Procurement of materials, setting up of basic rabies laboratory Changchun Tu

Transcription

1 Procurement of materials, setting up of basic rabies laboratory Changchun Tu Diagnostic Laboratory on Rabies and Wildlife-associated Viral Zoonoses, CVRI OIE Reference Laboratory for Rabies

2 FAT, Cell culture and FAVN Cell culture product & Reagent MEM Thermo Scientific (Gibco) Corning Sigma-Aldrich GE Healthcare Life Sciences NBCS Corning Thermo Scientific (Gibco) Penicillin- Streptomycin Thermo Scientific (Gibco) Sigma-Aldrich BD Corning Trypsin Sigma-Aldrich BD Thermo Scientific (Gibco)

3 FAT, Cell culture and FAVN FAT and FAVN product & Reagent PBS Thermo Scientific Corning Solarbio Disinfectant Virkon S Evans Blue Amresco Thermo Scientific Yuanye FITC Anti-Rabies Monoclonal Globulin Fujirebio Diagnostic, Inc Merk SIFIN

4 FAT, Cell culture and FAVN Instrument CO 2 biochemical incubator Thermo Scientific BINDER SHELLAB Panasonic Inverted Microscope Olympus Leica Carl Zeiss Nikon Fluorescence microscope Olympus Leica Carl Zeiss Biological safety cabinet Esco BAKER Thermo Scientific NUAIRE

5 FAT, Cell culture and FAVN Instrument Pipettes Thermo Scientific Eppendorf Gilson Sartorius Cryogenic refrigerator Thermo Scientific Panasonic Haier HARRIS Tissue grinder QIAGEN TIANGEN OMNI Bio Spec Products Inc

6 FAT, Cell culture and FAVN Instrument Liquid nitrogen container Thermo Scientific ( Locator) Taylor-Wharton Chart-MVE C.B.S. Scientific Company Micro Centrifuge Eppendorf Thermo Scientific Beckman Coulter Sigma Water bath TIANGEN Thermo Fisher Scientific IKA Memmert

7 FAT, Cell culture and FAVN Consumable item Freezing Tube & containers Thermo Scientific(NUNC) Corning (Axygen) Greiner Bio One well cell culture plates, Cell Culture Flask, etc. Corning Thermo Scientific Greiner Bio One BD Slide & tongue blade

8 RNA Extraction and RT-PCR Reagent RNA Extraction Kit TIANGEN QIAGEN Promega TAKARA Reverse Transcription TIANGEN Promega Amresco TAKARA PCR mix TIANGEN TAKARA Amresco Promega

9 RNA Extraction and RT-PCR Reagent DNA polymerase TIANGEN TAKARA NEB Agarose Sangon BIOWEST Sigma-Aldrich DNA Marker TIANGEN TAKARA Promega Sangon Nucleic acid dye TIANGEN TAKARA Sangon

10 Instrument RNA Extraction and RT-PCR Water bath TIANGEN Thermo Fisher Scientific IKA Memmert Micro Centrifuge Eppendorf Thermo Scientific Beckman Coulter Sigma Mini Plate Spinner TIANGEN Eppendorf Bio-rad PCR system Eppendorf Applied Biosystem Bio-rad

11 Instrument RNA Extraction and RT-PCR Pipettes Eppendorf Thermo Scientific Gilson Sartorius Nucleic Acid isolation machine TIANGEN Eppendorf QIAGEN Tissue grinder TIANGEN Bio Spec Products Inc QIAGEN OMNI Ice maker Panasonic Hoshizaki

12 Instrument RNA Extraction and RT-PCR Gel imaging system Bio-rad UVP UVItec FUJIFILM Microwave oven Haier Galanz Midea

13 Real-time qpcr Reagent FQ PCR Master Mix TIANGEN Thermo Scientific Sigma-Aldrich TAKARA Instrument Real-time qpcr system Applied Biosystem Bio-rad MJ Research LightCycler Agilent Technologies

14 Ab ELISA ELISA kit Haitai Alpha Diagnostic Intl DRG International Greenspring diagnostic Corporation Lvshiyuan Biotechnology

15 Setting up rabies laboratory WHO manual for microbiological Laboratory Guideline: risk assessment, BSL1-4, ABSL1-4 Equipment: BSC, Equipment-related hazards, and designed to reduce biohazards. Good microbiological technique Chemical, fire and electrical safety Safety organization and training

16 Setting up rabies laboratory OIE manual: Chapter BIOSAFETY AND BIOSECURITY: STANDARD FOR MANAGING BIOLOGICAL RISK IN THE VETERINARY LABORATORY AND ANIMAL FACILITIES This chapter is concerned exclusively with risks from infectious agents To prevent disease in humans in the laboratory; To prevent the release of the pathogen into the environment and causing disease in animals or humans.

17 Setting up rabies laboratory Biosafety and biosecurity what s the difference? Biosafety The consistent application of safety measures to minimize or prevent exposure to the person handling a biological agent, lab and building occupants, the community and the environment keeping nasty pathogens away from people Biosecurity Maintaining a biological agent a secure way, either by physical or procedural means, so as to ensure it does not constitute a hazard to man or his environment keeping nasty people away from pathogens

18 Setting up rabies laboratory Reducing/negati ng the risk Training Quality Physical containment Legislation Inspection Procedures Transport Handling storage Prophylaxis & health monitoring

19 Setting up rabies laboratory purpose: 1. To protect employees and their families from acquiring laboratoryassociated infectious diseases; 2. To prevent contamination of the environment and promote environmental quality; 3. To comply with all National, International and local guidelines and regulations for the use of potentially hazardous and/or regulated biological materials; 4. To conform to prudent biosafety practices

20 Setting up rabies laboratory Biosafety Program Elements: Biosafety Organization & Committee SOP Registration & Inventory Control Risk Assessment & Control of Biohazards Application & sanction Biosafety Training Facility Maintenance Program Emergency Response Medical Surveillance Auditing & Re-certification Program Documentation

21 Setting up rabies laboratory

22 PEP: Pre-Exposure Prophylaxis All lab staff must accept vaccination against rabies before working with the infectious materials 3-shot regime at day 0,7,28 should be adminstrated Ensure efficient protective NT Ab titer ( > 0.5 u/ml) by annual check using FAVN etc.

23 Setting up rabies laboratory RISK ASSESSMENT OF RABIES VIRUS Pathogens designated as one of four groups, based on their attributes (transmissibility, invasiveness, virulence and lethality, preventive measures ), risk1= lowest; risk4=highest. These are defined as four biosafety levels: BSL1 = lowest; BSL4 = highest Rabies virus (street) is risk 3 pathogen, i.e.highly pathogenic, but preventable activity Original infectious sample (without artificial culture) FAT,RT-PCR. FAVN,RFFIT (CVS-11) Ab tests (ELISA etc.) Cell isolation (street) Animal challenge(street) BSL BSL-2 BSL-2 BSL-2 BSL-1 BSL-3 ABSL-3

24 BSC Autoclave BSC Autoclave Doulb-door entry

25 Biological Safety Cabinets Protect human, sample and environment. Class I, II, III

26 Biological Safety Cabinets Class Ⅰ :protect person and enviroment, but not samples 100% exhausted

27 Biological Safety Cabinets Class Ⅱ :A1, A2, B1,B2. protect person and enviroment and samples Class Ⅱ A: 30% exhausted, 70% recirculated

28 Biological Safety Cabinets Class Ⅱ B1: 70% exhausted, 30% recirculated B2 100% exhausted

29 Biological Safety Cabinets Class Ⅲ : 100% exhausted

30 Biological Safety Cabinets

31 Molecular methods Room partition for PCR: Four-room principle Room 1: Reagent storage and pre-mixture prep Room 2: Prep of viral nucleic acid and PCR reaction Room 3: PCR Room 4:Gel running and documenting Room 1 Room 2 Room 3 Room 4

32 Use of FTA card Product of Whatman, GE Healthcare ( RT collection, shipment and purification of NA for PCR Impregnated with chemical forma that lyses cell and denature protein upon contact. Widely used for bacteria, viruses, variety of tissues in any form

33 Use of FTA card Drop homogenized brain tissues (about 100ul) onto each circle. Each card for one sample to avoid cross contamination of samples.

34 Use of FTA card Envelope the cards and send in Express Mail to: Dr. Tu Changchun Or Dr. Feng Ye Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences 666 Liuying West Rd, Jingyue Economic Development Zone, Changchun , China Used for the joint research Also used for confirmatory diagnoses of your sample in RL.

35 Thanks