The working principle of monkeypox virus rapid detection reagents is mainly based on molecular biology or immunology principles to detect the presence of the monkeypox virus.

I. Principle Based on Nucleic Acid Detection

1. Nucleic Acid Extraction

• Firstly, viral nucleic acids are extracted from the collected samples (such as skin vesicle fluid, pharyngeal swabs, etc.). This process usually employs specialized nucleic acid extraction reagents and methods. For example, magnetic bead-based or column-based methods can be used to extract nucleic acids. These methods can effectively separate the viral nucleic acids from impurities such as cell debris and proteins in the samples, providing a pure template for subsequent detection.

2. Nucleic Acid Amplification

• The extracted nucleic acids will be amplified through polymerase chain reaction (PCR) or other nucleic acid amplification techniques. Taking PCR as an example, specific primers are added to the reaction system. These primers are short DNA fragments designed according to the nucleic acid sequence of the monkeypox virus. Under appropriate conditions such as temperature, the action of enzymes, and the presence of buffers, the primers will bind to specific regions in the monkeypox virus nucleic acid. Then, with the help of DNA polymerase, replication will be carried out using the viral nucleic acid as a template. After multiple cycles of amplification, the originally trace amount of viral nucleic acid can be replicated in large quantities, making the detection more sensitive. For instance, after 20 to 30 cycles of PCR amplification, even if there is only a very small amount of viral nucleic acid in the sample initially, a sufficient amount of products can be generated for detection.

3. Detection of Amplified Products

• The amplified nucleic acid products can be detected in multiple ways. One common method is fluorescent quantitative PCR. Fluorescent-labeled probes are added to the reaction system. When the probes bind to the amplified products, fluorescent signals will be emitted. By detecting the intensity of the fluorescent signals, it can be determined whether there is monkeypox virus nucleic acid in the sample and the amount of the viral nucleic acid. If the fluorescent signal exceeds the set threshold, it indicates that the sample contains monkeypox virus nucleic acid and the test result is positive. Another method is gel electrophoresis. The amplified products are subjected to electrophoresis in an agarose gel. According to the different sizes of nucleic acid fragments, different bands will be formed in the gel. If a band that matches the expected size of the monkeypox virus nucleic acid appears, it can also be judged as a positive result.

II. Principle Based on Antigen Detection

1. Antigen-Antibody Reaction

• Antigen detection reagents utilize the immunological principle of specific binding between antigens and antibodies. The reagents contain antibodies specific to certain antigens of the monkeypox virus. These antibodies are usually obtained as monoclonal or polyclonal antibodies through genetic engineering techniques or from the serum of immunized animals. When the monkeypox virus antigens are present in the sample, the antibodies will bind to them to form antigen-antibody complexes. For example, some structural proteins of the monkeypox virus (such as viral envelope proteins) can serve as antigens to be recognized and bound by the antibodies.

2. Signal Detection

• In order to facilitate the observation of the results of antigen-antibody binding, the reagents also include a signal generation system. One common method is immunochromatographic technology. There are different areas on the test strip, such as the sample area, the conjugate area, and the detection area. When the sample is added, during the liquid flow process, if there are antigen-antibody complexes, they will be recognized and bound by the secondary antibody labeled with a chromogenic agent (such as colloidal gold) in the detection area. In this way, a colored band will be formed in the detection area. The presence or absence and the color intensity of the band are used to judge whether the sample contains monkeypox virus antigens and the approximate content of the antigens. If a distinct colored band appears in the detection area, it can be judged as a positive result; if there is no colored band or the color is very faint, it is a negative result. This method is simple to operate, does not require complicated instrument equipment, and can obtain results within a relatively short time, making it very suitable for on-site rapid detection.