What is the "nucleic acid test" that we are talking about every day?

Q1:What is nucleic acid testing?

"Doctor, I want to do nucleic acid!"

"Are you doing the nucleic acid test for the new coronavirus?"

"Ah! Sure, is there any other nucleic acid test?"

"Nucleic acid test = new coronavirus test"? In fact, the nucleic acid test for NSCV is only one of the nucleic acid tests. Nucleic acid testing methods have been widely used in pathogenic microbiological testing as an important means of diagnosing disease and monitoring viral load since before the New Crown epidemic, for example, the familiar quantitative DNA test for hepatitis B virus, the human papilloma virus (HPV) test, and the human immunodeficiency virus (HIV) RNA test. Therefore, in terms of testing scope, "nucleic acid testing > new crown testing".

So, what is nucleic acid?

Nucleic acid is the genetic material of almost all living things on earth. Nucleic acid is divided into deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Each virus contains only one type of nucleic acid, DNA or RNA, located on the inside of the virus and a "protective protein shell" on the outside.

Novel coronavirus is a virus of RNA. If RNA of a novel coronavirus can be detected in a patient sample, it suggests that the patient may be infected by a novel coronavirus.

Q2: Picking the mouth? Poke the nose? Why not take blood?

Currently, the routine sample collection methods for nucleic acid testing of novel coronaviruses include: throat swabs and nasopharyngeal swabs. This is also commonly known as "stabbing the nose" and "stabbing the throat".

"Doctor, which one is more comfortable?"

"Well ...... this varies from person to person, the sensitivity of different parts of each person is different, but they are not very comfortable. For testing, the nasopharyngeal swab is a little more accurate."

"Doctor, can you take blood for this nucleic acid test?"

"No. ......"

The reason is that the new coronavirus, after infecting the human body, will first "take root" in the epithelial cells of the respiratory tract and multiply. Therefore, respiratory specimens (nasopharyngeal swabs, pharyngeal swabs, sputum and alveolar lavage fluid) have the highest positive rate of testing. Since the virus may not have invaded the patient's bloodstream at the beginning of the infection, testing for viral nucleic acid in the blood is likely to be negative at this time, and such false-negative results can lead to missed tests.

Q3: How does the nucleic acid test "catch" the new coronavirus?

Step 1: Storage and transportation

After sampling, the swab is placed in virus preservation solution and transported to the nucleic acid testing laboratory for the new coronavirus. The virus preservation solution maintains the activity and integrity of the virus. At the same time, the preservation solution contains a variety of antibiotics, so that the "competition" can be defeated and the virus can live "freely" in this paradise in a "comfortable" environment all to itself. The second step: shock.

Step 2: Shock.

Virus sampling tube sent to the testing laboratory, first removed in the biosafety cabinet, full shaking, the swab adhering to the respiratory epithelial cells and new coronavirus can not be less "shake off" to the preservation solution.

Step 3: Nucleic acid extraction

(1) Lysis

Before the nucleic acid test, the protein "vest" must be removed with guanidine salt in order to release the nucleic acid of the virus and reveal the "true face" of the virus.

(2) Binding

Magnetic beads are added to bind to the nucleic acid. Say goodbye to all kinds of cell fragments and proteins ......!

(3) Washing

Binding once to catch the nucleic acid? It's not that easy! Yes, we can catch the nucleic acid, but there are still a few fragments and proteins that are stuck together with the nucleic acid. How to improve the purity of nucleic acid? We have two main advantages: adding salt and adding acid.

"Adsorption of nucleic acid - high salt and low pH"

"Elution of nucleic acid - low salt and high pH"

The nucleic acid is extracted and purified by repeated adsorption and elution two or three times. The rest is the "naked" nucleic acid.

Step 4: Nucleic acid detection

PCR was invented in 1983 by the famous American chemist Kary Banks Mullis and was awarded the Nobel Prize in Chemistry in 1993. PCR is a technique that selectively amplifies a specific region of DNA in vitro by mimicking the way DNA replicates in an organism.

The PCR reaction is like a large assembly plant. To amplify a viral fragment, you first design a pair of primers based on its characteristics. The primers are the most powerful "grabbers" for identification and localization, with the precision of "pointing and hitting". In a group of viral nucleic acids, he can catch new coronavirus, influenza, rhinovirus, respiratory syncytial virus, etc., only what you can't think of, nothing he can't catch.

When he identifies the "characteristics" of the new coronavirus - specific fragments, immediately one after another "capture", and then add dNTP, Mg2+, Taqase and other raw materials, adjusting The viral nucleic acid assembly is completed in three steps: denaturation-annealing-extension. The DNA yield increases exponentially with each amplification. The amplification of a new coronavirus typically takes 40 cycles, which theoretically yields 240=1,099,511,627,776 copies for one copy. Even if the viral content in the sample is very low, it can be detected by PCR amplification. Thus PCR reaction has high specificity and sensitivity.

240 copies of nucleic acid fragment is a lot, but it is colorless, odorless, and invisible, so what if the enemy is in the dark? What if the enemy is in the dark? Then we let him be in the light! Real-time fluorescence PCR reaction is based on PCR reaction with the addition of a probe, each amplification of the target fragment will emit fluorescence, captured by the fluorescence detector.

As the number of amplifications increases, the amount of fluorescence accumulates, which is the graph we see, and we use it to determine whether there is a new coronavirus.

"Hey, doctor, why is there a positive curve inside the positive and negative curves? Is the negative contaminated?"

"Don't panic, that is a human housekeeping gene, that is a gene that is stably expressed in all human cells."

"I am checking the new coronavirus, do what this "housekeeper" to do?"

"This is used to monitor the sampling, transportation and testing process!"

"Is a positive housekeeper gene means that the sampling site is fine?"

"This is not necessarily, butler gene, only on behalf of the human tissue, as to whether or not to pick up the virus latent site, it is not necessarily."

"Oh! I finally know why we need to get the nucleic acid report the next day, I thought you took the sample and put it on the machine to get the results, so there are so many steps!"