Demystifying the P3 Lab

Economic Daily, China Economic Network reporter Zheng Yang

The P3 laboratory is one of the secret weapons of the "Hand-Tear Virus" of the Second People's Hospital of Guangdong Province. Every day, more than 400 new crown pneumonia nucleic acid tests are carried out here. How exactly was the virus "pull out" in the P3 laboratory? Let the Economic Daily reporter take you to unveil the mystery of the hospital's P3 laboratory.

"Here is the place where it comes into contact with the virus" zero distance ". The entire laboratory is completely sealed and the room is under negative pressure. We like to use the" three flows "to describe it, that is, the one-way flow of people, logistics, and airflow. Pollutants will not leak to the outside and cause pollution. "Said Lin Jianguo, director of the P3 laboratory of Guangdong Second People's Hospital.

"Here" refers to the P3 laboratory. Every day, more than 400 new crown pneumonia nucleic acid tests are carried out in the Second People's Hospital of Guangdong Province. As the first provincial emergency hospital in the country, the Second People's Hospital of Guangdong Province has the WHO International Emergency Medical Team and has extensive experience in fighting viruses. The P3 laboratory is one of the secret weapons of the hospital's "hand tear virus".

Past and Present

P3 laboratory is the abbreviation of the third-level laboratory of biosafety protection, and P is the abbreviation of Protection. The entire laboratory is completely sealed, and the room is under negative pressure, so that the internal gas will not leak to the outside and cause pollution, which is the one-way flow of "airflow".

According to the degree of harm of microorganisms and their toxins, the biosafety protection laboratory is divided into 4 levels: the lowest level and the highest level. The first-level laboratory is suitable for microorganisms that have no pathogenic effect on healthy adults; the second-level laboratory is suitable for microorganisms that have medium potential harm to humans and the environment; the third-level laboratory is suitable for people who are infected with serious or even lethal diseases mainly through the respiratory route. Pathogenic microorganisms or their toxins; Level 4 is applicable to pathogenic microorganisms or their toxins that are highly dangerous to the human body, are transmitted through the aerosol route or the transmission route is unknown, and there is no effective vaccine or treatment.

The New Crown Virus nucleic acid test of Guangdong Second People's Hospital was carried out in the P3 laboratory. The slogan of this P3 laboratory is 3P, Peacetime war, Precaution, Progression. Combining peacetime with wartime: peacetime is mainly based on scientific research and regular emergency drills are conducted. Once an epidemic situation occurs, it immediately enters the emergency work state. Alarm bells: keep a clear head, pay close attention to global and domestic research trends in infectious diseases, and prevent problems before they occur. Keeping up with the times: follow up with new international knowledge, new technologies and new equipment in time to build a laboratory with an international perspective.

Pull cocoon

Nucleic acid testing is the "gold standard" for the diagnosis of new coronary pneumonia, and every link in the testing process is delicate and can not be allowed to be sloppy. During the operation, the experimenter faced the samples in front of them and needed to "remove the lid one by one and add the nucleic acid and extract the nucleic acid." This was undoubtedly a confrontation with the virus.

So, how did the P3 laboratory experimenters "overplay" the virus? According to reports, the testing process in the laboratory can be divided into 5 steps: the sample enters the "pass-through window"-inactivation processing-open lid addition and core extraction-judgment result of amplification reaction-autoclaving.

The first step is for the sample to enter the laboratory. Before the collected test samples enter the P3 laboratory, they must first enter a special window-the "transmission window", also known as the air lock. It has double doors that cannot be opened at the same time to ensure that the air taken in during sample transfer is isolated in the double doors, so that the environment outside the laboratory is not polluted. The sample will open here for a brief lifetime in the laboratory.

The second step is inactivation before detection. The samples will be inactivated in a water bath at 56 ° C for half an hour, making the viral proteins no longer physiologically active and losing their ability to infect, cause disease and reproduce. This makes the test relatively safe, but the gene sequence of the viral protein is not affected.

The third step is the core part of the test—open the lid and add the nucleic acid.

Hu Liangshan, deputy chief technician of the laboratory medical department of the hospital, was the first person to enter the P3 negative pressure laboratory to complete the preliminary preparations such as equipment debugging, program setting, and reagent trial. "In the specimen processing room, when the experimenter got the inactivated sample, the first action was to shake, try to let the virus on the swab elute in the medium solution, and let it stand for 5 minutes to precipitate. The cap sample addition must be performed manually, and requires the high cooperation of two people to complete. One person unscrews the lid of the sample tank, and the other holds the micropipette to suck the trace solution in the sample tank, put it into another extraction tube, and screw it Close the tube cap. "She said, just like this, more than 400 samples have to be opened to remove the bottle cap and sucked and transferred more than 400 times, each time the experimenter" opens tears "face to face with the virus.

"During the process of opening the lid, the virus is very easy to be exposed to the laboratory air." Hu Liangshan said, "When opening the lid to add samples, the hand holding the micropipette is often awkward because of wearing double gloves. You have to be careful, plus the triple barriers of the blindfold, face screen and glass windows of the biosafety cabinet, all movements appear slow, and the hands tremble from time to time because of the slow movements, often with the left hand holding the right hand to complete the addition. "

The steps of artificial nucleic acid extraction are more complicated, testing the patience and caution of the experimenter. "Nucleic acid extraction can be assisted by instruments, but due to the comparison, manual operations are often required, and more than 10 steps such as centrifugation, adding reagents, and washing are required. Among them, the operation of opening the tube cover has been performed more than 7 times. An artificial nucleic acid extraction takes about 50 minutes. "Hu Liangshan said.

The fourth step is to judge the results. The extracted nucleic acid is sent to the nucleic acid detection chamber, and it is about to be used in the instrument. The experimenter added the extracted nucleic acid to the amplification reagent, and performed the RT-PCR reaction with a fluorescent PCR instrument. The analysis took about 110 minutes to determine whether the result was negative or positive.

Finally, the autoclaving of pollutants. After the contamination produced in the experimental process is autoclaved, it is treated as ordinary medical waste.

Security

Let's take a look at how the staff works.

First, wear intermediate protective clothing in the clean area-enter the negative pressure work room in the laboratory through the buffer room, process the sample here, nucleic acid extraction and detection-after the space and surface disinfection, remove the protective clothing in order, After changing clothes in the shower, return to the cleaning area. None of these steps is necessary.

Nucleic acid testing is designed to be efficient, orderly, and safe. Among them, the safety of staff is of paramount importance. To ensure safety, the hospital has adopted various measures. First, personnel screening. After issuing anti-epidemic mobilization orders, many people enthusiastically signed up. After a lot of screening, what remained were healthy and well-trained young "veterans". Second, pre-job safety training, strictly according to It is required to wear protective clothing and be familiar with the operation process. Third, strictly implement the disinfection and sterilization system. Yan Xiukui, the P3 laboratory administrator in the hospital, is responsible for daily disinfection. Fourth, a sensory supervisor system is established to detect negative pressure areas in time. Discomfort or improper operation of personnel to ensure personnel physical and mental safety.

So far, the hospital's P3 laboratory has not experienced any biosecurity issues.

Fake the truth

In the P3 laboratory, any problem in any link may lead to "false negatives" or "false positives". In order to ensure the quality of the test, the test results will undergo heavy comparison and review.

According to reports, measures to ensure the quality of testing have begun before testing. The medical staff for sample collection must go through strict training and assessment before they can get on the job. The quality of the sample is closely related to the collection technology, and each frame of action must be performed in accordance with regulations. The escort work after the samples are collected must be delivered to the laboratory in accordance with strict procedures.

During the test, the experimenter took every test carefully and did not miss any suspicious samples. For suspicious samples, different reagents will be used to confirm the determination to prevent “false negatives”. At the same time, more than 3 blank controls will be randomly inserted for each batch of experiments, and viral genes will be sequenced and compared for positive samples to prevent A "false positive" appears.

After the test, the laboratory will periodically randomly sample the test samples and send them to a third-party laboratory for comparison and analysis of the data to ensure the accuracy of the test.

Invincible

The P3 testing team of the hospital, known as the "Scout", currently has 21 people, more than 70% of whom have a master's degree or above, have undergone molecular biology training and have a PCR employment certificate.

"Unlike the medical staff fighting in the ward, they are hidden in the laboratory and come into close contact with the virus every day, which is the" frontline "invisible to others." Lin Jianguo said.

It is reported that the laboratory strictly complies with the "New Coronavirus Pneumonia Diagnosis and Treatment Program" and biosafety requirements. The testers need to wear intermediate protective equipment in a P3 laboratory negative pressure confined space. They will not eat or drink for 5 hours. Dizziness and vomiting caused by pressure difference and tight equipment.

Not only that, the inspectors have to overcome psychological fear. Pharyngeal swab samples were collected from the nasopharynx of a suspected patient and may contain more neocoronavirus. When inspectors took samples from the sampling tanks, the virus could easily spread through the air in a closed environment, but they still fought on the front line with no regrets and took the first pass.

Zheng Yang