The detection principles of rapid detection reagents for monkeypox virus mainly include the following:

Detection Principle of Nucleic Acid

• Real – time Fluorescent Quantitative PCR
  Based on the unique nucleic acid sequence of the monkeypox virus, specific primers and probes are designed. In the PCR reaction system, the primers will bind to a specific region of the monkeypox virus nucleic acid. Under the action of DNA polymerase, the virus nucleic acid is used as a template for amplification. At the same time, the probe will specifically bind to the amplified product. With the action of the fluorescent group and the quenching group, when the probe is intact, the fluorescence emitted by the fluorescent group is absorbed by the quenching group. However, when the probe binds to the amplified product, the fluorescent group and the quenching group are separated, and the fluorescent signal is released. By monitoring the progress of the PCR reaction in real time through the intensity and growth rate of the fluorescent signal, it is possible to determine whether the monkeypox virus nucleic acid exists in the sample and the content of the virus.
• Isothermal Amplification Technology
  For example, the loop – mediated isothermal amplification (LAMP) technology uses 4 – 6 specific primers. Under the action of the strand displacement DNA polymerase, the rapid amplification of the monkeypox virus nucleic acid is achieved under isothermal conditions. The LAMP technology specifically recognizes and amplifies multiple regions of the monkeypox virus nucleic acid. After the reaction is completed, the color change of the reaction solution can be observed with the naked eye or the fluorescent signal can be detected using a fluorescence detection device to determine whether the monkeypox virus nucleic acid exists. This technology has the advantages of simple operation, fast reaction speed, and no need for complex instruments.

Detection Principle of Antigen

• Immunochromatography
  Specific anti – monkeypox virus antibodies are fixed on carriers such as nitrocellulose membranes. When the monkeypox virus antigen in the sample encounters the antibody on the test strip, a specific binding occurs. Then, another anti – monkeypox virus antibody with a marker (such as colloidal gold, latex particles, etc.) further binds to the antibody that has already bound to the antigen, forming an antigen – antibody – labeled antibody complex. If the monkeypox virus antigen exists in the sample, the marker will accumulate at the detection line, showing a colored band. The test result is judged by observing the presence or absence of the band with the naked eye.
• Enzyme – Linked Immunosorbent Assay (ELISA)
  The specific antibody against monkeypox virus antigen is coated on the wells of an enzyme – linked plate. The sample to be tested is added. If the monkeypox virus antigen is present in the sample, the antigen will bind to the coated antibody. Then, an enzyme – labeled another anti – monkeypox virus antibody is added to form an antibody – antigen – enzyme – labeled antibody complex. A substrate is added, and the enzyme catalyzes the substrate to undergo a color reaction. The absorbance value is detected by an enzyme – linked immunosorbent assay instrument. According to the size of the absorbance value, the content of the monkeypox virus antigen in the sample is judged. The higher the absorbance value, the higher the antigen content in the sample.

Detection Principle of Antibody

• Indirect Immunofluorescence Assay
  The monkeypox virus antigen is fixed on a slide. The serum sample to be tested is added. If the serum contains antibodies against the monkeypox virus, the antibodies will bind to the antigen. Then, a fluorescently labeled anti – human immunoglobulin antibody is added, which will bind to the human antibody bound to the antigen. Observed under a fluorescence microscope, if specific fluorescence appears, it indicates that antibodies against the monkeypox virus exist in the sample, and thus it is determined whether the tested person has been infected with the monkeypox virus.
• Chemiluminescence Assay
  Chemiluminescent substances are used to label anti – monkeypox virus antibodies or antigens. When the antibodies in the sample specifically bind to the labeled antigen or labeled antibody, under the action of a chemical reaction, the chemiluminescent substance will emit a light signal. The intensity of the light signal is detected by a chemiluminescence detector to determine the content or presence of monkeypox virus antibodies in the sample, thereby assisting in the diagnosis of monkeypox virus infection.