ICL Experience | Read the key points of PCR laboratory construction in one article

Author: Independent Medical Laboratory Information

On April 26, the National Health Commission held a national video conference, which clearly proposed that “all disease control institutions at or above the county level and general hospitals above the second level should step up their transformation and form nucleic acid detection capabilities in a short period of time”.

Independent medical laboratories can not meet the needs of public institutions in various places while enriching the testing force, while giving full play to their rich testing management operation concepts, as well as the experience advantages of regional coverage, integrated into the construction of the “national team” nucleic acid testing capacity, follow the trend, take advantage of the trend.

With the establishment of PCR laboratories in various places, this paper sorts out several key points of PCR laboratory construction from the perspective of hardware foundation, and independent medical laboratories can use their rich operational experience to carry out construction guidance, personnel echelon establishment, quality control system construction, regional service support, etc.

How to build a PCR laboratory

There are four main guiding documents related to the construction of PCR laboratories:

Regulations on the Administration of Medical Institutions

Measures for the Administration of Clinical Laboratories in Medical Institutions

Interim Measures for the Management of Clinical Gene Amplification Testing Laboratories

Measures for the Administration of Clinical Gene Amplification Laboratories in Medical Institutions

Clinical PCR laboratories are required not only to meet the conditions for clinical testing in laboratory settings and hardware such as instruments and equipment, but also to have documented working procedures in daily work.

The following are a few key control points during the construction of PCR laboratories:

01 Establish a PCR laboratory quality management system

According to the specific situation of the laboratory, prepare quality system documents, “quality manual”, “procedure documents”, “operation instructions”, and ensure the effective operation of the management system.

The characteristics of the management system are that it should have a clear purpose, standardized management, effective constraints, efficient mechanism, and self-development as a whole.

Establish a sound SOP document, stipulate the method of accomplishing various quality activities, and each SOP should describe a activity or set of activities that are related to each other.

Each SOP file should describe the documents, materials, personnel, records required for the input, conversion and output of each aspect of the quality and their interface relationship to the relevant activities.

Clarify the requirements of each factor in the conversion process of each link, that is, who does it, what procedures are done, what requirements are met, how to control, what records and reports are formed, and the corresponding approval procedures.

Corrective actions for exceptions or special situations that require attention in quality activities. SOPs should be concise, clear and understandable, and staffed and strictly adhered to.

02 PCR laboratory settings

To complete a set of PCR experiments, it is usually necessary to go through four experimental processes: reagent preparation, sample processing, nucleic acid amplification and product analysis.

The experimental rooms of these four experimental procedures should be arranged next to each other to form an independent PCR experimental area. Standard PCR experimental areas include: reagent preparation area, specimen preparation area, amplification area, product analysis area, buffer zone and common corridor in each area.

Overall layout of PCR laboratory

The layout of a PCR laboratory in a hospital is shown in Figure 1, with a common corridor throughout the area and a dedicated buffer zone for each individual experimental area. Through differential pressure control, reagents and specimens are protected from aerosols throughout PCR experiments and amplification products are reduced to personnel and the environment.

Figure 1, floor plan of a hospital’s PCR laboratory

Reagent preparation area

The main operations in this experimental area are the preparation of storage reagents, the dispensing of reagents and the preparation of the main reaction mixture.

Reagents and materials used in specimen preparation should be shipped directly to the area and should not pass through other areas. The reagent raw materials must be stored in the area and prepared into the required storage reagents in the area.

The reagent preparation area is equipped with a 2~8°C refrigerator and a -80°C refrigerator, which need to be taken into account when calculating the cooling load.

The area of the room should be controlled at 15m2~20m2. The pressure gradient requirement in this area is: relative positive pressure state to prevent the outside air containing nucleic acid aerosols from entering and causing pollution.

Specimen preparation area

The main operations in this area are the preservation of clinical specimens, nucleic acid (RNA, DNA) extraction, storage and synthesis of cDNA when added to amplification tubes and RNA determination.

The specimen preparation area is equipped with a 2~8°C refrigerator and a -20°C refrigerator, which need to be taken into account when calculating the cooling load. In the specimen preparation area, a biosafety cabinet is also required for the extraction of nucleic acids.

In order to avoid repeated circulation of extracted nucleic acids in the cabinet, causing cross-contamination between specimens and false positive results, the biosafety cabinet equipped in this area must be type B2. The vertical airflow in the biosafety cabinet work area comes entirely from the laboratory, and the exhaust air is filtered by a HEPA filter and discharged directly to the outside, and is not allowed to return to the cabinet and laboratory.

According to experience, if the laboratory is equipped with a biological safety cabinet, the area of the laboratory increases by 10m2 for each one; The area of the specimen preparation area should be between 25m2~30m2. The pressure gradient requirement for this zone is positive pressure relative to the adjacent area to avoid aerosol contamination from the adjacent area entering the area.

Amplification chamber

The main operations performed in this region are 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.

In nested PCR assays, the reaction tube must usually be opened after the first round of amplification, so nested amplification has a high risk of contamination and the second dose must be performed within the region.

The amplification room is mainly equipped with the core instrument PCR instrument of the PCR laboratory, and two types of PCR instruments, ABI7500 and ABI9700, are used in this example.

During the construction of the laboratory, the PCR instrument needs to be equipped with a special UPS power supply to ensure its normal operation. The area of the area is controlled at 15m2~20m2.

The pressure gradient requirement in this zone is negative pressure relative to the adjacent area to avoid aerosol leakage from the area. To avoid contamination caused by aerosols, unnecessary movement in the area should be minimized. Individual operations such as dosing should be carried out in the clean table. It is recommended to use 5~10Pa pressure difference, which is easier to achieve in control.

Product analysis area

The main operation in this region is the determination of amplified fragments. If testing with a fully automatic closed analytical instrument, this area can be dispensed with.

Equipped with a fume hood indoors to ensure the relative negative pressure in the room, air flows from the outside to the room. The area of this area is controlled at 15m²~20m².

This area is the most important source of amplification product pollution, so the pressure gradient requirement for this area is negative pressure relative to the adjacent area to avoid the diffusion of amplification products from this area to other regions. It is recommended to use 5~10Pa pressure difference, which is easier to achieve in control.

03 Setting of buffer chambers in positive and negative pressure areas

According to the requirements of the pressure gradient, the reagent preparation chamber and sample processing room are relatively positive pressure, the amplification chamber and the product analysis room are relatively negative pressure, and the pressure design in the buffer chamber of the area requiring positive pressure and the area requiring negative pressure is very different.

The positive pressure buffer chamber conforms to the buffer principle of the general positive pressure clean room, mainly to prevent aerosols in the outdoor ambient air from entering the room. The arrangement of the positive pressure buffer chamber is shown in Figure 2.

Figure 2, layout drawing of positive pressure buffer chamber

The negative pressure buffer chamber requires the buffer chamber to maintain positive pressure on the nucleic acid amplification chamber and product analysis area, and also maintain positive pressure on the buffer room (mainly to meet the needs of indoor purification, if there is no indoor cleanliness requirement, here can be 0 pressure). The arrangement of the negative pressure buffer chamber is shown in Figure 3.

Figure 3, layout diagram of negative pressure buffer chamber

04 Prevention and control of pollution

The core issue of PCR laboratory design is how to avoid contamination. In practice, the following types of pollution are common: contamination of amplification products; contamination of natural genomic DNA; Contamination of reagents and between specimens. Therefore, to avoid pollution, the first thing should be prevention, not elimination.

The work area should be strictly divided, and each experimental area should be clearly marked (such as eye-catching door numbers or different ground colors, etc.) to avoid confusion of equipment items, reagents, etc. in different experimental areas.

Reasonable system settings, reasonable air conditioning ventilation system settings, try to use the air conditioning system of full delivery and full row; Strict air flow pressure control ensures different pressure requirements in different experimental areas.

Standardized operation, the technical staff of the clinical gene amplification testing laboratory must be trained, and the operator must wear gloves and change frequently during the experimental operation. Cleaning is timely and correct. After the experimental work, the area must be cleaned immediately.

Strict management: Strictly control the personnel entering and leaving the laboratory. Use overalls with distinctive marks (e.g. different colors) in each experimental area, and do not take the overalls of the area with other areas when the staff leaves; Minimize unnecessary movement within the experimental area to reduce the possibility of cross-contamination. Amplification product analysis area is the most important source of amplification product pollution, waste liquid can not be dumped in the laboratory, must be soaked in disinfectant and discarded away away from the laboratory, used suction tips and other disposable materials should also be soaked and disinfected by disinfectant solution and uniformly treated, such as incineration;

In short, laboratory construction is a complex problem, here does not discuss the problem of personnel, laboratory personnel training and precipitation, also need a process, in the process of construction to adapt to local conditions, do not apply rigid sets, combined with their own reality slowly explore the construction ideas suitable for their own development. Build laboratories scientifically and rationally, and better play the role of laboratories.


[1] Ministry of Housing and Urban-Rural Development of the People’s Republic of China, General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China. GB50591-2010, Code for clean room construction and acceptance[S].Beijing:Guangming Daily Press,2010.

[2] Xu Zhonglin. Principles of Clean Air Technology (3rd Edition)[M].Beijing:Science Press,2004.

[3] Interim Measures for the Administration of Clinical Gene Amplification Testing Laboratories (Wei Yifa [2002] No. 10).

[4] LIU Shuqing,XIE Fengjie. Establishment and implementation and maintenance of quality management system[J] Industrial Engineering, 2003.

[5] In 2010, the General Office of the Ministry of Health issued the “Measures for the Management of Clinical Gene Amplification Laboratories in Medical Institutions”.

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