Alat rapid antigen: basic knowledge of nucleic acid detection - part 2

Alat rapid antigen: know nucleic acid detection

Real time fluorescence quantitative PCR

Real time fluorescence quantitative PCR technology refers to the method of adding fluorescent groups to the PCR reaction system, using fluorescent signals to monitor the whole PCR process in real time, and finally quantitatively analyzing the unknown template through the standard curve.

The principle of this technology is to mark the probe with fluorescent group, mark the fluorescent group R at the 5 'end and mark the quenching group Q at the 3' end. When there is no PCR amplification, the fluorescent group is quenched and does not emit fluorescence due to the close spatial distance between the fluorescent group and the quenching group;When PCR is amplified, the primer and the fluorescent labeled specific probe are combined on the template at the same time. The binding position between the fluorescent labeled probe and the template is located between the upstream and downstream primers. The fluorescent probe is hydrolyzed by using the 5 ′ 3 ′ exonuclease activity of Taq enzyme, and the fluorescent group is released. Because it is separated from the quenching group in space, it emits fluorescence.The fluorescence emitted can be detected by the fluorescence probe, amplified and detected at the same time, so as to realize "real-time" detection.This technology not only realizes the leap of PCR from semi quantitative to quantitative, but also has the characteristics of strong specificity, high degree of automation and effectively solving the problem of PCR pollution compared with conventional PCR.

Branched strand DNA detection -- bDNA

BDNA is a method for quantitative detection of HIV type 1 RNA in plasma.BDNA refers to a synthetic DNA fragment with side chains. Each side chain can be labeled with an excited marker.The RNA of HIV is released from the virus particles by centrifugation, and then captured into the micropores with capture probe 

Specifically binds to another part of viral RNA and also binds to pre amplified probe.The latter is then hybridized with the amplification probe, bDNA probe.The two sets of target probes specifically bind to different regions of pol gene of viral RNA.Hivrna oligonucleotide complexes are formed in micropores.The chemiluminescence signal can be amplified after incubation with a chemiluminescence substrate.It is quantified by luminous intensity, which is proportional to the content of hivrna in the sample.BDNA has no cross contamination of amplicons, which is a great progress compared with PCR.BDNA has dozens of probes covering the whole genome, which can easily detect some HIV variants, but the sensitivity is not as sensitive as PCR. Improving the sensitivity of bDNA is a major difficulty.

Detection steps

The detection process of HIV nucleic acid qualitative detection technology is divided into nucleic acid extraction, reverse transcription synthesis of cDNA, PCR amplification reaction, qualitative analysis of amplification products, result judgment and completion report.