Detection principle flow chart
Through continuous attempts, we finally established a method for rapid detection of 9 genotypes of 3 polymorphic loci in one tube without extraction, which we named RPCR-MC. As shown in Figure 1, the first step is to add the sample to be extracted to the extraction-free reagent at a ratio of 1:30 and simple shock mixing. The whole sample extraction process is simple and fast, requiring only 5 s to complete. Next, the amplification reaction solution is prepared, and the extracted samples are added to the reaction solution for detection. We used asymmetric amplification to amplify the initial template. The ratio of upstream and downstream primers was 1:4. The excess downstream primers will bind with the probe in the melting stage. The designed probe involves two adjacent probes. The 3’ end of the first CYP2C19 * 2 probe is modified with the FAM fluorescent group, the 5’ end of the second probe of the adjacent interval 1 base is modified with the BHQ1 quenching group, and the 3’ end is modified with phosphate group P to block the extension. Similarly, the fluorophore of the first probe of CYP2C19 * 17 is ROX, and the quenching group of the second probe is BHQ2. During the study, it was found that the presence of multiple adjacent probes in one tube leads to mutual interference between the primers and probes. However, the method of adjacent probe detection can finally accommodate two polymorphic sites in one tube. Therefore, we combined the Taqman probe with the adjacent probe. CYP2C19 * 3 is designed as a TaqMan probe: the 5’ end is modified by the HEX fluorophore, and the 3’ end carries the BHQ1 quenching group. In the melting heating stage, which is conducted originally under low temperature conditions because the two adjacent probes are close to each other, the fluorescent group and the quenching group interact with each other and do not emit fluorescence. With the gradual increase in temperature, the adjacent probes gradually become separated such that the fluorescent group and the quenching group are separated. For adjacent probes, during the dissolution phase, as the temperature increases, the adjacent fluorophore and quencher separate, thereby releasing fluorescence; For Taqman probes, during the dissolution phase, as the temperature increases, the ability of the probe and template to combine decreases, and gradually falls off the template, that is, from the state of stretching and straightening gradually to the curled state, and the fluorescence intensity changes. As the binding force of the probe and the template chain is reduced with the increase in temperature, a melting peak occurs. Similarly, due to the difference of a base between the homozygous wild-type and mutant samples, the Tm value is different in the melting stage, and the final test results show different Tm values.