How to evaluate a PCR machine?

When it comes to PCR (Polymerase Chain Reaction) technology, practitioners in the field of life science will certainly be familiar with it. It has been more than 30 years since the advent of PCR technology, during which innovative biotechnology has constantly emerged, but the status of PCR technology is still unshakable - more than 30 years later, as an indispensable technical means, PCR is still widely used in gene cloning, gene expression analysis, pathogen detection, disease diagnosis, gene sequencing, biopharmaceutics, gene/cell therapy, molecular breeding, species identification Forensic expertise and other fields.

In 2020, the COVID-19 will spread all over the world, and it is PCR technology that plays a central role in the laboratory detection of COVID-19.

What exactly is PCR technology?

In short, PCR technology is a technology that uses the principle of DNA double strand replication to massively amplify specific DNA fragments in vitro.

After the completion of the traditional PCR experiment, the product needs to view the amplification bands through electrophoresis and imaging to obtain qualitative results. In the mid-1990s, fluorescence quantitative PCR (qPCR for short) was born on the basis of traditional PCR technology.

QPCR can detect the starting template in real time. It has the advantages of good specificity, high detection sensitivity, intuitive results, time-saving, safety, etc., and is more used in the detection and diagnosis field. In basic scientific research, qPCR is also the main method for gene expression analysis.

At the beginning of the 21st century, PCR technology ushered in the third innovation. Digital PCR technology emerged, which can achieve accurate quantification without standard curve. However, because its instrument platform and reagent consumption costs are much higher than qPCR, and the operation is cumbersome, traditional PCR and fluorescent quantitative PCR are still the most widely used technologies.

Corresponding to the three generations of PCR technology, there are mainly three types of PCR instruments on the market, namely, ordinary PCR instrument, fluorescent quantitative PCR instrument and digital PCR instrument.

What are the key components of a PCR instrument?

From the perspective of instrument composition, the ordinary PCR instrument is mainly composed of a thermal cycle system. In addition to the thermal cycle system, the fluorescent quantitative PCR instrument and the digital PCR instrument also contain an optical detection system and supporting operational analysis software.

The performance of temperature control determines the success or failure of PCR reaction

Temperature control performance is one of the most important factors affecting PCR reaction

It can also be seen from the PCR principle that the PCR reaction is a process of constant heating and cooling. It is required to be able to control the temperature in the three key steps of denaturation annealing extension. Temperature control involves many aspects such as temperature accuracy, homogeneity, temperature rise and fall rate, temperature gradient, etc.

Temperature accuracy is an important performance index of PCR instrument. If the temperature accuracy of the instrument is poor, and the actual temperature of the sample tank cannot reach the set temperature, the expected amplification results will not be obtained, that is, either amplification cannot be achieved, or non-specific amplification or even false amplification will occur.

Temperature uniformity is the guarantee for stable amplification. If the temperature uniformity of the PCR instrument is poor, it is impossible to determine whether the different samples or the poor performance of the instrument are the cause of different amplification results.

The rate of temperature rise and fall not only directly affects the time of PCR experiment, but also affects the work efficiency and the specificity of amplification products. The faster the rate of temperature rise and fall, the better the specificity.

The gradient temperature function of PCR instrument is very helpful for the optimization of reaction conditions (especially the optimization of annealing temperature). For the new experiment, the optimal annealing temperature can be obtained quickly through the temperature gradient function.

The sample tank material affects the temperature control performance

The material of the sample tank of the PCR instrument will affect the performance of these temperature controls. At present, most PCR instruments use aluminum alloy sample tanks, while high-end PCR instruments use silver sample tanks with better thermal conductivity.

The optical inspection system determines the result

For the fluorescent quantitative PCR instrument, the quality of the optical detection system will have a great impact on the detection results. The optical detection system mainly includes excitation light source, detector, signal acquisition mode, number of detection channels, etc.

The excitation light source shall consider the spectral range and light intensity. Although most of the excitation light sources of the new generation of products are LED lights, monochrome LED lights and white LED lights with composite light have their own advantages and disadvantages in terms of excitation effect. It is better to use their respective advantages to form a wide and strong light excitation effect.

The detector mainly focuses on detection sensitivity. Of course, the higher the sensitivity of the detector, the better. The higher the sensitivity, the lower the signal strength can be detected, that is, the lower the sample concentration can be detected. The detectors used in qPCR instrument mainly include PMT and CCD. The detection sensitivity of PMT with photomultiplier tube is higher than that of CCD.

Each company's products have their own unique signal acquisition methods. Some products have the problem of optical path difference of signals, and it is troublesome to add additional correction dyes.

The number of detection channels will determine how many times the instrument can perform fluorescence detection at most. Different application fields have different requirements for the number of detection channels. For example, users who do gene expression analysis only need one detection channel. For example, COVID-19 detection usually requires triple or quadruple detection, which will have different requirements for the configuration of the instrument.

In addition to the above temperature control and optical detection systems, the sample flux, the diversity of sample tanks, the anti evaporation effect of the hot cover, the noise level during operation, the applicability of the platform to reagent consumables, and the friendliness of the operating interface are also factors to consider the quality of a PCR instrument. In addition, since the PCR instrument is a very frequently used instrument, the durability and after-sales service of the instrument are also the concerns of users.

With the outbreak of COVID-19 this year, people's demand for automated work platforms has become more urgent and strong. Automated PCR detection platform is essential for government detection departments, hospitals, biopharmaceutical enterprises and some large laboratory center platforms with many samples and limited staff.

At present, more and more personal laboratories are actively building automation systems, which can be predicted to usher in the rapid growth of full-automatic detection platforms in the next few years.