Presentation on theme: "Experiment five Isolation and Identification of influenza virus."— Presentation transcript:
1Experiment fiveIsolation and Identification of influenza virus
2Isolation and Identification of viruses in lab SpecimenIsolation and identification of virusSerological diagnosis of viral infectionRapid diagnosis of virus infections
3Isolation and Identification of viruses in lab Isolation of virusEmbryonated EggsLaboratory AnimalsTissue CultureQuantitation of virus50% tissue culture infectious dose (TCID50)Plaque forming assay
4Isolation and Identification of viruses in lab Serological diagnosis of viral infectionNeutralization test(NT)Complement fixation test(CF)Titration of complement
5Isolation and Identification of viruses in lab Rapid diagnosis of virus infectionsIdentification virus particleDetection of viral antigensDetection of viral nucleic acidDiagnosis of virus infections by detecting IgM antibodiesOther methods
6Isolation and Identification of influenza virus Purpose：To obtain information about microbiological diagnosis methods of influenza virusProcedureAcquiring and managing specimenIsolation and Identification of influenza virusserological diagnosis of influenza virus
7Isolation and Identification of influenza virus Acquiring and managing specimenThroat washing or gargling are obtained within 3 days after infectionTest at once or stored frozen.Centrifuge the washings 15 min at 3000 rpm, Harvest 1 ml of supernatant.Add antibiotic to prevent bacterial contamination.
8Embryonated egg inoculation Patient’s serumThroat washings or garglingTreated with penicillin and streptomycinHemagglutination testEmbryonated egg inoculation(amniotic or allantoic route)Harvest amniotic and allantoic fluidsHemagglutination test+-Hemagglutination inhibition testcomplement fixation testInoculate to fresh embryos after two such passagesthe result is negative
9Isolation and Identification of influenza virus Isolation of influenza virusMATERIALS1.Throat washings or garglings2.Embryos (within 9 to 10-day-old)3.0.5% chicken RBC suspension, physiologicalsaline4. Scissors, forceps, drill, syringe, tubes, egg-checking box, 75% ethanol, tincture of iodine
10Mark the location of embryo air sac under egg-checking box, sterilize the shell with tincture and 75% ethanol, drill a small hole near the marked shell , inoculate 0.2 ml of virus inoculum into the allantoic cavity by pushing the needle just below the surface of the shell, seal the hole with melton wax.Incubate the inoculated embryo at 35C, check it daily. After incubating for 72 h,place the embryo at 4C overnight. If the embryo dead within 72 h, it should be discard.Harvest allantoic fluid.Assessing HA titer refers to Experiment 25.
12Hemagglutination test, HT Materials well No.(ml)SalineVirusDilution :10 1:20 1:40 1:80 1:160 1:3201:6401:12801:2560 con0.5%RBCincubate the plate at room temperature for 30 to 60 min.Check the agglutination.
13Hemagglutination test, HT Analysis of the results
15Hemagglutination test (HT) RESULTRecord the hemagglutination as the followings:++++ : All the RBC had been agglutinated.+++ : 75% of RBC had been agglutinated++ : 50% of RBC had been agglutinated.+ : 25% of RBC had been agglutinated.: No RBC was agglutinated.
16Hemagglutination inhibition test (HIT) MATERIALSPatient‘s serum: deactivated 30 min at 56C;influenza virus: 4 HA units;0.5% chicken RBC suspension;physiological saline20-well microtiter plates, pipettes and tubes.
17Materials well No.(ml)SalineSerumDilution :10 1:20 1:40 1:80 1:160 1:320 1:VirusMix evenly incubation at 37C for 1h0.5%RBCMix evenly incubation at 20c for 1 hResult
19Hemagglutination inhibition test (HIT) RESULTAssess hemagglutination as above, the hemagglutination inhibition (HI) titer is the reciprocal of the highest dilution of the patient's serum which shows complete inhibition of agglutination.1:40 dilution in Table is the HI titer.