But this method is not suitable for large-scale application, nor for the diagnosis of this suspected case. This is because the virus culture takes a long time and requires high site conditions (P3 laboratory), and the timeliness is far behind the clinical needs.
As one of the detection methods of etiology, nucleic acid detection is very efficient, and the results can be known in a few hours under normal circumstances. Therefore, in this epidemic, the main way to obtain pathogenic evidence in clinic is not virus culture, but nucleic acid detection.
Nucleic acid is the carrier of genetic information and the general name of DNA and RNA. The virus is mainly composed of genetic material and protein. Novel coronavirus is an RNA virus, and its genetic material is single-stranded RNA. The principle of nucleic acid detection is to detect whether there is viral RNA in samples such as throat swabs of subjects. If it exists, it means it is infected by a virus. So, do we know the RNA of the virus?
△RNA and DNA (image source network)
In the early stage of Wuhan epidemic, China Center for Disease Control and Prevention completed the identification of the virus in a short time by using metagenomics technology, and obtained the whole genome sequence. Real-time fluorescent reverse transcription polymerase chain reaction (RT-PCR) reagents developed rapidly after the whole genome sequence of the virus was published.
Subsequently, the nucleic acid detection kit was applied in clinic, which provided the etiological basis for the diagnosis of COVID-19. So, what is the process of nucleic acid detection using the kit?
First, inspectors need to extract nucleic acids from samples with the help of reagents. Then the separated nucleic acid is added to the nucleic acid amplification reagent, and then put into the nucleic acid amplification instrument, and generally the results will be produced within two hours.
In fact, amplification is a very important step in the operation steps. The significance of amplification lies in the exponential increase of trace genetic information. As long as there is a nucleic acid gene fragment in the sample, it can be amplified by PCR, so as to compare it with the whole genome sequence of the virus more clearly.
Real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR) has been used in clinical laboratories for many years. The detection system is mature, with the advantages of strong specificity, high sensitivity, short detection period and quantitative detection, and can also dynamically monitor the degree of virus infection and therapeutic effect.
Chen Xin, chief physician of the Department of Respiratory and Critical Care Medicine of China-Japan Friendship Hospital, said in an interview that nucleic acid detection is very sensitive. As long as a part of viral nucleic acid is found in the patient's secretion sample, it can be integrated into the sequence by RT-PCR amplification.
"Although it is not certain whether the nucleic acid comes from a live virus, for patients or suspected patients with respiratory infection symptoms, if their throat swabs have this nucleic acid, it is generally considered to be part of a live virus, which indirectly proves that there is virus reproduction and infection in their bodies." Chen Xin told reporters.
Nucleic acid detection is prone to "false negative"
Although nucleic acid detection has the advantages of high sensitivity and short detection period, it also has some shortcomings. Different from the way of isolating live virus directly from human body, nucleic acid detection is an indirect way, which may cause false negative.
Because the kit has the problem of the lowest detection limit, it can only be detected if the virus content in the collected samples reaches a certain level. In a few cases, the recovered person may not be able to recover, but due to the low viral load in the sample, the nucleic acid test result of the recovered person is negative, which is what we often call "false negative".
Therefore, in the "novel coronavirus Diagnostic and Treatment Standard (Trial Seventh Edition)", one of the discharge criteria is that respiratory tract samples such as sputum and nasopharyngeal swab are negative for two consecutive times (sampling time interval is at least 24 hours).
The reason why respiratory tract samples are selected for nucleic acid detection is that COVID-19 is a respiratory infectious disease, so it is preferred to obtain samples from respiratory secretions. Respiratory tract is divided into upper respiratory tract and lower respiratory tract, the former mainly includes nose, pharynx and larynx; The latter mainly includes trachea, main bronchus and bronchi at all levels in the lung.
Therefore, at present, there are two kinds of samples collected for detecting viral nucleic acid in suspected patients, namely upper respiratory tract samples and lower respiratory tract samples.
Upper respiratory tract specimens mainly include oropharyngeal swab, nasopharyngeal swab (NPS) and nasopharyngeal aspirate (NPA). Lower respiratory tract specimens mainly include sputum, tracheal aspirate and bronchoalveolar lavage fluid (BALF).