Author: Long Qiuyue, Zheng Yali, Gao Zhancheng
Unit : Department of Respiratory and Critical Care and Sleep Medicine, Xiang'an Hospital Affiliated to Xiamen University; Department of Respiratory and Critical Care and Sleep Medicine, Peking University People's Hospital
Quote this article: Long Qiu Yue, Zheng Yali, Gao Zhancheng. New Research progress of coronavirus detection technology [J]. Chinese Journal of Tuberculosis and Respiratory , 2022, 45(8): 819-825. DOI: 10.3760/cma.j.cn112147-20220326-00240.
Abstract
despite natural infection or Vaccination has initially established a population protection barrier, but with the continued emergence of a variety of new coronavirus variants, breakthrough infections cannot be completely avoided, and new coronavirus testing is still the key to discovering the source of infection and interrupting the transmission chain. At present, the new coronavirus detection forms based on nucleic acid, antigen, , and antibody detection are developing diversifiedly. In the post- new crown era in the early stages of the epidemic, resumption of work and production and epidemic prevention and control have become normalized, and we need to choose appropriate testing methods based on the prevention and control needs of different scenarios. This article summarizes the principles and applicable characteristics of existing coronavirus detection technology, in order to provide precise decision-making basis for clinical and public health personnel.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, referred to as the new coronavirus) is a 21st century after severe acute respiratory syndrome (SARS) and Middle East Hope. (Middle East Respiratory Syndrome , MERS) the third β-genus coronavirus that triggered major public health incidents. As a positive-strand RNA virus, its genome can encode 16 non-structural proteins involved in RNA transcription and replication, as well as spike (S) protein, envelope (E) protein, membrane (M) protein, 4 main structural proteins including nucleotide (nucleotide, N) protein. At the end of 2019, a sudden outbreak of pneumonia in Wuhan was found, and the new coronavirus was identified for the first time through virus isolation and culture and nucleic acid identification technology. In the early stage of the epidemic, with the spread of the new coronavirus and the surge in the number of new coronavirus pneumonia, the real-time fluorescence quantitative polymerase chain reaction (RT-PCR) targeted by nucleic acid is the most accurate diagnosis technology for detecting positive individuals. Cutting off the transmission chain and formulating prevention and control measures have played a key role. In the post-COVID era in the early stages of the epidemic, natural infection or vaccination has gradually established an immune defense line to resist viral invasion. A considerable number of infections are attributed to asymptomatic infections, and rapid screening of large-scale suspected patients and close contact groups has become a concern. Focus. In this context, the diagnosis technology of new coronavirus based on nucleic acid, antigen and antibody detection is diversified, and emerging detection methods such as clustered regularly interspaced short palindromic repeats (CRISPR) and biosensor principles have been developed. application. This article reviews the principles and applicable characteristics of the reported new coronavirus detection technology, in order to help readers understand and reasonably choose diagnostic methods.
1. Virus isolation and culture
Virus isolation and culture can be used to identify and amplify live viruses with replication capabilities. It has long been regarded as the gold standard for laboratory diagnosis of viral infection. Inoculation of virus suspensions from clinical samples in experimental animals or viral phenophils can achieve purification and amplification of isolated strains for subsequent identification and typing and etiological characterization. The isolation and culture of the new coronavirus must be carried out in a level 3 biosafety laboratory. Given that the angiotensin converting enzyme 2 (ACE2) receptor is the key to the new coronavirus invading the host cell [1] , it is now used for culture. The main cell lines of the new coronavirus are Vero or Vero E6 monkey kidney cells [2] that express rich ACE2 receptors. The endpoint titration method or plaque method can be used to quantify the virus infection titer. Endpoint titration method [3] determines the 50% tissue culture infection dose by observing the cytopathic effect of infected cells (TCID50); plaque method [4] is a single cell infected by viruses. The single phage formed plaque, that is, the number of plaque-forming units (PFU).Although high sensitivity and specificity molecular detection technology has become the mainstream method for viral pathogenic diagnosis, for the purpose of identifying and studying emerging pathogens (such as the new coronavirus), the status of virus isolation and culture technology is still irreplaceable, mainly reflecting the fact that it is the main manifestation of the virus isolation and culture technology. In [5, 6, 7]: (1) as a reference for performance evaluation of nucleic acid and serological detection methods; (2) Verify the antiviral effect of preventive and therapeutic drug preparations and neutralizing antibodies; (3) To cause Pathogenesis research, epidemiological investigation, and inactivated virus vaccine research and development provide virion resources; (4) To clarify whether the sample is contagious. However, the isolation and culture time of the new coronavirus can reach 4~6 d [8], and the safety protection requirements are high, and it cannot be a routine method to screen suspected patients as soon as possible under the epidemic situation.
2. Viral nucleic acid detection
(I) Whole genome sequencing
metagenome (Metagenomic next-generation s Equencing, mNGS) technology is currently the most commonly used high-throughput and whole-genome sequencing in clinical practice. Pathogenic diagnostic methods can sequence all DNA or RNA nucleic acid molecules from the sample source. Without target enrichment and amplification, parallel characterization of microbial genomes such as bacteria, viruses, fungi and parasites in the sample can be achieved, with "no" Characteristics of partially panoptic pathogen detection [9]. The detection of the new coronavirus is performed by the mNGS program based on RNA [10]. At the beginning of the epidemic, with the help of mNGS technology, Chinese scientists have clarified within 5 days that the pathogenic agent of pneumonia of unknown cause is a new β genus of the Sarbe subgenus The evolutionary analysis results of coronavirus [11] show that its genome sequence is closest to the bat source strain, and its homology is as high as 79% with the SARS coronavirus. In addition, Mostafa et al. [12] reported that among the 40 nasopharyngeal swab samples identified as positive by mNGS, RT-PCR could only detect 77.5% (31/40). At the same time, it was also found that the patient's upper respiratory tract microorganisms were found. Differences in diversity were significantly correlated with disease severity. Therefore, mNGS is not only a basic means to track the evolutionary lineage of viruses, characterize the gene sequence of variant strains and subunit vaccine development, but also an effective tool to accurately evaluate the characteristic changes in the respiratory microecology of patients with COVID-19. However, there is no unified standard for the overall workflow of this technology. The complexity of its procedure determines that the detection cycle takes at least 24 h [13], with an average of about 48 hours or even longer. The premature diagnosis of patients with COVID-19 does not account for Advantages.
(II) nucleic acid detection based on PCR
Based on the amplification of the genetic material of the virus, nucleic acid sequence, RT-PCR can detect the presence of a very small amount of viruses from the mixture, and is the first choice for diagnosis and diagnosis of new coronary pneumonia. Standard . This technology includes the reverse transcription step and the polymerase chain reaction [14] . The reverse transcription product amplifies and is accompanied by the generation of fluorescence signals, thereby forming a quantifiable reaction system captured by the thermal cycler. The number of cycles when the fluorescence value reaches the defined threshold is called the threshold cycle [15] (Cycle threshold, Ct), which is an important parameter for judging the nucleic acid detection results. The lower the Ct value , the higher the virus load in the sample. WHO (WHO) announced primer and probe targeting sequences designed by authoritative institutions in many countries in mid-2020. Subsequently, Jung et al. [16] studies showed that these The primer -probe combination is highly specific for the novel coronavirus, and no cross-reaction with other respiratory viruses was observed. At present, my country recommends that the N gene and the ORF1ab gene be used as the target of nucleic acid detection [17] . If the same sample meets positive dual targets or positive repeated detection of single targets, it can be determined as a positive result. Relatively speaking, the S gene sequence belongs to the mutation-prone region in the reported variants [18] . Although it is not suitable as a general target for the diagnosis of novel coronavirus variants, it is used for the identification of mutation sites and various variants. Identification is of great benefit.Xiong et al. identified the characteristic mutation C1709A of the S gene of Alpha and Delta virus strains through database comparison analysis, thus designing targeted primers so that RT-PCR can be used for specific diagnosis of the above two variants [19].
It is worth mentioning that digital PCR (Droplet digital, dPCR) is a third-generation PCR technology based on finite dilution, based on Poisson distribution as the principle, and with absolute quantification as the core. It is not affected by reaction efficiency and primer specificity, and absolute quantification is obtained without external control. Under the same experimental conditions, the detection limits of dPCR for the ORF1ab and N genes of the new coronavirus were 2.1 and 1.8 copies/ml, respectively, while RT-qPCR was 1039 and 873.2 copies/ml [21], respectively. Related clinical studies show that [22, 23], although the copy number of dPCR is highly correlated with the Ct value of RT-PCR, the former reports fewer false negative results, and is more sensitive to the detection of low viral load samples. Accurate tools. my country's "2019 Novel Coronavirus Nucleic Acid Testing Expert Consensus" [24] mentioned that when the nucleic acid detection Ct value is in the grayscale area, a dPCR method with higher sensitivity can be further determined. However, given the limitations of high cost and low throughput, dPCR has not been popularized in clinical etiological diagnosis.
(III) Nucleic acid detection based on isothermal amplification
Compared with PCR technology, isothermal amplification nucleic acid detection simplifies the thermal cycle reaction process and shortens the detection cycle. Taking Loop-mediated isothermal amplification (LAMP) [25] as an example, this method uses 4 to 6 primers to identify different regions of the target sequence at a constant temperature (60 to 65 ℃) Next pass The stem ring structure was repeatedly amplified, and the target gene was synthesized in 1 h and up to 10 9 copies. LAMP technology used for nucleic acid detection of the new coronavirus has added a reverse transcription step. On this basis, the turbidity changes caused by the by-product magnesium pyrophosphate are amplified [26], and the amplification can be judged by the turbidity meter . As a result, the same study also confirmed that LAMP and RT-PCR have similar sensitivity. Other studies [27, 28] also stated that LAMP method can achieve 90%~100% sensitivity and 95%~99% specificity for the detection of new crown samples, and at the same time, multiple targets also improve the accuracy of amplification. , but the detection time only takes 30 min. In addition, the new coronavirus variants Alpha, Beta, Gamma, Delta and Omicron are highly conserved in the 12-213 base sequence region of the N gene. Luo et al. optimized the primer design [29], making LAMP technology for emerging variants Diagnosis is compatible. Other isothermal amplification nucleic acid detection methods also include recombinant polymerase amplification, rolling ring amplification and nucleic acid sequence amplification [30], but applications in the diagnosis of new coronavirus are relatively rare. In summary, isothermal amplification technology has the advantages of high specificity, high sensitivity, high efficiency and equipment simplification, but its multi-primer and multi-enzyme reaction system needs also increase the complexity of detection design and research and development. my country's Only two of the new coronavirus nucleic acid testing kits approved by the Drug Administration use isothermal amplification technology.
(IV) Nucleic acid detection based on CRISPR
CRISPR The principle of CRISPR is the Cas protease nucleic acid scissors under the guidance of guide RNA (guide RNA, gRNA). Cut function. Specifically, specific targeting gRNA can be designed according to the RNA sequence of the new coronavirus. Theoretically, as long as there is a virus-targeting sequence in the sample, gRNA can accurately identify it and activate Cas protease to perform reporter nucleic acid molecule cleavage function, so that Signal molecules are released and captured.Zhang Feng's team developed SHERLOCK technology using the CRISPR-Cas 13a system [31] is a typical representative of this field. Combined with the recombinant polymerase isothermal amplification technology, the average detection time is about 70 min in preoperative patient screening. The lower limit is 42 copies/ml, the specificity and sensitivity of fluorescence readings are both 100%, and the lateral flow readings are 100% and 97% [32] , respectively; to simplify the process, the team also used the LAMP method to transfer RNA The amplification and Cas enzyme cleavage steps are combined into one, and the nucleic acid detection is completed in less than 1 hour. The detection limit of is one-thirty of RT-PCR (33 copies/ml and 1 000 copies/ml) [ 33]. On this basis, de Puig et al. designed gRNAs targeting the mutation sequence of the new coronavirus variant, and expanded the application of this technology in the instant identification of mutation sites related to variants such as N501Y and E484K [34] . In addition, Broughton and colleagues reported the first DETECTR technology [35] based on the CRISPR-Cas 12 system, achieving qualitative detection of the new coronavirus within 45 minutes. Liang et al. used this technology to develop multiple detection of S genotyping of the new coronavirus, achieving rapid identification of Alpha, Beta, and Delta variants [36]. The enzyme reaction characteristics and gRNA selection of CRISPR give the technology the advantages of high sensitivity, high specificity and flexible targeting, but its excellent efficacy is still to be proven.
3. Viral antigen detection
nucleic acid detection and antigen detection are both detection methods that directly target the new coronavirus. The former is used to detect viruses that are actively replicated in the early stages of infection, while the latter uses specific antibodies to identify virus-specific proteins. N protein is highly evolutionarily conserved and is the main target for antigen detection of new coronavirus [37]. The antigen detection reported today is mostly based on the principle of Lateral flow immunoassay (LFIA), which is the most mainstream form of on-site instant detection (Point-of-care tests, POCT). The results can be obtained in about 15 minutes. ]. This technology drives sample droplets through to chromatography on html strips (Figure 1). The molecules to be tested (specific antigen) in the sample will first be connected to a part of the labeled molecules in the binding region to detect the band (T) region The molecule-labeled molecule joint can be captured and reacted with it to convert it into a color signal to qualitatively detect a specific antigen; while the QR band (C) region can react with the unlinked marker molecule to reflect the quality control level of the same detection [39]. The first batch of antigen self-test kits for new coronavirus that were launched in my country in March 2022 are based on the LFIA principle. The difference is that the labeling molecules and signal reading devices they use are different. You can choose colloidal gold and latex. Particles or fluorescent molecules. The sensitivity of rapid antigen detection is related to viral load, and the evidence is still limited in [40] . The study found that [41] has a sensitivity of 97% to 100% in samples with Ct values below 25; in samples with Ct values between 25 and 30, the sensitivity is 50% to 81%; and in samples with Ct values between 25 and 30; In samples with Ct values above 30, the sensitivity is as low as 12% to 18%. Correspondingly, the WHO file on rapid antigen detection of the new coronavirus [42] pointed out that the results of rapid antigen detection are the most reliable in areas where community transmission exists. Therefore, antigen testing cannot replace nucleic acid testing and becomes the diagnosis standard. However, for grassroots health institutions that do not have the ability to detect nucleic acid, community residents with relatively high infection risk, and people who resume work and production on a large scale, antigen testing is a cheap and convenient effective screening. The inspection tool helps to detect cases early and interrupt transmission early.
Figure 1 lateral flow immune response (LFIA) principle diagram
4. Antibody detection
After the new coronavirus invades the human body, the innate immunity and adaptive immune systems are activated one after another. Usually within 1 to 2 weeks after infection, the virus Specific IgM antibodies appear It then slowly declines. After about 2 weeks of infection, the IgG antibody is produced and can be maintained in the body for a long time. Therefore, IgM can be used to indicate recent infections, and IgG can be used to indicate previous viral infections.The N protein and S protein S1 subunits have strong immunogenicity and are the most common biomarker in the serological detection of the new coronavirus [43]. The ninth edition of the diagnosis and treatment guide in my country points out that [8] , and those with positive specific IgM antibodies and IgG antibodies can be diagnosed with new coronary pneumonia. However, new crown vaccine vaccinated with or those who have been infected with the new crown virus will also undergo serological conversion, so this indicator has no diagnostic reference significance for it and can only be used as an important tool for evaluating humoral immune response after the establishment of an immune barrier, such as determining antibody levels. Decay over time and antibody effectiveness against mutant strains. Common methods of serological detection include enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA) and PO based on LFIA principle. CT equipment. ELISA is a microplate-based assay technology that can realize qualitative or quantitative detection of peptides, proteins, antibodies and other molecules, with a detection period of 1~5 h [44]. The viral antigen coated in the well plate can specifically bind to the specific antibodies in the sample, and then the antigen-antibody complex is connected to the labeled molecule to produce a colorimetric or fluorescence signal that is positively correlated with the antibody concentration. The CLIA method uses chemical luminescence intensity to quantify the antibody level in the sample, which has the advantages of high signal intensity, wide detection range, and no scattered light interference [45]. The previous 4,000 detections of can be performed every 24 hours. 6 ]. Studies have shown that [47, 48], CLIA is used to detect multi-antigens with higher specificity than monoantigens, and is used to detect total antibody levels with the best time-effect. As for LFIA products, they are portable, efficient and require only a few microliters of sample size, which is suitable for fingertip sampling [49]. However, no matter what kind of serological detection technology, its efficacy is closely related to the sampling time. Nicol et al. [46] used ELISA, CLIA and LFIA technologies to evaluate the IgG of 293 samples, and found that the above methods were confirmed by 14 The sensitivity was highest after d, reaching 100%; Pickering et al. [50] also reported that antibody detection had the highest sensitivity and the narrowest confidence interval among the samples collected 20 days after the first symptoms. Therefore, given the serological conversion time window of about 1 to 2 weeks, serological testing has limited utility in the early stages of the disease.
5. Detection of host respiratory metabolites
Detection of host exhaled gas metabolites is called respiratory analysis. It is a non-invasive and real-time POCT method with the rise of metabolomics . It is detected by mass spectrometry -chromatography technology The key volatile organic compounds (VOC) are realized, with the potential for clear diagnosis and large-scale screening. In the fields of respiratory infection , obstructive pulmonary disease and lung cancer, the important role of the host metabolic microenvironment in the development of lung diseases has been clarified. [51]. Grassin-Delyle et al. [52] characterized the exhaled air metabolites in patients with COVID-19, and found that four VOCs (methylpentadienal, 2,4-octidine, 1-chlorheptane and nonane Aldehyde) can be used to effectively distinguish between COVID-19-related acute respiratory distress syndrome and non-COVID-19 acute respiratory distress syndrome. Among the 40 patients tested, the sensitivity and specificity reached 90% and 94% respectively. In addition, Chen et al. [53] successfully distinguished new coronavirus patients from healthy controls through 12 VOCs dominated by acetone , and this diagnostic model can detect infected people with false negative nucleic acid tests. The U.S. Food and Drug Administration (FDA) issued the first emergency use authorization for the judging of new coronavirus infection in April 2022, which mainly detects the characteristic characteristics of new coronavirus infection in exhaled gas 5 ketones and aldehyde VOCs, the metabolite detection limit is 10 ppb, and the results can be obtained within 3 min [54].In a large cohort of 2,409 confirmed COVID-19 patients (PCR positive rate is only 4.2%), the technology was proven to have a sensitivity of 91.2% and a specificity of 99.3%, with a negative predictive value of up to 99.6% ; In addition, for the detection of Omicron variant, this technology has a similar sensitivity of 90.9% (10/12) [55]. It should be noted that the FDA pointed out that this test can be used to screen asymptomatic individuals with unclear exposure history, but it cannot be used as the only decision-making basis for patient management and treatment. The results should be combined with the individual's clinical symptoms, signs, and molecular diagnostic reports. consider.
6. Emerging biosensor
Biosensor is an instrument that is sensitive to biological matter and converts its concentration into electrical signals through physical and chemical transducers and signal amplifier for detection, aiming to optimize existing biomolecular detection methods. : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : For nucleic acid detection, Peter et al. developed a wearable biosensor based on CRISPR-SHERLOCK technology, integrating this technology into a standard mask to detect the new coronavirus in the exhaled aerosol , and gave it within 90 min. Accuracy is comparable to gold standard RT-PCR. Recently, Wang et al. [57] also reported that microelectromechanical sensing systems based on graphene field effect transistors do not require nucleic acid extraction and amplification steps, and in 33 nasopharyngeal swab samples (Ct values 24.9~41.3) are less than The new coronavirus can be identified in 4 minutes; at the same time, no false negative results appeared in 54 negative samples, and the detection limit was as low as 10~22 copies/ml. For antigen detection, Raziq et al. [58] used molecule imprinted polymer as a biological target recognition element for the first time, and developed an electrochemical sensor for the new coronavirus N protein. The reaction time was 15 minutes, with the detection limit and quantitative limit respectively reaching 15%. fM and 50 fM (0.7~2.2 ng/L) match the antigen concentration in real-world samples. For serological detection, Elledge et al. reported an antibody detection method based on the split luciferase-target antigen fusion protein [59], targeting anti-coronavirus S protein (S-Sensor) and N protein (N- Sensor) antibodies can achieve quantitative analysis of low-level antibodies in blood and saliva within 30 minutes. In the verification of clinical samples, the specificity of N-Sensor and S-Sensor reached 99.2% and 100%, the sensitivity reached 98% and 89%, respectively, and there was no cross-reactivity with the seasonal coronavirus samples. The above modules can be equipped with portable devices to be modified in the direction of POCT equipment. Therefore, based on the comprehensive timeliness, simplicity, specificity and sensitivity, biosensors are expected to develop into an efficient tool for community screening and even clinical laboratory diagnosis.
7. Summary and Outlook
With the large-scale vaccination of the new crown vaccine, the herd immunity barrier is basically established. However, new mutant strains adapted to the host change from time to time, and the occurrence of breakthrough infections is difficult to completely avoid. In the situation of normalization of epidemic prevention and control. Next, testing for the new coronavirus remains the cornerstone of discovering sources of infection and deploying public health strategies. Different detection methods have their own emphasis (Table 1 [19, 27, 28, 29, 33, 34, 36, 46, 50, 60, 61, 62, 63, 64]), which complement each other, but in practical applications It is still necessary to consider comprehensive considerations in combination with epidemiological history, clinical manifestations and underlying diseases to avoid misjudgment.From the perspective of biological targets, nucleic acid detection and antigen detection target virus genetic material and functional molecules, respectively, while antibody detection and respiratory analysis target human virus-specific immune response or metabolic changes; from the perspective of detection timeline, nucleic acid detection targets The rapid antigen test can be used to diagnose new coronavirus infection in the acute phase. The former has a relatively lower detection limit, while the antibody test has the best effect after serological conversion 1 to 2 weeks of onset. From the applicable characteristics, See: (1) The molecular diagnostic technology based on nucleic acid amplification detection has high sensitivity and strong specificity, and is still the first choice for COVID-19 infection. LAMP technology has the potential to become a replacement for RT-PCR; (2) Antigen detection and respiratory analysis It has an advantage in terms of operability and timelines, and is a convenient way for the diversion of primary medical institutions and large-scale population screening, which helps ensure the normal operation of social activities; (3) Serological testing is an important diagnostic tool in the later stage of the disease course. It is also a basic means to monitor antibody response; (4) Emerging CRISPR technology and biosensor strategies have excellent comprehensive strength and broad application prospects, which need to be verified in large-sample clinical cohorts.
htmlPositive cases in the post-0 new coronavirus era may be common asymptomatic or latent infections. The detection rate of detection methods with low sensitivity (such as rapid antigen detection) may decline under low viral load. At the same time, given the new coronavirus The continuation of adaptive mutations, universal detection based on highly conserved sequences and variant diagnosis based on characteristic mutation sites, need to be coordinated and taken into account. Therefore, a single detection technology cannot become an ideal diagnosis method that has both high efficiency and high efficiency. The combined detection strategy of virus-oriented and host-oriented can provide case monitoring and epidemic prevention and control with site change, disease period transition and variant change with the change of the disease. Multiple guarantees are the evolutionary trend of new coronavirus testing in the post-COVID era. References (omitted)
