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The Pcr laboratory, also known as the Gene Amplification Laboratory. PCR is the abbreviation for Polymerase Chain Reaction. It is a molecular biology skill used to amplify specific DNA fragments, which can be seen as a special DNA imitation of organisms in vitro. Through the DNA gene tracking system, we can quickly grasp the virus content in patients, with an accuracy of up to nanometer level. We can accurately detect the number of hepatitis B virus in patients, whether it is imitated, whether it is infected, how infectious it is, whether it is necessary to take drugs, whether liver function is abnormal, and timely identify which antiviral drugs are most suitable for patients to use, and how effective the drugs are Providing a reliable examination basis for clinical treatment
1. It is necessary to have a standardized PCR fluorescence laboratory;
Real time fluorescence quantitative PCR technology was launched by Applied Biosystems in the United States in 1996
Because this skill not only completes the qualitative to quantitative leap of PCR, but also compared to conventional PCR, it
It has stronger specificity, useful handling of PCR contamination questions, and advanced level of initiative, which has been widely recognized
Universal application. This article attempts to introduce its quantitative principles, methods, and reference questions
2. It is necessary for the testing equipment to meet the requirements of setting up a standardized PCR fluorescence laboratory;
The PCR DNA/RNA real-time fluorescence quantitative detection system is composed of a fluorescence quantitative PCR instrument, real-time fluorescence quantitative reagents, universal computers, and active analysis software.
3. It is necessary to undergo national clinical examination and certification in the middle;
4. It is necessary for testing personnel to undergo national temporary inspection intermediate business training and obtain qualification certificates;
It is necessary for PCR laboratory staff to participate in clinical gene amplification training courses held by the National Ministry of Cleanliness or provincial clinical testing centers, and work with certificates. The PCR laboratory has been inspected and it is necessary for at least two of them to hold a "Clinical Gene Testing Certificate". It is necessary to establish a strict laboratory management system, establish standardized operating procedures (SOPs), and establish a series of quality management documents in the PCR laboratory to ensure that the normal operation of the laboratory meets the needs of the National Ministry of Cleaning, ensure accurate testing results, ensure laboratory cleanliness and safety, and ensure long-term stable operation of the laboratory
5. It is necessary to operate in a sterile and dust-free environment
PCR laboratory certification certificate
It can control the patient's condition scientifically, accurately and in real time, and contact anti HBV and T-cell immunity to break the immune tolerance, block the treatment of imitation of liver disease virus and useful differentiated hepatitis virus, deal with medical problems such as hepatitis B virus mutation and drug resistance, virus imitation template is difficult to cure, and human immune tolerance is not easy to break. It can quickly eliminate clinical manifestations, and effectively suppress hepatitis B virus imitation, Significantly accelerate the serum transformation speed of e antigens and antibodies, kill viruses in blood and liver cells, provide long-term maintenance to avoid reinfection, and effectively block and reverse the progression of liver fibrosis and cirrhosis.

1. Establish sample preparation area
This area is specifically used for sample preparation, and precautions should be taken when preparing and operating reagents for nucleic acid acquisition: ⑴ PCR products and DNA clones with the desired amplification sequence cannot be operated in this room. ⑵ Arrange cultures, specimens, and serum samples to be brought into the sample preparation room for disposal, in order to obtain DNA or RNA according to the needs of the application Things used for sample disposal cannot be used for general molecular cloning, nor can they be used as operation target sequences. ⑷ The DNA sample may be operated with a special protective or positive pressure piston pipette to avoid aerosol transmission during sample extraction. ⑸ Large volume samples may be taken from sterile disposable pipette packed separately. (6) Before opening the pipe, it should be roughly centrifuged to reduce the occurrence of aerosols, and the pipe should not be forcefully collapsed, as this can cause aerosols Always wear test clothes and gloves, and gloves should be replaced frequently, especially between every step of the extraction process. The test suit should be specifically used in the sample preparation room and cleaned frequently.
2. Sample preparation and rated process requirements for RNA PCR and RNA PCR require rated sample operations,This increases the opportunity for contamination between samples. To avoid this doubt, the reverse transcription step can be performed in the sample preparation region. The use of UNG in RNA PCR to avoid contamination has also been reported.
3. Establish a pre PCR area.
It should be specifically used to prepare for various reactions, and it is necessary to maintain cleanliness in this area without contamination from cloning and sample preparation. It is necessary to have reagents and preparations in the pre PCR area, especially the positive pressure piston pipette specially used in the pre PCR area.
4. Operation of PCR laboratory reagents.
⑴ All solutions used are probably free from nucleic acid and/or nuclease (DNase and RNase) pollution. ⑵ The water used in all PCR reagents is approximately high-quality - fresh distilled deionized water, using 0.22 μ It is filtered and sterilized under high pressure Reagents stored at 20 ℃ to 25 ℃ advocate the addition of antimicrobial agents such as sodium azide, and the addition of 0.025% sodium azide in amplification reagents or sample preparation reagents does not suppress amplification reactions The reagents used are generally manufactured in large volumes, tested to see if they meet the requirements, and then packaged into quantities that are only sufficient for one use for storage All reagents and sample preparation processes should use disposable sterilized bottles and tubes Newly manufactured reagents should be roughly tested before being used to prepare new specimens The pipette used in sample preparation and pre PCR area should be carefully reserved when not in use.
5. Establish a PCR mixture in the pre PCR area.
(1) It is useful to prepare, package, and retain immediately available "main mixture" solutions at -20 ° C or 4 ° C, when only one or a small number of specific sequences are involved in amplification in the laboratory. (2) If your laboratory uses multiple sets of primers, making it uneconomical to produce a single reaction mixture containing all reagents, you can consider packaging and retaining enough PCR components for a day As a rule, a set of negative, weakly positive, and strongly positive control samples should be retained to analyze the power and cleanliness of the sample manufacturing and PCR process. And you also expect to verify your sample buffer by using a known weakly positive sample to prove that it does not contain amplification inhibitors Negative samples should be taken together with each group of samples to analyze whether there is contamination between samples and whether there is contamination from PCR products. The negative control roughly includes all reagents except nucleic acid When used as a positive control, there are two reasons to determine the approximate use of the minimum amount of nucleic acid Because it is necessary to have a comparative response and roughly consider the characteristics of the template.
6. Methods for manipulating pollution.
A powerful enzymatic approach has been planned to eliminate pollution in one way - using UNG, this skill can effectively eliminate pollution caused by PCR products. Another way to manipulate pollution is to use ultraviolet radiation, which cannot completely eliminate pollution concerns, but can reduce pollution by several orders of magnitude.
7. After the completion of PCR in the post PCR area,Analyze the samples and explain the data, and roughly set aside a dedicated area for handling the samples after response. The reagents, disposable consumables, and instruments used in post PCR activities must be specifically designed for this purpose, and reagents or instruments in this area of the laboratory must not be used for any pre PCR activities.

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PCR laboratory, HIV laboratory

IntroductionDesign BasisKey Notes