Quality Control of PCR Detection

The quality control of PCR testing is a systematic project, which generally involves three aspects: first, test instruments; second, test reagents and consumables; third, personnel operations. Problems in any one aspect will affect the quality of PCR detection.

Test Instruments

PCR machines and UV gel imaging systems (UV analyzers or portable UV, etc.) and pipettes may affect the quality of PCR assays. The PCR instrument controls the temperature change of the PCR reaction, and the accuracy of its temperature and control time may affect the quality of PCR detection. The accuracy of pipette extraction affects whether the reaction system can achieve the best reaction environment. UV analysis systems have a significant impact on detection sensitivity. There are 3 types of instruments for UV analysis systems: UV analyzers, UV gel imaging systems, and portable UV lamps. The UV analyzer directly observes the DNA amplification band on the agarose gel by eye. The UV gel imaging system is to observe the DNA amplification band on the agarose gel through the camera system on the computer. The portable UV lamp can directly observe the DNA amplification band on the agarose gel during electrophoresis, which is convenient to use and has a relatively low resolution. According to the resolution of these instruments from high to low order is UV analyzer, UV gel imaging system, portable UV lamp. Weak positive amplification bands can sometimes be seen on UV analyzers, but not on UV gel imaging systems. The portable UV lamp can only be seen when the positive amplification band is relatively bright. It is recommended that the portable UV lamp should not be used to ensure the detection quality. It can be used for temporary electrophoresis result observation.

The quality control of the instrument should be implemented through laboratory quality certification, and the validity of the instrument should be carried out on a regular basis. Laboratories without quality certification can regularly ask instrument manufacturers to conduct verification and maintenance to ensure that all instruments are in normal working condition.

2. Test reagents and consumables

In the quality control of PCR detection, the selection of reagents plays a very important role, and it is also the detection quality control factor that experimenters pay the most attention to. Reagents must be selected from manufacturers with good quality and reputation. Once selected, it is best not to change them easily. If it must be replaced, a strict comparison test should be carried out with the original product, including tests of sensitivity, specificity, and shelf life of reagents. And often pay attention to the differences in the quality of different batches of products. For example, the quality of TaqDNA polymerase produced by different manufacturers is different. Although they are all marked with the same activity unit, the difference in the calibration method, the composition of the preservation solution, and the transportation process often lead to differences in the actual activity unit. Sometimes, the actual activity unit is different. Due to its low activity, weak positive samples are missed; sometimes non-specific amplification occurs due to its high activity. This is similar to the role of enzyme conjugates in ELISA assays. In fact, there are many reagents that affect the results of PCR detection. It can be said that all the reagents used in PCR detection may affect the detection effect. Controlling the quality of reagents is a prerequisite for PCR detection. We believe that it is best to choose PCR kits to control the quality of the reagents.

To evaluate a PCR kit, in addition to considering its sensitivity, specificity, stability, and ease of operation, it should also include: the extent to which the reagents provided by the kit cover the entire PCR detection test. If the kit provides the reagents for the entire PCR detection test, it indicates that the kit has the whole-process control performance for the reagents of the PCR detection test. On the contrary, the quality control degree of the reagents for PCR detection is relatively low, which means that the quality control degree of the detection is low. For example, ethidium bromide only acts as a fluorescent dye. If it fails under sunlight for a long time, the fluorescence efficiency will decrease under ultraviolet light, which may cause some weak positive samples to be missed and false negatives. Problems with other reagents also affect the quality of the test. During the production process of the kit, there is a set of reagent production quality control system. Each reagent component has been tested for sensitivity, sensitivity and specificity quality control, and the quality of each reagent can be guaranteed within the validity period. Therefore, choosing a kit for PCR detection is an ideal control method. In PCR detection, it is best to use all kits to provide reagents, which is beneficial to the quality control of PCR detection.

Experimental consumables are also one of the factors that affect the quality of PCR detection. The thickness of the PCR reaction tube and the heat conduction performance directly affect the PCR detection effect. Mineral oil generally does not need to be added to the hot-lid PCR instrument, but some PCR reaction tubes are not tightly capped, resulting in the evaporation of water in the reaction solution during the amplification process, which seriously affects the detection sensitivity. The PCR reaction system is a small amount. When the production quality of the tip of the pipette is not good, sometimes the liquid residue is large or the flooding effect is strong, resulting in insufficient reagent volume in the kit and inaccurate configuration of the PCR reaction system, resulting in a decrease in the detection quality. .

In each PCR test, a negative control sample and a positive control sample must be set up. When the negative control sample is detected as negative, it indicates that the reagents in the whole process of the test are not contaminated; when the positive control sample is detected as positive, it indicates that the working system of DNA extraction (RNA extraction, RNA reverse transcription), DNA amplification and electrophoresis identification is normal. The test samples can only be judged on the premise that the test results of the negative control samples and positive control samples are established. Therefore, each test should set up controls indicating DNA extraction (RNA extraction, RNA reverse transcription), DNA amplification and electrophoresis identification. Only by setting up controls throughout the test can the test results be proven. This is very important for PCR and RT-PCR detection assays.

A positive control is a must-have component in the kit. There are three types of positive controls: the first is to use the detected pathogenic microorganisms as positive controls. The advantage of such a direct comparison is that it is intuitive, accurate, and the condition of the experiment is complete, which can indicate whether the experiment is established. The disadvantage is that it increases the index of PCR contamination during the detection process. In addition, it cannot be shown that each test sample control holds. The second is to set up a microorganism not related to the detection of the virus as a positive control. The advantage is that the risk of PCR contamination is reduced and the biosafety of the kit is improved. The disadvantage is that it is only a reference to the positive control, which is not directly and completely reflects the establishment of this test, nor can it show that the control of each test sample is established. It is very suitable for the detection of pathogenic microorganisms in laboratories that are afraid of contamination or requiring high biosafety levels. The third is to set up a reference control in each reaction tube. In addition to the positive amplification band, another amplification band is set up to indicate the detection situation in each reaction tube. When the test is positive, there are two amplified bands of different sizes, and when the test is negative, one amplified band appears. This kind of control establishment has high technical requirements and high cost, and the control effect is the most ideal, which can eliminate the influence of the operation error of each test sample or the problem of the reagent on the test. Since two templates are amplified at the same time in one reaction tube, there is a certain amount of interference between each other, so the sensitivity of the detection of pathogenic microorganisms is more or less affected. This control method may become the development trend of PCR control.

1. 3. Personnel Operation

Several of our laboratory personnel have tested the same sample at the same time, and the test results do vary. Frequent testers are 10 to 100 times more sensitive than other testers. It shows that the PCR detection test does have certain operating techniques that need to be familiar with and trained, and different personnel have a certain impact on the test results. It is necessary to strengthen the training of personnel's operating skills, and it is necessary for laboratory technicians to have a proficient process for PCR detection.

PCR contamination control is a content that PCR testing operators must pay great attention to. The laboratory is set up in the distribution area, template extraction area, amplification area, and electrophoresis area. The logistics should be in the order of distribution liquid area, template extraction area, amplification area, and electrophoresis area, and backflow is strictly prohibited. Personnel operation should also be very careful, such as regular cleaning and disinfection, and regular cleaning and disinfection of pipettes. Timely test operation is simple, fast and accurate.