In the post-epidemic era, the term "nucleic acid testing" is familiar to all of us, and it has become a part of our daily life.
So, do you know how nucleic acid testing detects viruses? And how does nucleic acid testing make invisible viruses "visible"?
01 What is nucleic acid
Nucleic acid is the general term for DNA and RNA.
Nucleic acid is an essential component of all known life on earth. The DNA we often say is deoxyribonucleic acid. The genetic material of the new coronavirus is RNA - ribonucleic acid.
Nucleic acid amplification testing
02 What exactly does nucleic acid testing detect?
The substance for nucleic acid detection is the nucleic acid of the virus.
The new coronavirus is a virus that only contains RNA, and the specific RNA sequence in the virus is a marker that distinguishes the virus from other pathogens. Nucleic acid detection is to find out whether there is nucleic acid of foreign invading virus in the patient's respiratory specimen or blood to determine whether he is infected by the new coronavirus. The vast majority of the current nucleic acid detection of the new coronavirus uses the fluorescent quantitative PCR method to amplify the target sequence of the viral RNA by PCR after reverse transcription.
PCR, or polymerase chain reaction, is a molecular biology technique used to amplify specific DNA fragments; it refers to the DNA polymerase-catalyzed reaction of specific genes or The reaction of rapid in vitro amplification of DNA sequences is also called gene in vitro amplification. Fluorescent quantitative PCR technology is to add a fluorescent reporter system to the PCR reaction system, and use the change of fluorescent signal to monitor the PCR process in real time, so as to realize the quantitative detection of the initial template.
To detect nucleic acid, the first step is to collect a sample. At present, the most conventional sampling method is: use throat swabs or nasal swabs to wipe and collect the upper respiratory tract (pharynx or nasal cavity); the collected samples will be strictly sealed, and then sent to the laboratory for gradual testing.
1. Extraction of viral RNA
The RNA of the virus is first extracted from the sample. Add nucleic acid extraction reagents to the sample to destroy the virus and release the nucleic acid.
2. "Reverse transcription" of viral RNA into cDNA
Through reverse transcription technology (RT), the RNA of the virus is "reversed" into a specific DNA that is more convenient to detect, that is, cDNA.
3. Amplification and detection of cDNA
In fact, whether it is throat swab, nasal swab or even blood sampling, the virus content in the sample is relatively low, even in severely infected patients (the viral load is large); because the virus particles are too small and the sampling volume is limited, The number of viruses contained in the sample is still too small for nucleic acid detection.
Therefore, by using PCR technology, the inspectors amplify the unique "password" of the new coronavirus in the sample to the point where the equipment can detect it, so as to judge whether it is a positive infection. The whole process of grabbing the genetic material of the virus is like fishing. The hook will identify and grab it, so that the virus has nowhere to hide.
Simply put, it is to amplify the number of cDNA, so that the cDNA is continuously replicated, so that the number increases exponentially.
The standard PCR process is divided into three steps:
Denaturation: The use of high temperature to separate the double strands of DNA. The hydrogen bonds between DNA double strands are broken at high temperature (93 - 98°C).
Annealing: After the DNA double strands have separated, the temperature is lowered to allow the primers to bind to the single stranded DNA.
Extension: DNA polymerase synthesizes a complementary strand along the DNA strand from the primer bound when the temperature is lowered. When elongation is complete, one cycle is completed and the number of DNA fragments doubles. Repeat these three steps 25-35 times, and the number of DNA fragments will increase exponentially.
PCR double-stranded template
While the cDNA is being amplified, the fluorescent probes in the kit are working simultaneously. It will release a fluorescent signal, and every time the cDNA is amplified, the fluorescent signal will increase a little, and the PCR detector can record a Ct value of a fluorescent signal increase.
The Ct value is an important indicator for judging whether the virus nucleic acid is negative or positive. The less initial DNA, the more PCR cycles required to reach a certain threshold, and the higher the Ct value. Conversely, the more initial DNA, the lower the Ct value. The initial amount of DNA reflects the amount of viral RNA in the sample, which in turn reflects the amount of virus in the sample. Therefore, if the Ct value is not detected or is higher than a certain value, then the suspected patient is judged as negative; if it is lower than this value, then the suspected patient is judged as positive.
During the PCR reaction, the selection of PCR consumables is also one of the most important factors affecting the results of nucleic acid detection.
