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[hot spot] PCR laboratory construction knowledge and cost estimation
Time:2020-07-13
At present, the international epidemic situation is still grim, which is not optimistic, and China is facing severe risks of overseas import. Recently, the National Health and Health Commission issued the Notice on Further Perfecting the Appointment Diagnosis and Treatment System and Strengthening the Construction of Smart Hospitals, guiding all localities and hospitals to further establish and improve the appointment diagnosis and treatment system, strengthen the construction of smart hospitals, speed up the establishment of a new online and offline integrated medical service model, and continuously enhance people's sense of medical treatment. Therefore, it is extremely urgent to establish a PCR laboratory. So, what do you know about the construction knowledge and cost estimation of PCR laboratory? Below, Hai Si 'an Xiaobian will come one by one.
PCR laboratory is also called gene amplification laboratory, and PCR is short for Polymerase Chain Reaction. It is a molecular biology technique, which is used to amplify specific DNA fragments and can be regarded as special DNA replication in vitro. Through the DNA gene tracking system, we can quickly grasp the virus content in patients, and its accuracy is as high as nanometer level. It can accurately detect the number of hepatitis B virus in patients, whether it is replicated, whether it is contagious, whether it is necessary to take medicine, whether there is abnormal change in liver function, and it can timely judge which antiviral drugs are most suitable for patients and how effective the drugs are, thus providing a reliable test basis for clinical treatment.


PCR is a conventional method of molecular biology research and experiment, which is widely used in various fields of biology, such as detection and diagnosis of AIDS, hepatitis B, avian diseases, oncogene, DNA fingerprint, individual identification, paternity test and forensic material evidence, animal and plant quarantine, animal and its derivative products detection, animal feed, cosmetics, food hygiene detection, transgenic crops and transgenic microorganisms detection, etc.
On average, major outbreaks of infectious diseases occur once every 1-2 years, such as severe acute respiratory syndrome (SARS coronavirus) in 2003, avian influenza virus (HSN1 avian influenza virus) in 2005, hand-foot-mouth disease (enterovirus) in 2008, fever with platelet syndrome (New Bunia virus) in 2011 and influenza virus (H7N9 virus) in 2013. Avian influenza (H10N8 virus) and Ebola virus in 2014, avian influenza virus (H5N6 virus) in 2015, Sika virus in 2016 and novel coronavirus (COVID-19 virus) at present. Outbreak is characterized by acute onset, rapid progress and strong infectivity, so rapid and accurate identification of infectious disease pathogens is the key factor to control the epidemic. Most of these diseases are diagnosed by nucleic acid amplification, and this process needs to be completed by professionals in high-standard PCR laboratories. Therefore, PCR laboratories have become an indispensable force for disease diagnosis and service in the prevention and control of major infectious diseases. In addition to viral infectious diseases, the detection of pathogenic microorganisms by PCR laboratory includes the diagnosis of bacteria, fungi and protozoa, such as cytomegalovirus, Epstein-Barr virus, Mycobacterium tuberculosis, influenza A and B virus pneumonia, mycoplasma and chlamydia infection, hepatitis B, hepatitis C and hepatitis E. PCR laboratory plays an extremely important role in disease diagnosis and control.

The standard PCR laboratory is divided into four areas: reagent storage and preparation area, sample processing area, nucleic acid amplification area and product analysis area. In order to avoid cross-contamination, entering each working area must strictly follow a single direction, that is, the transfer of reagents and samples between each experimental area can only be carried out from reagent storage and preparation area → specimen preparation area → expansion reaction mixture preparation and amplification area → amplification product analysis area through transfer window.
PCR laboratories can be in the form of dispersion or combination. To complete a set of PCR experiments, there are usually four experimental processes: reagent preparation, sample processing, nucleic acid amplification and product analysis.
1. PCR laboratory in dispersed form:
The so-called decentralized PCR laboratory means that the experimental rooms for completing the above-mentioned experimental process are far away from each other and are in a decentralized arrangement. For the PCR laboratory with this arrangement, it is not easy for each experiment to interfere with each other, so there is no need for special conditions.
2. combined PCR laboratory:
The so-called combined PCR laboratory refers to the form that the experimental rooms for completing the four experimental processes of PCR are adjacently arranged to form independent experimental areas. For the combined PCR laboratory, it is easy to interfere with each other because of the centralized layout of each laboratory. Therefore, there are certain requirements for the overall layout and specific pathogen free. Buffer rooms are set at the entrance of each room to reduce indoor and outdoor air exchange.
Main functions of PCR laboratory
1. The functions of reagent storage and preparation area are:
Preparation of stored reagents, sub-packaging of reagents and preparation of amplification reaction mixture, and storage and preparation of consumables such as centrifuge tubes and suction heads. Materials such as stored reagents and consumables used for specimen preparation should be directly transported to the reagent storage and preparation area, and cannot pass through the amplification detection area. The positive reference substance and quality control substance in the kit should not be stored in this area, but should be stored in the specimen processing area.
2. The functions of specimen preparation area are:
Extraction and storage of nucleic acids (RNA, DNA) and their addition to amplification reaction tubes. For operations involving clinical samples, the requirements of protective equipment, personal protection and operation specifications of biosafety level II laboratories should be met. Because the pollution caused by aerosol may occur in the process of sample mixing and nucleic acid purification, the aerosol pollution entering this area from the adjacent area can be avoided by establishing positive pressure conditions in this area. In order to avoid cross contamination between samples, the reaction tube containing the reaction mixture must be covered after adding the nucleic acid to be tested. For materials with potential infectious risk, the cover must be opened in the biosafety cabinet, and there must be clear procedures for sample processing and inactivation.
3. The functions of the nucleic acid amplification region are:
DNA synthesis, DNA amplification and detection. In order to avoid the pollution caused by aerosol, we should minimize the movement in this area. It must be noted that all tested reaction tubes must not be opened in this area.
4. The functions of the amplification product analysis area are:
Further analysis and determination of amplified fragments, such as hybridization, enzyme digestion electrophoresis, denaturing high performance liquid chromatography, sequencing, etc. There are many analytical methods for products after nucleic acid amplification, such as probe hybridization on membrane or microplate or chip (radionuclide labeling or non-radionuclide labeling), agarose gel electrophoresis after direct or enzymatic digestion, polyacrylamide gel electrophoresis, Southern transfer, nucleic acid sequencing, mass spectrometry and so on.
The amplification product analysis area is the main pollution source of amplification products in PCR laboratory, so attention should be paid to avoid carrying out amplification products through articles and overalls in this area. When using PCR-ELISA method to detect amplification products, the plate must be washed with a plate washer, and the waste liquid must be collected in 1 mol/L HCl, and should not be dumped in the laboratory, but should be discarded away from the PCR laboratory. Used tips must also be soaked in 1 mol/L HCl and then put into garbage bags for treatment according to procedures, such as incineration. Because some toxic substances such as ethidium bromide, acrylamide, formaldehyde or radionuclides may be used in this area, attention should be paid to the safety protection of laboratory personnel.


Main basis and standard of PCR laboratory design
The production and inspection environment of PCR reagents are clearly defined in the clauses of Appendix in Vitro Diagnostic Reagents of Good Manufacturing Practices of Medical Devices and Guiding Principles of On-site Inspection of in Vitro Diagnostic Reagents of Good Manufacturing Practices of Medical Devices. In addition, the current regulations, standards and related literatures in the health industry all provide for PCR testing laboratories, which mainly involve the relevant requirements of national clinical testing regulations (third edition), management measures for clinical gene amplification in medical institutions, working guidelines for clinical gene amplification testing laboratories in medical institutions and application of polymerase chain reaction (PCR) technology in clinical diagnosis.
Key points of PCR laboratory construction
1, the main structure
The main body is clean color steel plate and aluminum alloy profile. The inside and outside corners of the room are made of aluminum alloy 50 with inner rounded corners, which solves the problems of easy pollution, dust accumulation and difficult cleaning. Solid structure, simple and elegant lines and good sealing performance.
2. Standard three-zone separation and pressure difference control
The PCR process is divided into three independent experimental areas: reagent preparation, sample preparation and PCR amplification detection. The whole area has a whole buffer corridor. Each independent experimental area has a buffer.
Air conditioning and ventilation system design of PCR laboratory and pressure control PCR laboratory does not have strict purification requirements, but in order to avoid the possibility of cross-contamination among various experimental areas, it is advisable to adopt the air flow organization form of full delivery and full discharge. At the same time, the ratio of air supply and exhaust should be strictly controlled to ensure the pressure requirements of each experimental area.
Main basis and standard of PCR laboratory design
The production and inspection environment of PCR reagents are clearly defined in the clauses of Appendix in Vitro Diagnostic Reagents of Good Manufacturing Practices of Medical Devices and Guiding Principles of On-site Inspection of in Vitro Diagnostic Reagents of Good Manufacturing Practices of Medical Devices. In addition, the current regulations, standards and related literatures in the health industry all provide for PCR testing laboratories, which mainly involve the relevant requirements of national clinical testing regulations (third edition), management measures for clinical gene amplification in medical institutions, working guidelines for clinical gene amplification testing laboratories in medical institutions and application of polymerase chain reaction (PCR) technology in clinical diagnosis.
Key points of PCR laboratory construction
1, the main structure
The main body is clean color steel plate and aluminum alloy profile. The inside and outside corners of the room are made of aluminum alloy 50 with inner rounded corners, which solves the problems of easy pollution, dust accumulation and difficult cleaning. Solid structure, simple and elegant lines and good sealing performance.
2. Standard three-zone separation and pressure difference control
The PCR process is divided into three independent experimental areas: reagent preparation, sample preparation and PCR amplification detection. The whole area has a whole buffer corridor. Each independent experimental area has a buffer.
Air conditioning and ventilation system design of PCR laboratory and pressure control PCR laboratory does not have strict purification requirements, but in order to avoid the possibility of cross-contamination among various experimental areas, it is advisable to adopt the air flow organization form of full delivery and full discharge. At the same time, the ratio of air supply and exhaust should be strictly controlled to ensure the pressure requirements of each experimental area.
3. Disinfection
Ultraviolet lamps are installed in three experimental areas, the tops of three buffer zones and transfer windows for disinfection. Mobile ultraviolet lamps are also set in reagent preparation area and specimen preparation area to disinfect the test bench locally.
4. Mechanical interlocking stainless steel transmission window
Reagents and specimens are transmitted through mechanical interlocking stainless steel (electronic interlocking is not recommended) transmission window, so as to ensure that the reagents and specimens are not polluted during transmission (diversion of people).
5. Ground
Generally, PVC coiled material floor or gravity floor is used for the ground, which has good integrity. Easy to clean and corrosion resistant. Terrazzo floor can also be used, or joints of large tiles (at least 800mm×800mm) need to be less than 2mm. Try to avoid dust production and accumulation.
6. Lighting
Clean lamps shall be selected for lamps, which are easy to clean and dust-free.
Main instruments and equipment of PCR laboratory
1. Instruments and equipment in the reagent preparation area mainly include refrigerators, ultra-clean tables, sample feeders, mixers, scales and centrifuges, etc. Besides being dedicated, the sample feeders and scales should be calibrated regularly. Reagent subpackaging should be carried out in ultra-clean table. The mixed solution should be of molecular biological grade. After the reagent is prepared, it should have quality inspection: first, whether there is pollution; second, test the amplification effect of the reagent;
2. The instruments and equipment in the specimen preparation area mainly include biosafety cabinets, sample feeders, centrifuges, incubators, mixers, etc.
3. The main instruments in the amplification area are nucleic acid amplifiers, ultra-clean tables, micro-samplers, and mobile ultraviolet lamps of centrifuges. The expander needs to be calibrated. UPS power supply is provided to prevent power failure from affecting amplification effect due to voltage fluctuation.
4. Instruments and equipment used in the analysis area mainly include microplate reader, plate washer, sample feeder, water bath box, DNA sequencer, etc.
For example, as shown below:


Main cost evaluation of decoration construction of PCR laboratory
PCR laboratories have different requirements for cleanliness and constant temperature and humidity, so they are generally divided into the following categories:
1. The overall decoration cost of PCR laboratory without clear requirements for cleanliness is about 2,500 ~ 3,000 yuan/㎡;
2. The overall decoration cost of the PCR laboratory with a cleanliness of 100,000 grade and a constant temperature and humidity is about 3,000 ~ 4,000 yuan/㎡;
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