min for 15 minutes. The cell pellet is washed 1 to 2 times with PBS. After the last centrifugation, the supernatant is removed, and an appropriate amount of pH7.2 PBS is added to resuspend the cells, and the cells are dropped onto the glass slide with a ring, dried naturally, and fixed with cold acetone for 10 minutes. C1.3 Indirect immunofluorescence method
C1.3.1 Block the specimen slide with 10% fresh sheep or bovine serum and place it in a humidified box at 37℃ for 30 minutes. C1.3.2 Add appropriate dilutions of anti-influenza A and B type-specific monoclonal antibodies respectively, and place it in a humidified box at 37℃ for 1 hour. Wash twice in PBS for a total of 10 minutes.
GB15994-1995
C1.3.3 Add rabbit anti-mouse fluorescein conjugate of appropriate dilution and place at 37℃ for 1h. Wash three times in PBS for a total of 10min, and rinse each time with distilled water.
C1.3.4 Observe the results under a fluorescence microscope. If more than three fluorescent positive cells in the cytoplasm or nucleus are observed, it can be judged as positive. C2 Detect antigens after the specimen is proliferated by sensitive cells for one generation C2.1 Principle
After the patient's respiratory specimen is inoculated with sensitive cells (dog kidney passage cells MDCK) and proliferated for 1 to 3 days, the cells are digested and dispersed to make antigen slices, and then the intracellular antigens are detected by immunofluorescence or immunoenzyme staining. Since the virus in the specimen is detected for antigen after the sensitive cells proliferate, the sensitivity of its diagnosis can be significantly improved. The antigen can be detected before the cytopathic effect appears, and it can be directly typed. It is much faster than the conventional chicken embryo virus isolation method, and the diagnosis can be made in 24~~72 hours. The inspection method can be indirect immunoenzymatic staining (C2.5) or indirect immunofluorescence (C2.4). C2.2 Collection and processing of specimens
For children, throat swabs can be used, preferably negative pressure aspiration method; for adults, throat gargle can be used, preferably throat wash. The specimens are processed according to method A2.3 in Appendix A (Standard Appendix). C2.3 Inoculation, culture and preparation of specimens
Take 0.2mL of the treated specimen solution and inoculate it into MDCK cells grown into a monolayer. Inoculate 3 tubes of each specimen, place at 37°C for adsorption for 1~~2h, then discard the specimen solution, add 199 maintenance solution containing 2ug of trypsin per milliliter to 1ml, culture at 35°C for 24, 48 and 72h, take out 1 tube respectively, collect the supernatant for use, digest the cells with digestion solution (1 part 0.25% trypsin + 4 parts Versene), wash once with PBS, then drop it on the glass slide with a ring, dry naturally, and fix with cold acetone. Prepare uninfected normal MDCK cell slices in the same way as normal control cells. C2.4 Indirect immunofluorescence method
C2.4.1 Adding monoclonal antibodies is the same as C1.3.2. C2.4.2 Adding immune anti-mouse fluorescein conjugate is the same as C1.3.3. C2.4.3 Observe the results under a fluorescence microscope. If fluorescence is observed in the cytoplasm or nucleus, but no fluorescence is observed in normal cell sheets, it can be determined as positive.
C2.5 Indirect immunoenzymatic staining method
C2.5.1 Draw the monoclonal antibody as in C1.3.2. C2.5.2 Add sheep anti-mouse enzyme-labeled conjugate at an appropriate dilution, place in a wet box at 37C for 1 hour, and wash twice with PBS for 10 minutes. C2.5.3 Add o-phenylenediamine substrate solution, the preparation method is: 4mg o-phenylenediamine + 10mL pH5.0 phosphate-citric acid buffer + 15μL hydrogen peroxide. The preparation method of pH5.0 phosphate buffer is: first prepare 0.1mol/L citric acid (C.HgO,·H,0) (21g/1000ml.) and 0.1mol/L disodium hydrogen phosphate (NazHPO,) (28.4g/1000mL), respectively, mix them according to 24.3mL and 25.7mL, and then add an equal amount (50mL) of double distilled water to mix to obtain pH5.0 phosphate-citric acid buffer. Add the substrate solution for 3~~5min to observe the results. C2.5.4 Observe the results under an inverted microscope (cells should be wet when observing the results. If the cells are dry, add a drop of tap water). If brown-red dark-stained cells are observed, record the number of dark-stained cells as 10, ...
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