Portable Real-Time fluorescent quantitative PCR

I. Overview of Portable Real-Time Fluorescence Quantitative PCR

Portable real-time fluorescence quantitative PCR (Polymerase Chain Reaction) is a miniaturized and portable molecular biology detection device developed based on the traditional real-time fluorescence quantitative PCR technology. It combines the high sensitivity of PCR technology with the real-time quantitative characteristics of fluorescence detection, enabling the amplification and quantitative analysis of specific nucleic acid fragments in a short period of time. It is widely used in multiple fields such as medical diagnosis, food safety testing, environmental monitoring, epidemic prevention and control, and biological scientific research, providing powerful technical support for on-site rapid detection.

II. Working Principle

1. PCR Amplification Principle

• PCR is an in vitro nucleic acid amplification technique that simulates the process of DNA replication in vivo. It utilizes DNA polymerase to add four deoxynucleotides (dNTPs) one by one to the 3′ end of primers according to the principle of base complementary pairing, with single-stranded DNA as a template, under specific temperature conditions, to synthesize new DNA strands. The whole process includes three basic steps: denaturation, annealing, and extension. Through multiple cycles (usually 20 – 40 cycles), the target DNA fragment is amplified exponentially.
• In portable real-time fluorescence quantitative PCR, the nucleic acid sample to be detected is first added to a reaction system containing specific primers, Taq DNA polymerase, dNTPs, buffer solution, and other components. Then, the heating module of the instrument is used to rapidly change the temperature of the reaction system in cycles to achieve DNA amplification.

2. Fluorescence Detection Principle

• One or more fluorescent dyes or fluorescent probes are added to the reaction system. These fluorescent substances can bind to the amplification products and emit fluorescent signals. As the PCR reaction progresses, the amount of amplification products increases continuously, and the intensity of the fluorescent signals also increases accordingly.
• Common fluorescence detection methods include the SYBR Green I dye method and the TaqMan probe method. SYBR Green I dye can non-specifically bind to double-stranded DNA. When DNA is amplified, the amount of bound dye increases, and the fluorescence intensity is enhanced. The TaqMan probe is a specific oligonucleotide probe with a fluorescent reporter group labeled at the 5′ end and a fluorescent quencher group labeled at the 3′ end. During the PCR amplification process, when Taq DNA polymerase extends to the position where the probe binds, its 5′ – 3′ exonuclease activity will cut off the probe, separating the reporter group from the quencher group, thereby generating a fluorescent signal.
• The instrument can achieve quantitative analysis of the target nucleic acid by detecting the changes in the intensity of the fluorescent signals in real time and comparing them with known concentrations of standards, and determine the initial copy number of the target gene in the sample.

III. Characteristics

1. Portability

• Compared with traditional large PCR instruments, portable real-time fluorescence quantitative PCR devices are compact in size and lightweight. They usually adopt a compact integrated design, with built-in batteries or the ability to be connected to mobile power supplies, which is convenient for use in on-site environments such as the wild, primary medical institutions, customs ports, and farms. They are not restricted by the venue and power supply, and can quickly respond to the detection needs of various emergency situations.

2. Rapid and Efficient

• They have a fast temperature rise and fall rate and can complete multiple cycles of the PCR reaction in a relatively short period of time, greatly shortening the detection time. Generally, conventional PCR detection may take several hours, while portable real-time fluorescence quantitative PCR can shorten the detection time to dozens of minutes or even less, significantly improving the detection efficiency and making it possible for early diagnosis of diseases and rapid screening of epidemics.

3. High Sensitivity and Specificity

• It can detect extremely low concentrations of target nucleic acid sequences and has high sensitivity. This is due to the exponential amplification ability of PCR technology and the high sensitivity of fluorescence detection. Even if the content of the target nucleic acid in the sample is extremely low, after multiple amplifications, a strong enough fluorescent signal can be generated to be detected by the instrument. Meanwhile, through the design of specific primers and probes, the target gene sequence can be accurately identified, avoiding non-specific amplification and false-positive results, ensuring the accuracy and reliability of the test results.

4. Real-Time Quantitative Analysis

• It can monitor the changes in fluorescent signals in real time during the PCR reaction process, obtain the amplification status of the target nucleic acid in real time, and perform quantitative analysis on it according to the standard curve. This real-time quantitative ability enables it not only to determine whether the target nucleic acid exists in the sample but also to accurately measure its content, providing an important quantitative basis for disease diagnosis, disease condition monitoring, and pathogen load analysis.

5. Easy to Operate

• Equipped with a simple and intuitive operation interface and automated control programs, operators only need to add samples and reaction reagents to the reaction tubes, put them into the instrument, and start the detection according to the preset program. The instrument will automatically complete the temperature cycle control, fluorescent signal collection, and data analysis processes and directly display the test results. There is no need for professional molecular biology technicians to perform complicated operations and data analysis, reducing the technical requirements for operators and facilitating its promotion and application in grass-roots units and on-site environments.

IV. Application Scenarios

1. Medical Diagnosis Field

• In the diagnosis of infectious diseases, it can be used to rapidly detect the nucleic acids of pathogens such as viruses (such as COVID-19 virus, influenza virus, hepatitis B virus, hepatitis C virus, etc.), bacteria (such as Mycobacterium tuberculosis, Streptococcus pneumoniae, etc.), and fungi (such as Candida, etc.), assisting clinicians in diagnosing diseases accurately and in a timely manner, formulating treatment plans, and effectively preventing and controlling the spread of diseases. For example, during the COVID-19 pandemic, portable real-time fluorescence quantitative PCR devices were widely used for the on-site rapid detection of the COVID-19 virus, enabling the screening of a large number of suspected samples in a short period of time and timely detection of infected individuals, which played a crucial role in epidemic prevention and control.
• In the diagnosis of genetic diseases, it can be used to detect gene mutations related to genetic diseases, such as the genetic diagnosis of thalassemia, cystic fibrosis, and other diseases, helping doctors conduct prenatal diagnosis, genetic counseling, and early screening of diseases, and reducing the incidence of genetic diseases.

2. Food Safety Testing Field

• It can detect pathogenic microorganisms in food (such as Escherichia coli, Salmonella, Staphylococcus aureus, etc.), transgenic components, and food allergens. For example, during the food production and processing process, rapid detection of raw materials, semi-finished products, and finished products can ensure the quality and safety of food and prevent food poisoning and disease transmission events caused by consuming contaminated food. In the inspection and quarantine of imported food, portable real-time fluorescence quantitative PCR can quickly screen out food containing transgenic components or with excessive pathogenic microorganisms, safeguarding the health and rights of domestic consumers.

3. Environmental Monitoring Field

• It is used to detect microbial pollution in the environment, such as the detection of pathogens such as bacteria, viruses, and parasites in drinking water to ensure the safety of drinking water; the analysis of the structure and functional genes of microbial communities in soil and water bodies to evaluate the health status of the ecological environment; and the monitoring of pathogens in the air, such as in hospitals and public places, to monitor in real time whether there are pathogenic microorganisms in the air and provide a basis for preventing and controlling airborne diseases.

4. Epidemic Prevention and Control Field

• In the early stage of an epidemic outbreak, it can be quickly deployed to the epidemic site to conduct large-scale nucleic acid screening for suspected patients, close contacts, and other populations, timely discover the source of infection, cut off the transmission route, and strive for precious time for epidemic prevention and control. Meanwhile, it can also be used to monitor and analyze the epidemic trend, evaluate the effectiveness of prevention and control measures, and provide scientific data support for epidemic prevention and control decisions.

5. Biological Scientific Research Field

• In molecular biology research, it can be used for gene expression analysis, gene mutation detection, gene cloning verification, and other experiments, helping researchers quickly and accurately obtain experimental data and promoting the progress of scientific research projects. For example, when studying the mechanism of tumor occurrence and development, by detecting changes in the expression of tumor-related genes, a deeper understanding of the biological behavior of tumor cells can be achieved, providing a theoretical basis for the development of new tumor treatment methods.