Principles and procedures of nucleic acid detection for COVID-19

The most common method to detect the specific sequence of the COVID-19 is real time quantitative PCR (polymerase chain reaction). Because the PCR reaction template is only the DNA, the COVID-19 (RNA) should be reverse transcribed into DNA before the PCR reaction.In the PCR reaction system, it contains a pair of specific primers and a Taqman probe. The probe is a specific oligonucleotide sequence with a reporter fluorophore and a quencher fluorophore labeled at both ends.When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quencher group;

If there is a target sequence in the reaction system, the probe binds to the template during the PCR reaction, and the DNA polymerase uses the exonuclease activity of the enzyme to cut and degrade the probe along the template, and the reporter group is separated from the quencher group and emits fluorescence.Every time a DNA strand is amplified, a fluorescent molecule is produced.The real time quantitative PCR instrument can monitor that the number of cycles (Ct value) at which the fluorescence reaches the preset threshold is related to the concentration of viral nucleic acid. The higher the concentration of viral nucleic acid, the smaller the Ct value.The products of different manufacturers will determine the positive judgment value of this product based on the performance of their own products

Inspection process

For nucleic acid testing of the COVID-19 , first sample collection is performed according to the "sample requirements" part of the kit instructions. The conventional sample types include throat swabs, nasal swabs, sputum, bronchial lavage fluid, and alveolar lavage fluid

After obtaining patient samples, testing should be carried out as soon as possible. If the samples that need to be transported cannot be tested immediately, they should be packaged at low temperature according to the instructions and sent to a special testing agency for testing.After receiving the sample, the testing institution shall perform nucleic acid extraction on the sample, and the nucleic acid extraction reagent shall use the nucleic acid extraction kit specified in the approved product manual.

Viral RNA needs to be reverse transcribed into cDNA first, and then amplified and tested.PCR amplification and detection should use the fluorescent quantitative PCR instrument specified in the approved product manual. The Ct value of the sample obtained by fluorescent quantitative PCR can be used to determine whether the patient sample contains the COVID-19

In the above process, the collection, storage, and transportation of samples, the extraction and detection of sample nucleic acid, and the interpretation of results must be carried out in strict accordance with the requirements of the kit instructions.