What should be paid attention to in a standardized PCR laboratory

The registration process for the PCR certificate is as follows:1. Register at the Clinical Laboratory Center.

2. Take the test at the designated location.

3. Pass the exam.

PCR nucleic acid detection.

Qualifications for registration:

1. Be at least 18 years old.

2. Abide by laws and regulations, and abide by professional ethics.

3. Have good conduct.

4. Have the physical conditions to perform duties normally.

5. Have a certain knowledge of biology.

The training courses for the PCR certificate are:1. The basic principles of PCR and related detection technologies and progress.

2. Quality assurance of clinical gene amplification and tung testing.

3. Processing, template preparation and preservation of clinical PCR specimens.

4. Analysis of fluorescence PCR results and common problems.

5. Clinical gene amplification laboratory.

biosecurity and anti-pollution measures.

6. Problems and solutions in PCR laboratory acceptance.

7. PCR quality control in molecular pathological diagnosis.

1. Reagent storage and preparation areaThe main operations in this experimental area are the preparation of storage reagents, the sub-packaging of reagents and the preparation of the main reaction mixture. Reagents and materials used for specimen preparation should be transported directly to this area and should not pass through other areas. The raw materials of the reagents must be stored in the zone and prepared in the zone to make the required storage reagents.

For the control of air pressure, the district does not have strict requirements.

2. Specimen preparation area The main operations in this area are the preservation of clinical specimens, nucleic acid (RNA, DNA) extraction, storage and the synthesis of cDNA when it is added to the amplification reaction tube and the determination of RNA. The pressure gradient in this area is required to be positive relative to the adjacent area to avoid aerosols entering the area from the adjacent area.

Contaminate. In addition, unnecessary movement in the area should be avoided due to contamination caused by the sol of the scale during the dispensing operation.

3. Preparation of amplification reaction mixture and amplification area The main operation in this region is DNA or cDNA amplification. In addition, the addition of prepared DNA templates and synthesized cDNA (from the sample preparation area) and the preparation of the main reaction mixture (from the reagent storage and preparation area) into the reaction mixture can also be carried out in this area. PCR in the nest

In the assay, the reaction tube must be opened after the first round of amplification, so nested amplification has a high risk of contamination, and the second injection must be performed in the local area. The pressure gradient in this area is required to be negative relative to the adjacent area to avoid aerosol leakage from the area.

To avoid aerosol-induced pollution, unnecessary movement in the area should be minimised. Individual operations such as sample filling should be carried out in the clean room.

4. The main operation in the amplification product analysis area is the detection and sensitivity of the amplified fragment. If a fully automatic closed analytical instrument is used, this area can be omitted. This zone is the most important amplification product contamination**, so the requirements for the pressure gradient in this zone are:

Negative pressure is applied relative to adjacent areas to avoid the spread of amplification products from this area to other areas.

1. The material of the template is mainly based on the PCR increase object, which can be pathogen specimens such as viruses or pathophysiological specimens such as cells.

2. The basic requirement of specimen processing is to remove impurities and partially purify the nucleic acids in the specimen. Most samples are treated with SDS and proteinase K. Bacteria that are difficult to break can be treated with lysozyme plus EDTA.

3. The primer is best designed in the conserved region of the template cDNA, the primer length is generally between 15 and 30 bases, and the primer length is commonly used to be 18 27 bp, but it should not be greater than 38, because too long will cause its elongation temperature to be greater than 74, which is not suitable for Taq DNA polymerase reaction.

4. The GC content of primers is between 40% and 60%, and the TM value should be close to 72, and the GC content is too high or too low is not conducive to initiating the reaction. The TM value of the upstream and downstream primers is the melting temperature of the oligonucleotide, that is, the temperature at which 50% of the oligonucleotide double-stranded is thawed at a certain salt concentration.

5. The 3' end of the primer should avoid the 3rd position of the codon, the 3rd end of the primer should not terminate at the 3rd position of the codon, the 3rd end of the primer can not choose A, it is better to choose T, and when the 3rd end of the primer is mismatched, there is a great difference in the initiation efficiency of different bases. <>

PCR Experiments:

1. DNA denaturation (90-96): Under the action of heat, the hydrogen bonds of the double-stranded DNA template are broken to form single-stranded DNA.

2. Annealing (refolding) (40-65): The system temperature decreases, and the primers bind to the DNA template to form a local double strand.

3. Extension (68 -75): Under the action of Taq enzyme (the best activity at about 72), DNTP is used as raw material to extend from the 5 ends and 3 ends of the primer to synthesize DNA strands that are complementary to the template. Each cycle is denatured, annealed, and elongated, and the DNA content is doubled.