The detection methods for new coronaviruses mainly include nucleic acid testing, antibody testing, and antigen testing. Due to the low detection rate of antigen testing, the current testing for new coronaviruses is focused on antibody and nucleic acid testing. Nucleic acid testing is currently the "standard" for new coronavirus detection, with early diagnosis, high sensitivity and specificity. However, antibody testing is convenient and rapid, and can be used as a complementary tool to nucleic acid diagnosis, but due to the limitations of "false positive" and "false negative" antibody testing, it is not applicable to the screening of the majority of people who return to work and school, and is not applicable to epidemiological surveys in low-prevalence areas. It is also not suitable for epidemiological investigation in low prevalence areas.
Principle of detection.
Basically, all organisms contain nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), except for prions. In contrast, novel coronaviruses belong to the genus Beta coronavirus, which is a protein-coated single-stranded positive-stranded RNA virus that parasitizes and infects higher animals (including humans). The specific RNA sequence in the virus is a marker to distinguish this virus from other pathogens.
After the emergence of novel coronaviruses, our scientists have successfully discovered specific nucleic acid sequences in novel coronaviruses. If the specific nucleic acid sequence of a new coronavirus can be detected in the sample of a suspected patient, it is considered that the patient may be infected by the new coronavirus.
There are two commonly used nucleic acid diagnostic methods for novel coronaviruses: viral nucleic acid-specific gene testing and viral genome sequencing. The most common method for detecting nucleic acid sequences specific for novel coronaviruses is fluorescent quantitative PCR (polymerase chain reaction). Since novel coronaviruses are RNA viruses, the kits basically use reverse transcription plus real-time polymerase chain reaction (RT-PCR) to amplify the pathogenic nucleic acid (RNA), while detecting the amplification products in real time by fluorescent probes.
The PCR reaction system consists of a pair of specific primers and a Taqman probe, which is a specific oligonucleotide sequence labeled with a reporter fluorescent group and a quenched fluorescent group at each end.
When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quenched group; if the target sequence is present in the reaction system, the probe binds to the template during the PCR reaction, DNA polymerase degrades the probe enzymatically along the template using the exonuclease activity of the enzyme, and the reporter group separates from the quenched group and emits fluorescence. For each DNA strand amplified, a fluorescent molecule is produced. The number of cycles in which the fluorescence reaches a predetermined threshold (CT value) can be monitored by a fluorescence quantification PCR instrument is related to the viral nucleic acid concentration; the higher the viral nucleic acid concentration, the smaller the CT value. Different manufacturers' products will determine the positive value of this product based on the performance of their own products.
Since each RT-PCR reaction takes about 2 hours to produce results, kits are generally developed to target 2-3 sequences that are highly conserved in the viral sequence. For example, primers are designed for nucleic acid sequences encoding replicase (replicase) or necleocapsid (nucleoprotein coat, nucleosheath), but the sequences used are not identical from method to method. Some methods use multiplex, which means that multiple target sequences are amplified in a unified reaction; others use distribution, where one target sequence is used to screen first and then another sequence is used to confirm after a positive result, which can effectively shorten the whole process time and speed up the detection.
In addition RNA viruses mutate rapidly, and using multiple conserved target sequences can prevent false negatives caused by viral mutations.
Currently approved products are selected based on the open reading frame 1a/b (ORF1ab), envelope protein (E) and nucleocapsid protein (N) in the novel coronavirus genome. The detection principles of different products are basically the same, but the primers and probes are designed differently, and there are differences in the detection and interpretation of single target (ORF1ab), dual target (ORF1ab, N protein) and triple target (ORF1ab, N protein and E protein). Generally, two targets located on the ORF1ab and N genes of the virus are detected, and positive viral nucleic acid can be confirmed only when the same specimen meets the double-target positive or repeatedly tested as single-target positive or both specimens meet the single-target at the same time.
Detection process.
The testing procedure requires 5 steps, sampling, sample retention, preservation, nucleic acid extraction, and on-line testing. The sample is first collected according to the kit instructions, and the sample types include pharyngeal swab, nasal swab, sputum, etc. After obtaining the patient samples, they need to be tested as soon as possible. If immediate testing is not possible, they need to be cryo-encapsulated and sent to a specialized testing facility for testing. After receiving the sample, the testing facility performs nucleic acid extraction of the sample. The nucleic acid extraction reagents should use the nucleic acid extraction kit specified in the approved product specification. Finally, there is the fluorescent PCR nucleic acid test, which is the on machine test. The extract is subjected to a fluorescent PCR amplification reaction, which takes 70 to 80 minutes.
