1. There is no Ct value in the fluorescence quantitative PCR experiment

(1) Incorrect sequence or primer: The test sample does not contain the gene to be tested. Carefully search for the sequence of the gene to be tested in the NCBI database, and redesign the primers.

(2) Degradation of nucleic acid templates: To avoid the introduction of impurities and repeated freezing and thawing in nucleic acid sample preparation, consumables such as centrifuge tubes and tips used in the operation are all disposable.

(3) Insufficient amount of nucleic acid template: For samples of unknown concentration, start with a high concentration of serially diluted samples, but it should be noted that high concentration of nucleic acid will lead to non-specific amplification and aerosol contamination.

(4) The number of cycles of the reaction is not enough: the number of cycles in the fluorescence quantitative PCR reaction is generally more than 35 cycles, but too many cycles can increase the background value (recommended not to exceed 45 cycles).

(5) Degradation of primers or probes: The integrity of the primers can be detected by PAGE electrophoresis. If the electrophoresis bands are diffuse, resynthesis of primers or probes can be considered. It is recommended to repackage them before use.

(6) Incorrect steps to detect fluorescent signals: Fluorescence quantitative PCR generally collects signals at the end of annealing or extension. If the acquisition steps are wrong, the results cannot be accurately interpreted.

2. The Ct value appeared too late in the fluorescence quantitative PCR experiment

Cause analysis and solution:

(1) The amplification product is too long: the product length of 80-150bp is generally used, and the three-step amplification method is recommended for fragments longer than 200bp.

(2) The structure of the detected gene is complex: the detected gene contains complex structures such as high GC, complex secondary structure and long fragments, which will affect PCR amplification.

(3) Low amplification efficiency: optimize the reaction conditions, redesign the primers or probes, the fluorescent dye is degraded, there are inhibitors in the system, and the three-step method is used for the reaction.

(4) The template is degraded or the template concentration is too high: Check its integrity by PAGE electrophoresis. If the template is degraded, re-extract the nucleic acid template, and if the concentration is too high, dilute it.

(5) Poor sensitivity of the reagents: The detection performance of the reagents (especially the premix) is not good, the sensitivity is low, and the anti-interference ability is poor. It is recommended to replace the reagents with higher sensitivity and stronger anti-interference.