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I. Standardized setting and management of clinical gene amplification testing laboratory<br> The standardization setting of clinical gene amplification testing laboratory is detailed in the “Interim Measures for the Management of Clinical Gene Amplification Testing Laboratory†(Health Medical Development [2002]10 No.) Attachment "Basic Setting Standards for Clinical Gene Amplification Laboratory". In order to avoid contamination, the clinical gene amplification testing laboratory must be set up strictly in accordance with the "Clinical Gene Amplification Laboratory Setting Standards".
The clinical gene amplification test laboratory must have dedicated equipment in each of the four separate work areas. Each area must be clearly marked to prevent equipment items such as samplers or reagents from being removed from their respective areas, resulting in confusion of equipment items between different work areas. Access to each work area must strictly follow a single direction sequence, that is, only from the reagent storage and preparation area, the sample preparation area, the amplification reaction mixture preparation and amplification area (abbreviated as amplification area) to the product analysis area to avoid cross-contamination. In different work areas, work clothes with different colors or marked signs should be used for identification. In addition, when workers leave the work area, they must not bring out specific work clothes from each area.
Improper cleaning methods are also a major cause of contamination, so laboratory cleaning should be performed in the direction of reagent storage and preparation areas to the amplification product analysis area. Different experimental areas should have their own cleaning appliances to prevent cross-contamination.
(1) Reagent Storage and Preparation Zone <br> The following operations are carried out in this zone: preparation of a storage reagent, dispensing of a reagent, and preparation of a main reaction mixture.
Storage reagents and materials used for specimen preparation should be shipped directly to the reagent storage and preparation area and not through the product analysis area. Centrifuge tubes or tubes containing the reaction mixture for a few seconds before opening them. The reagent raw materials must be stored in the area and prepared into the required storage reagents in the area. When the stored reagent solution is available for inspection, it should be stored and stored in reserve to avoid contamination due to frequent opening of the reaction tube.
Centrifuge tubes or tubes containing the reaction mixture should be centrifuged for a few seconds before freezing. Most reagents used for amplification should be stored frozen. In order to avoid frequent freezing and thawing of the main storage reagent due to a single reaction, the frozen storage reagent solution should be dispensed. The dispensing volume of the storage reagent is determined by the number of amplification reactions typically required for one measurement in the laboratory.
The suitability and stability of the components of the main reaction mixture, especially the polymerase, are checked by pre-tests and the results must be reported in writing. For "hot start" techniques (adding enzyme after the first high temperature denaturation step), the polymerase may also not be included in the main reaction mixture.
During the entire experimental operation in this area, the operator must wear gloves and replace them frequently. In addition, the use of disposable caps in operation is also a measure to effectively prevent contamination.
It is strictly forbidden to suck the liquid with the mouth, and the sampler and the tip must be treated with high pressure.
The work area must be cleaned immediately after the work is over. The surface of the bench in this work area should be resistant to the disinfection and cleaning of chemicals such as sodium hypochlorite. Ultraviolet irradiation on the surface of the test bench should be convenient and effective. Since the distance and energy of ultraviolet irradiation are critical to the effect of decontamination, a movable UV lamp (wavelength of 254 nm) can be used, and after the work is completed, it is irradiated to 60 to 90 cm on the test bench. Since the amplified product is only a few hundred bp and is not sensitive to ultraviolet damage, the ultraviolet irradiation of the amplified fragment must be extended by irradiation, preferably overnight. The use of the laboratory and its equipment must be recorded on a daily basis.
(2) Specimen preparation area <br> The following operations are carried out in this area: preservation of clinical specimens, extraction and storage of nucleic acids (RNA, DNA), and addition of cDNA to the amplification reaction tube and determination of RNA.
To use the sampler correctly. Since aerosol-induced contamination may occur during the loading operation, unnecessary movement in the area should be avoided. Aerosol contamination from the adjacent area into the area can be avoided by establishing positive pressure conditions in the area. In order to avoid cross-contamination between samples, after adding the nucleic acid to be tested, the reaction tube containing the reaction mixture must be covered. For materials with potential infectious risks, there must be clear sample handling and inactivation procedures.
The used applicator tip must be placed in a special sterile (eg sodium hypochlorite solution) container. The surface of the laboratory table and chair should be cleaned after each work. The experimental materials (original blood specimen, serum specimen, mixture of specimen and reagent in the extraction, etc.) must be treated separately and recorded if there is any splash.
Appropriate UV irradiation (254 nm wavelength, close to the work surface) for the bench is suitable for inactivation and decontamination. Movable UV tube lamps can be used to ensure adequate exposure to the test bench after work.
Sample processing has a great impact on nucleic acid amplification, and efficient nucleic acid extraction methods must be used to evaluate extraction methods prior to clinical specimen testing.
After the sample for RNA amplification detection is prepared, cDNA synthesis should be performed immediately because the cDNA strand is stable compared to RNA and is relatively easy to store. To ensure the need for a reverse transcription reaction, more than one incubation device should be placed in the specimen preparation area.
The ideal temperature for cDNA synthesis depends on the enzyme used, and tends to use a one-step method: reverse transcription using a thermostable DNA polymerase with reverse transcription activity under amplification reaction buffer conditions, which is more synthetic than cDNA. There is a reduced likelihood of contamination by opening the lid to adjust the buffer or adding a polymerase for amplification.
The cDNA copy of the RNA to be tested must be stored in the specimen preparation area, and the sample should not be PCR amplified in this area.
(III) Amplification zone <br> The following work is carried out in this zone: DNA or cDNA amplification.
Further, the addition of the prepared DNA template and the synthesized cDNA (from the sample preparation zone) and the main reaction mixture (from the reagent storage and preparation zone) to prepare a reaction mixture or the like can also be carried out in the region. In the nested PCR assay, the reaction tube must usually be opened after the first round of amplification, so nested amplification has a higher risk of contamination, and the second addition must be performed in the region.
It is not possible to re-enter any “upstream†area from this area, which can reduce the pressure in this area to prevent aerosols from leaking out of this area. In order to avoid contamination caused by aerosols, movement in the area should be minimized. If there is a sample, it should be carried out in a clean room. Liquid precipitating must be prevented when opening the pretreated reaction mixture, especially between nested amplification steps. An easy way is to centrifuge quickly for a few seconds before opening the reaction tube. A smaller centrifuge can be used, which is easy to handle with one hand because it has a small test bench and is suitable for most clean benches. Moisture barriers such as paraffin oil or light mineral oil also have anti-pollution effects, but it must be noted that mineral oil itself may also be a persistent source of pollution. Used samplers must be cleaned and disinfected.
The laboratory benchtop must be cleaned and disinfected after the operation and daily work, and the UV irradiation method is the same as in the front area. If a solution spills, it must be processed and recorded.
(4) Amplification product analysis region <br> The following operation is carried out in the region: measurement of the amplified fragment.
There are various methods for analyzing products after nucleic acid amplification, such as probe hybridization (isotopic or non-isotopic labeling) on ​​membrane or microplate, agarose gel electrophoresis, polyacrylamide gel electrophoresis, Southern transfer, nucleic acid Sequencing methods, etc. At present, most of the domestic commercial kits use non-isotopically labeled probe hybridization methods on the microplate, namely PCR-ELISA method, and also on-membrane probe hybridization method.
This area is the most important source of amplification product contamination, so care must be taken to avoid the amplification products from passing through the items and work clothes in this area. When using PCR-ELISA to detect amplification products, the plate must be washed with a washer. The waste must be collected into 1mol/L HC1 and cannot be dumped in the laboratory. Instead, it should be discarded away from the PCR laboratory. . The used tips must also be soaked in 1mol/L HCl and placed in a garbage bag and disposed of according to procedures such as incineration.
Since some genetically mutated and toxic substances such as ethidium bromide, acrylamide, formaldehyde or isotopes may be used in this area, the safety protection of the experimental personnel should be noted.
The cleaning, disinfecting and UV irradiation methods in this area are the same as in the previous area. This area can reduce the possibility of amplification products spreading from this area to the front area if under negative pressure conditions or under reduced pressure (such as installing an exhaust fan).
Second, the clinical gene amplification test laboratory quality assurance <br> clinical gene amplification test laboratory quality assurance involves all stages of the entire gene amplification test, that is, the sample collection and processing before the measurement analysis, the nucleic acid extraction, expansion Addition product analysis and report results after measurement, etc.
(1) Collection of specimens <br> Clinical specimens commonly used for gene amplification detection include EDTA or sodium citrate anticoagulated whole blood or bone marrow, serum or plasma, sputum, cerebrospinal fluid, urine and secretions. When taking samples such as blood, disposable closed containers, such as vacuum blood collection tubes, should be used. When using a non-closed sampling system, such as urine, secretions and bone marrow sampling, care must be taken to prevent contamination of the dander or secretions from the sampler. Disposable gloves must be worn when sampling.
Glassware should be treated with high pressure before use, as glassware often contains RNases that are not easily inactivated. Preferably, it is heat sterilized, and baking at 250 ° C for more than 4 hours can permanently inactivate RNase.
Whole blood and bone marrow specimens must be treated with anticoagulation. EDTA and citrate are the preferred anticoagulants. Heparin anticoagulation cannot be used because heparin is a strong inhibitor of the Taq enzyme and is difficult to remove in the subsequent nucleic acid extraction step.
Blood samples clinically used for amplification of RNA (eg, HCV RNA) are recommended for anticoagulation and plasma is separated as soon as possible (within 3 hours) to avoid RNA degradation. If anticoagulation is not performed, serum must be separated within 1 hour after blood draw.
(2) Stabilization of specimens <br> Specimens used for DNA amplification detection generally do not require special stabilization treatment after collection, but specimens should be sent to the laboratory in time.
Because RNA is susceptible to degradation by RNase, specimens used for RNA assays must sometimes be stabilized, such as on-site sampling of epidemiological investigations. Guanidine thiocyanate (GITC) can immediately inactivate DNase and RNase. Therefore, when collecting specimens, specimen materials such as serum or plasma can be added to a ratio of 1:4 to contain 5mol/L GITC. In the test tube, the RNase in the serum (slurry) is irreversibly inactivated. After the above stabilization treatment, the specimens are generally mailed without refrigeration. For the specific detection item, what is the effect of the above stabilization treatment method, and evaluation is performed using the corresponding reverse transcription PCR assay method.
(3) Shipping of specimens <br> Specimens must be sent to the laboratory as soon as possible after collection. Specimens that have been properly stabilized can be shipped by post at room temperature. For example, EDTA anticoagulated whole blood samples for DNA amplification detection and GITC stabilized samples for RNA amplification detection. Specimens should normally be loaded in containers that are not easily broken when transported. Specimens for RNA testing, if not stabilized, must be frozen and shipped in dry ice.
(4) Storage of specimens <br> Clinical fluid specimens such as serum/plasma can be stored at -70 °C for a long time. The purified nucleic acid sample for DNA assay can be stored at 10 ° C in 10 mmol/L Tris-1 mmol/L EDTA buffer (pH 7.5-8.0). Purified nucleic acid samples for RNA assays should be stored in buffer at -80 ° C or in liquid nitrogen. The nucleic acid sample precipitated with ethanol can be stored at -20 °C. RNA samples treated with GITC can be stored for 7 days at room temperature.
(5) Treatment of specimens (nucleic acid extraction)
Specimen processing, ie, nucleic acid extraction and purification, is a critical step in determining the success or failure of amplification assays. Before using a commercial nucleic acid extraction reagent to extract a nucleic acid template from a clinical specimen, it should be fully evaluated to verify the effectiveness of the extraction. In general, the quality of nucleic acid preparation is not high due to incomplete removal of inhibitors, which may be derived from the specimen itself (such as heme and its precursors or degradation products) or nucleic acid extraction process.