Nucleic acid detection is an important tool for diagnosing patients with novel coronavirus (COVID-19), assessing clinical treatment effects, population screening and epidemiological investigations in cases of epidemic situations. At present, the most commonly used specimen collection methods for nucleic acid detection are nasal swabs and throat swabs. The major difference between the two is the different sampling sites, nasal swabs mainly target the secretions from the nasal mucosa, which is directly accessed from the inside of the nasal cavity to extract samples. However, throat swabs need to enter from the oral cavity and extract secretions from the throat. During the first few days of infection, the patient's viral titer is high, and a single patient's nasopharyngeal swab may contain nearly 1 million SARS-COV-2 virus particles. Therefore, nucleic acid detection provides the earliest and most sensitive detection for the presence of SARS-COV-2.
Commonly used nucleic acid detection methods
The detection methods of viral nucleic acid usually include reverse transcription polymerase chain reaction (RT-PCR), isothermal amplification and high-throughput sequencing. Here, we introduce the two most common methods.
RT-PCR can efficiently amplify very small amounts of viral genetic material in a mixture of other nucleic acid sequences and is considered the gold standard technique for the detection of SARS-COV-2. The general workflow of RT-PCR detection includes five main steps: sample collection and transport, sample lysis, viral RNA extraction and purification, RT-PCR amplification, detection and analysis. It is important to note that RNA molecules are very susceptible to degradation. Therefore, samples must be processed under optimized protocols and strict laboratory conditions to avoid any contamination and reduce RNA degradation, thus avoiding erroneous results.
Figure 1. The general workflow of RT-PCR detection .
The requirement for sophisticated thermal cycling equipment is a limitation of RT-PCR technology . However, the use of an isothermal amplification platform helps to eliminate this requirement and allows amplification at a constant temperature. Reverse transcription‐loop‐mediated isothermal amplification (RT-LAMP) is the most common method to follow this strategy and it is also a powerful alternative to RT-PCR. Similar to RT-PCR, RT-LAMP first transcribes the target RNA into cDNA by reverse transcriptase and then performs an amplification reaction . This method can use different primers to amplify multiple target sequences in the same reaction exponentially, so it has the characteristics of high efficiency, specificity and sensitivity. In addition, expensive reagents and instruments are not required to perform the assay, making the method cost effective.
Figure 2. Molecular overview of the isothermal amplification technique .
The current COVID-19 pandemic has caused a serious public health crisis, and disease diagnosis and isolation of patients are effective methods to prevent and control the spread of COVID-19. Today, nucleic acid detection has become the most sensitive and early detection for COVID-19. Alfa Chemistry provides you with raw materials for nucleic acid detection to assist your research. If you do not find what you need, please contact us. We also offer product customization according to customer's detailed requirements.
- Esbin, M. N.; et al. Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection. Rna. 2020, 26(7): 771-783.
- Notomi, T.; et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000, 28, E63.
- Zhang, X.; et al. Advances in laboratory detection methods and technology application of SARS‐CoV‐2. Journal of Medical Virology. 2022, 94(4): 1357-1365.
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