1. Determine the detection target: determine the species and gene fragments of pathogenic microorganisms to be detected, understand their biological characteristics and genome sequence, and determine the detection target gene fragments.
2. Design primers: According to the sequence of the target gene fragment, use bioinformatics software to design specific primers. Primers should have high specificity, sensitivity and stability, which can effectively amplify the target gene fragment and avoid non-specific amplification.
3. Construction of standard substance: According to the sequence of the target gene fragment, the standard substance is synthesized, and its concentration gradient is diluted to make a standard curve.
4. Screening and optimizing reaction conditions: According to the designed primers and standards, screening and optimizing reaction conditions, including reaction temperature, reaction time, primer concentration, Mg2+ concentration, etc.
5. Verification kit: A verification experiment is conducted with a standard substance with a known concentration to check the accuracy and repeatability of the kit. At the same time, it is necessary to cross-verify the kit to check whether it can detect other pathogenic microorganisms.