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What are the nucleic acid detection methods for Mycoplasma haemofelis in cats?

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  1. Polymerase Chain Reaction (PCR) Detection
    • Principle: Specific primers are used to amplify the nucleic acid of Mycoplasma haemofelis, and then the amplification products are detected. If the nucleic acid of Mycoplasma haemofelis is present, nucleic acid fragments of a specific size will be obtained after PCR amplification, thus determining whether the pathogen is present in the sample.
    • Steps:
      1. DNA extraction of the sample to be tested: Nucleic acids are usually extracted from samples such as a cat’s blood, nasopharyngeal or conjunctival secretions. For example, protease K is first added to the sample for treatment, followed by the addition of specific buffers, incubation, centrifugation and other operations to remove impurities and obtain relatively pure DNA.
      2. PCR amplification: Amplification is carried out using specific primers for Mycoplasma haemofelis identification. The specific primers are designed according to the specific gene sequence of Mycoplasma haemofelis and can specifically bind to the target nucleic acid. Multiple cycles of denaturation, annealing, and extension reactions are carried out under appropriate temperature conditions to amplify the target nucleic acid fragments in large quantities.
      3. Product detection: The amplified products are detected by electrophoresis. The PCR amplification products are electrophoresed on an agarose gel. According to the size and migration speed of the nucleic acid fragments, it is observed under an ultraviolet detector whether nucleic acid fragments of a specific size appear. If they do, the tested sample is determined to be positive for Mycoplasma haemofelis nucleic acid.
  2. Real – time Fluorescent Quantitative PCR (qPCR) Detection
    • Principle: Fluorescent probes or fluorescent dyes are added to the PCR reaction system. As the PCR reaction proceeds, the fluorescence signal accumulates continuously. By detecting the change of the fluorescence signal, the progress of the PCR reaction can be monitored in real – time, and the content of Mycoplasma haemofelis nucleic acid in the sample can be quantitatively analyzed.
    • Advantages: Compared with ordinary PCR, qPCR has higher sensitivity and specificity, can more accurately detect pathogen nucleic acids at low concentrations, and can quantitatively analyze the quantity of nucleic acids, which is of great significance for disease diagnosis and evaluation of treatment effects.
  3. Loop – mediated Isothermal Amplification (LAMP) Detection
    • Principle: Nucleic acid amplification is carried out under isothermal conditions using specially designed primers and a DNA polymerase with strand – displacement activity. This method does not require temperature cycling like PCR, and the amplification reaction can be completed at a constant temperature, and the operation is relatively simple and fast.
    • Advantages: It has lower requirements for instruments and equipment, does not need expensive PCR instruments, and is suitable for use in basic – level laboratories or on – site testing. Moreover, LAMP has a high amplification efficiency, can produce a large number of amplification products in a short time, and has a high detection sensitivity.
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