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The magic of quantitative RT-PCR: Why do scientists love using this technology to detect viruses?
As scientific research increasingly relies on precise molecular biology techniques, quantitative RT-PCR (reverse transcription polymerase chain reaction) is widely used in virus detection and gene expression analysis and is favored by many scientists. Since its introduction in 1977, this technology has gradually replaced traditional techniques, such as Northern blot, as the gold standard for detecting RNA.
The simplicity, specificity, and sensitivity of reverse transcription polymerase chain reaction technology enable it to be used in a variety of studies, from quantifying wine yeast cells to diagnosing emerging infectious diseases.
Basic principles of RT-PCR
The process of RT-PCR involves converting an RNA template into complementary DNA (cDNA) and then amplifying it using polymerase chain reaction. This technology allows us to improve transcript detection of almost any gene. Among variations of RT-PCR, phase-detection real-time polymerase chain reaction (qPCR) introduces fluorescently labeled probes during the amplification process, allowing researchers to monitor DNA production in real time.
Why choose one-stage or two-stage RT-PCR?
When performing RT-PCR, you can choose a one-stage or two-stage strategy. One-stage RT-PCR completes cDNA synthesis and PCR amplification in a single reaction tube, helping to reduce the risk of contamination caused by sample handling. Two-stage RT-PCR performs these two steps separately in different tubes. Although this may make the technology more accurate, it also increases the complexity of the operation.
Attention needs to be paid to the quality of the RNA sample when performing RT-PCR, especially when using the one-stage method, as this may affect the final experimental results.
Application areas of RT-PCR
In terms of research methods, RT-PCR is widely used for the measurement of gene expression. For example, scientists use qRT-PCR to analyze gene expression in yeast cells to understand the impact of gene mutations on expression. In addition, RT-PCR has also shown its potential in the diagnosis of genetic diseases and cancer detection.
Not only that, RT-PCR technology has been used to quickly identify and detect certain viruses, such as SARS-CoV-2 and influenza viruses, which has had a profound impact on public health.
Challenges and future development
Although RT-PCR has many advantages, there are also some challenges, such as possible contamination problems caused by excessive detection sensitivity and the accuracy of quantitative results. To overcome these challenges, scientists are constantly improving experimental designs and techniques and developing more sophisticated control methods to reduce the impact of variables.
In the future, with the advancement of technology and reduction of costs, RT-PCR may become a mainstream technology for more research and clinical applications. Not only will it help us decode the mysteries of gene expression, it will also aid in rapid response during outbreaks, with the goal of improving public health and safety.
With the advancement of science and the expansion of applications, what new and unexpected solutions will quantitative RT-PCR bring us?
