Requirements of HIV nucleic acid testing laboratory for HIV nucleic acid testing

HIV nucleic acid testing should be carried out in appropriate laboratories, and standard operating procedures and strict management systems related to experiments and laboratory management should be established, and staff must fully understand these regulations and strictly abide by them. The laboratory should set up two independent working areas: pre-amplification area and post-amplification area. Nucleic acid preamplification package

Comprises a reagent preparation area and a sample processing area, which are located in different rooms or areas; The post-amplification region of nucleic acid includes amplification region and amplification product analysis region, which are located in different rooms or regions. The functions of each district are:

1. 1 reagent preparation area: storage, preparation and subpackaging of amplification reagents.

1.2 sample processing area: sample registration, processing and sub-packaging; Nucleic acid extraction, preservation and use, the first round of PCR amplification.

1.3 amplification region: nucleic acid amplification. If the second sampling of nested PCR is carried out in this area, it should be carried out in a PCR protective cover.

1.4 Analysis of amplification products: electrophoresis, hybridization, imaging, sequencing, result analysis, registration and reporting of amplification products.

On the premise of meeting the following requirements, the pre-nucleic acid amplification area or the post-nucleic acid amplification area can be set in one room:

1.4. 1 Before nucleic acid amplification, there were two experimental areas in the laboratory. Reagents are configured in the ultra-clean workbench, and sample processing is operated in the biosafety cabinet.

1.4.2 when a totally enclosed amplification detection system such as a real-time fluorescent PCR instrument is used in the region after nucleic acid amplification.

1.4.3 Each experimenter uses his own reagents, consumables, pipettes and containers containing pollutants.

1.4.4 Clean and disinfect the operation area and common appliances before and after the experiment.

1.4.5 The reagents, utensils, instruments and equipment in each area are for the exclusive use of this area and shall not be used interchangeably. 3. 1 Provide corresponding facilities and equipment according to the test items.

3.2 The facilities and equipment in each area are dedicated and should be configured in the corresponding area according to the experimental requirements.

3.3 Reagent preparation area: equipped with refrigerator, clean workbench, common centrifuge, sampler, oscillator, waste liquid container and movable ultraviolet lamp.

3.4 Sample processing area: equipped with -80℃ refrigerator, biosafety cabinet, high-speed refrigeration centrifuge, sample feeder, oscillator, ice maker or refrigeration module, constant temperature water bath or dry bath, water supply and drainage equipment, waste container and ultraviolet lamp.

3.5 amplification area: equipped with nucleic acid amplification instrument, ordinary refrigerator, laminar PCR operation cabinet or clean workbench, micro centrifuge, sample feeder, waste container and ultraviolet lamp.

3.6 Amplification product analysis area: equipped with electrophoresis instrument, electrophoresis tank, sampler, ultraviolet transmission instrument, photography/imaging equipment, ordinary refrigerator, ordinary centrifuge, constant temperature water bath, microwave oven, water supply and drainage equipment, waste container, ultraviolet lamp, etc. Sequencer can be placed in other special experimental areas.

3.7 All areas should use special or disposable work clothes, hats, masks, shoe covers and sleeves, and be equipped with anti-ultraviolet glasses. Must meet the general biosafety requirements of AIDS laboratories. 5. Measures to prevent residual pollution of nucleic acid 5. 1 Strictly implement the division system of laboratories and take measures to prevent residual pollution of nucleic acid in laboratories.

5.2 Each area is only used for specific operations, and no other work is allowed.

5.3 Special systems for instruments and materials

Instruments, equipment, materials and facilities shall be identified according to the working area and shall not be mixed.

5.4 One-way workflow system

5.4. 1 The airflow in the laboratory shall flow from the pre-amplification area to the post-amplification area, and shall not flow in reverse. It is suggested that the area before amplification should be kept in a weak positive pressure state, and the area after amplification should be kept in a weak negative pressure state.

5.4.2 One-way working flow direction of experimenters should be: reagent preparation area → sample processing area → amplification area → amplification product analysis area, and reverse flow is not allowed. In principle, the experimenter can't return to work in the front amplification area on the day after working in the rear amplification area.

5.4.3 The unidirectional working flow direction of experimental articles should be: reagent preparation area → sample treatment area → amplification area → amplification product analysis area, and reverse flow is not allowed. Experimental supplies include experimental materials (reagents, samples and amplification products), experimental equipment (containers, shelves, experimental clothes, hats, masks, gloves and shoe covers) and office supplies (recording paper, pens, etc.). ) and cleaning materials.

5.5 Laboratory procedures to prevent nucleic acid residue pollution

5.5. 1 Before the experiment, apply 70% ethanol or 0. 1 ~ 1% sodium hypochlorite solution to the surface of the console or instrument, and wipe it with a paper towel two minutes later.

5.5.2 Reagents, consumables, samples and containers needed to move pollutants before the experiment.

5.5.3 All reagents, consumables and pipettes used for qualitative and quantitative nucleic acid detection must be used separately. After the experiment, take out personal belongings to the storage area; Close the pollutant container and put it into the dirt bag; Take commonly used electrical appliances to the storage area; Apply 70% ethanol or 0. 1 ~ 1% sodium hypochlorite solution to the surface of the console or appliance, and wipe it with a paper towel after two minutes; Turn on the ultraviolet lamp to irradiate at least 1 hour.

5.5.4 The laboratory shall be thoroughly cleaned regularly (weekly). All indoor worktops, floors and surfaces of instruments and equipment should be cleaned with 70% ethanol or 0. 1 ~ 1% sodium hypochlorite, and the worktops and air should be disinfected with ultraviolet lamps. Clean the pipette regularly (monthly).

5.5.5 Regularly (monthly) monitor and report the residual pollution of nucleic acid in the laboratory, including laboratories and shared corridors in the area before and after nucleic acid detection.

5.5.6 After each test, the DNA sequence of the same batch of test samples and the DNA sequence of the previous batch of test samples shall be analyzed by genetic phylogenetic tree. 6. 1 All wastes should be treated as HIV-contaminated items.

6.2 Chemicals such as agarose gel treated with ethidium bromide should be treated according to relevant schemes.