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.