New Insights from Chemical Biology: Molecular Basis of Transmission, Diagnosis, and Therapy of SARS-CoV-2

Abstract

Open AccessCCS ChemistryMINI REVIEW1 Jan 2021New Insights from Chemical Biology: Molecular Basis of Transmission, Diagnosis, and Therapy of SARS-CoV-2 Zilong Zhao†, Yaling Wang†, Liping Qiu†, Ting Fu, Yu Yang, Ruizi Peng, Mengyu Guo, Lichun Mao, Chunying Chen, Yuliang Zhao and Weihong Tan Zilong Zhao† Molecular Science and Biomedicine Laboratory (MBL), State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Hunan University, Changsha 410082 †Z. Zhao, Y. Wang, and L. Qiu contributed equally to this work.Google Scholar More articles by this author , Yaling Wang† CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing 100190 †Z. Zhao, Y. Wang, and L. Qiu contributed equally to this work.Google Scholar More articles by this author , Liping Qiu† Molecular Science and Biomedicine Laboratory (MBL), State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Hunan University, Changsha 410082 †Z. Zhao, Y. Wang, and L. Qiu contributed equally to this work.Google Scholar More articles by this author , Ting Fu Molecular Science and Biomedicine Laboratory (MBL), State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Hunan University, Changsha 410082 The Cancer Hospital of the University of Chinese Academy of Sciences, Institute of Cancer and Basic Medicine (IBMC), Chinese Academy of Sciences, Zhejiang 310022 Google Scholar More articles by this author , Yu Yang Institute of Molecular Medicine (IMM), Renji Hospital, State Key Laboratory of Oncogenes and Related Genes, Shanghai Jiao Tong University School of Medicine, College of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200240 Google Scholar More articles by this author , Ruizi Peng Molecular Science and Biomedicine Laboratory (MBL), State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Hunan University, Changsha 410082 Google Scholar More articles by this author , Mengyu Guo CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing 100190 Google Scholar More articles by this author , Lichun Mao CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing 100190 Google Scholar More articles by this author , Chunying Chen *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 GBA Research Innovation Institute for Nanotechnology, Guangdong 510700 Google Scholar More articles by this author , Yuliang Zhao CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 GBA Research Innovation Institute for Nanotechnology, Guangdong 510700 Google Scholar More articles by this author and Weihong Tan *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Molecular Science and Biomedicine Laboratory (MBL), State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Hunan University, Changsha 410082 The Cancer Hospital of the University of Chinese Academy of Sciences, Institute of Cancer and Basic Medicine (IBMC), Chinese Academy of Sciences, Zhejiang 310022 Institute of Molecular Medicine (IMM), Renji Hospital, State Key Laboratory of Oncogenes and Related Genes, Shanghai Jiao Tong University School of Medicine, College of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200240 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.020.202000322 SectionsAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Coronavirus disease 2019 (COVID-19) is caused by a novel strain of coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has caused a global pandemic rapidly sweeping across all countries, bringing social and economic hardship to millions. Most countries have implemented early warning measures to detect, isolate, and treat patients infected with SARS-CoV-2. This minireview summarizes some of those steps, in particular, testing methods and drug development in the context of chemical biology, and discusses the molecular basis of COVID-19’s virulent transmissibility. Download figure Download PowerPoint Introduction Coronavirus disease 2019 (COVID-19), which emerged in December 2019, is caused by a novel strain of coronavirus (CoV), designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As of July 29, 2020, the virus had attributed to more than 16.5 million laboratory-confirmed cases of SARS-CoV-2 infection with 655,112 deaths, sweeping virtually every country worldwide.1 Initial COVID-19 cases presented pneumonia-like symptoms with abnormal lung scanned computed tomography (CT) images. These cases were determined initially to be a “pneumonia of unknown etiology” by clinicians, according to the following criteria: fever (≥38 °C), radiographic evidence of pneumonia, low or normal white cell count or low lymphocyte count, and no symptomatic improvement after treatment with antibiotics for 3–5 days, following standard clinical guidelines.2,3 On January 5, 2020, the causative agent of the “pneumonia of unknown etiology” was identified as a SARS-CoV-2 by deep sequencing and etiological investigations.4 The genome of the novel virus was about 80% identical to that of SARS-CoV, which caused the outbreak of severe acute respiratory syndrome in 2003.5–7 Based on phylogeny, taxonomy, and established practice, the novel virus was designated as SARS-CoV-2 by the International Committee on Taxonomy of Viruses (ICTV).8 The introduction of CoV CoVs belong to the subfamily Coronavirinae in the family of Coronaviridae of the order Nidovirales and are characteristic enveloped, single-stranded, and positive-sense RNA viruses.9 As observed under electron microscopy (EM), CoVs are named based on their pleomorphic or spherical shape (diameter 80–120 nm) and further characterization as bearing club-shaped peplomers on the surface (Figure 1a). CoVs possess the largest genomes (26.4–31.7 kb) in spherical capsid among all known RNA viruses. Based on serological and genetic properties, CoVs are subdivided into four genera: alpha-, beta-, delta-, and gammacoronavirus.10 Their host range includes humans and several other vertebrates, typically causing respiratory, digestive, and nervous system diseases. Up to now, seven human CoVs (hCoVs) have emerged, including hCoV-229E, hCoV-NL63, hCoV-OC43, hCoV-HKU1, SARS-CoV, Middle-East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2. Among these hCoVs, SARS-CoV-2, MERS-CoV, and SARS-CoV have crossed the species barrier to cause deadly pneumonia in humans in the first 20 years of the 21st century.1,11,12 Figure 1 | (a) Transmission electron microscopy (TEM) of SARS-CoV-2. Reproduced with permission from ref 7. Copyright 2020 China CDC. (b) Schematic of SARS-CoV-2, which is probably a fair representation. Download figure Download PowerPoint The genome and proteins of SARS-CoV-2 The genetic makeup of SARS-CoV-2 comprises ∼29.9 nucleotides, and its genome organization is similar to two representative members of the genus betacoronavirus: SARS-CoV Tor2, a CoV associated with humans, and bat SL-CoVZC45, a CoV associated with bats (Figure 2).13–15 The genome of SARS-CoV-2 is predicted to contain 14 open reading frames (ORFs) encoding 29 proteins. Its gene order is ORF1a/b, spike (S), envelope (E), membrane (M), and nucleocapsid (N) in 5′ to 3′ direction. The 265-nt 5′-terminal and 229-nt 3′-terminal sequences are characterized as betacoronaviruses. The ORF1a/b gene (21,291 nt), about two-thirds of the SARS-CoV-2 genome, encodes two viral polypeptides: PP1a and PP1 ab. These polypeptides are further processed by virus-encoded chymotrypsin-like protease (3CLpro) or main protease (Mpro, also called 3C-like protease), producing 16 predicted nonstructural proteins (NSPs), including nsp1 to nsp16. These NSPs are necessary for replication and transcription. Figure 2 | Genome organization of SARS-CoV-2 and two SARS-CoVs: bat SL-CoVZC45 and SARS-CoV Tor2. Reproduced with permission from ref 13. Copyright 2020 Springer Nature. Download figure Download PowerPoint Additionally, the SARS-CoV-2 comprises structural proteins, with genes located among the ORF genes, ORF10 and ORF11, predicted to encode four main viral proteins: S, E, M, and N (Figure 1b).14 The M is a matrix glycoprotein is the most abundant structural protein, characterized as a triple membrane-spanning protein with a short NH2 terminus outside the virus and a long COOH terminus inside the virion. S glycoprotein is a single-pass type I membrane-anchored protein and constitutes the peplomers, the glycoprotein spikes that stud the viral capsid. N protein is a nucleocapsid that binds and interacts with viral genomic RNA to form the helical nucleocapsid. The E protein is a small membrane envelope protein involved in the assembly of the virus and its budding, playing a role in the viral pathogenesis. A molecular interaction that might determine the formation and composition of the coronaviral membrane exists between the E proteins. The remaining genome part of SARS-CoV-2 produces eight predicted accessory proteins (ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9a, and ORF9b), which might be necessary for the viral virulence in vivo, but their actual biological functions are undefined. Since the outbreak of SARS-CoV-2, researchers have rapidly obtained the crystal structures of SARS-CoV-2-related proteins (e.g., S glycoprotein and Mpro). Ensuing research has produced valuable data facilitating our understanding of the infectivity mechanism and human-to-human transmission of SARS-CoV-2, effectively laying the foundation for the development of therapeutics and vaccines. Molecular basis of SARS-CoV-2 transmission SARS-CoV entry into host cells is mediated by the S (spike) glycoprotein which recognizes angiotensin-converting enzyme 2 (ACE2) on host cells via the formation of homotrimers.16–18 S glycoprotein of SARS-CoV consists of two functional subunits: S1 subunit and S2 subunit. The S1 subunit contains the receptor-binding domain (RBD) responsible for specific binding to ACE2 on host cells. The S2 subunit is responsible for virus-cell fusion. SARS-CoV-2 shares several highly homologous patches of sequences in the RBD domain with SARS-CoV; however, four of five key residues, which are reported to be critical for cross-species and human-to-human transmission of SARS-CoV, are mutated in SARS-CoV-2.6 Therefore, to determine whether SARS-CoV-2 infectivity is mediated by ACE2, Zhou et al.16 systematically investigated the entry of SARS-CoV-2 in HeLa cells that expressed or did not express ACE2 receptors from different species, including human, Chinese horseshoe bats, civets, pigs, and mice. The results confirmed that all ACE2 receptors, except mouse ACE2, mediate the entry of SARS-CoV-2 into HeLa cells (Figure 3). They also identified other CoV receptors, such as aminopeptidase (APN) and dipeptidyl peptidase 4 (DPP4), but these were not classified as cellular entry receptors of SARS-CoV-2. The results strongly supported the notion that SARS-CoV-2 infectivity in the host cell is mediated by ACE2. The binding affinity between SARS-CoV-2 and ACE2 was also investigated by different research groups.19–22 These results suggested that the binding affinity between SARS-CoV-2 and hACE2 is better than that of SARS-CoV and hACE2, although the exact dissociation constant (Kd) values vary depending on the specific experimental operations (Table 1). Figure 3 | Analysis of SARS-CoV-2 infectivity in HeLa cells that expressed or did not express ACE2 from various species. hACE2, human ACE2; bACE2, ACE2 of Rhinolophus sinicus (bat); cACE2, civet ACE2; sACE2, swine ACE2 (pig); mACE2, mouse ACE2. All ACE2 receptors with S tags can be detected using mouse anti-S tag monoclonal antibody. Green, ACE2; red, viral protein (N); blue, 4’,6-diamidino-2-phenylindole (DAPI) (nuclei). Scale bars, 10 μm. Reproduced with permission from ref 16. Copyright 2020 Springer Nature. Download figure Download PowerPoint Table 1 | Analysis of Binding Affinities Between RBD and hACE2 SARS-CoV-2 SARS-CoV Measuring Method References Kd (nM) Kon (M−1 S−1) Koff (S−1) Kd (nM) Kon (M−1 S−1) Koff (S−1) 1.2 ± 0.1 2.3 ± 1.4 × 105 1.7 ± 0.8 × 10−4 5.0 ± 0.1 1.7 ± 0.7 × 105 8.7 ± 5.1 × 10−4 Biolayer interferometry 20 44.2 1.75 × 105 7.75 × 10−3 185 2.01 × 105 3.70 × 10−2 Surface plasmon resonance 19 4.674 1.400 × 106 6.544 × 10−3 31.59 1.367 × 106 4.317 × 10−2 21 94.6 ± 6.5 4.0 ± 0.2e4 3.8 ± 0.1e-3 408.7 ± 11.1 2.9 ± 0.2e5 1.9 ± 0.4e-3 22 To elucidate the molecular basis for SARS-CoV-2 infectivity and virulent human-to-human transmission, many research groups have committed to discovering the crystal structure of the interacting interface between SARS-CoV-2 RBD and ACE2.19–24 These findings could shed light on the molecular mechanism of virulent human-to-human transmission, facilitate the understanding the viral infectivity, and provide targets for drug development. For example, Shang et al.19 designed a SARS-CoV-2 chimeric RBD, consisting of a core from SARS-CoV RBD as the crystallization scaffold, and the receptor-binding motifs (RBMs), containing most of the contacting residues of virus for ACE2 binding from SARS-CoV-2 as the functional unit. To enhance the crystallization of the complex, a short loop from SARS-CoV RBD maintaining a strong salt bridge between Arg426 from the RBD and Glu from hACE2 was retained. X-ray diffraction revealed that the overall structure of the chimeric RBD/hACE2 complex and that of the SARS-CoV RBD/hACE2 complex bear subtle similarities in conformational changes, despite the presence of mutations in some residues (Figure 4a). Both chimeric RBD and SARS-CoV RBD cradle hACE2 N-terminal helix by forming a gently concave surface with a ridge on one side. One structural difference between the RBMs of chimeric RBD and SARS-CoV RBD involves the conformation of loops in the hACE2-binding ridge. As shown in Figures 4b and 4c, the ridge loop of SARS-CoV RBD contains a three-residue motif, proline-proline-alanine, between the disulfide-bond-forming cysteines, allowing the loop to take a sharp turn. The ridge loop of chimeric RBD contains a four-residue motif, glycine-valine/glutamine-glutamate/threonine-glycine, allowing the loop to take a different conformation. The structural difference, together with an additional main-chain hydrogen bond between Asn487 and Ala475 in SARS-CoV-2 RBM, makes the conformation of the ridge more compact and draws the loop containing Ala475 closer to hACE2. Figure 4 | Structure of SARS-CoV-2 chimeric RBD/human ACE2 complex. (a) Crystal structure of SARS-CoV-2 chimeric RBD/hACE2 complex. ACE2, RBD core, RBM, a retained side loop from SARS-CoV, and a zinc ion in ACE2 are presented in green, cyan, magenta, orange, and blue, respectively. (b) Conformation comparison between the ridges in SARS-CoV-2 RBM (purple) and SARS-CoV RBM (orange). (c) Comparison of conformations between the ridges from another view angle. In SARS-CoV RBM, a proline-proline-alanine motif is shown. In SARS-CoV-2 RBM, a newly formed hydrogen bond, Phe486, and some interactions between the ridge and the N-terminal helix of ACE2 are shown. Reproduced with permission from ref 19. Copyright 2020 Springer Nature. Download figure Download PowerPoint Another structural difference between the RBMs of SARS-CoV-2 and SARS-CoV occurs near the two virus-binding hotspots at the RBM/hACE2 interface.19 Two hotspots were identified on hACE2: hotspot-31, consisting of a salt bridge between Lys31 and Glu35, and hotspot-353, consisting of a bridge between Lys353 and ASP38.25 Upon virus binding, the hotspots are buried in hydrophobic environments, providing significant energy to fuel virus/receptor interaction. In the SARS-CoV/hACE2 complex, the structures of hotspot-31 and hotspot-353 are supported by Tyr442 and Thr487 on SARS-CoV, respectively (Figure 5a). In comparison, Leu442 and Asn501 from SARS-CoV-2 RBM, corresponding to Tyr442 and Thr487 from SARS-CoV RBM, respectively, provided less support for the hotspot structures. Consequently, hotspot-21 was rearranged, and Lys31 and Glu35 from hACE2 both formed hydrogen bonds with Gln493 from SARS-CoV-2 RBM. Hotspot-353 also takes a slightly different conformation, and Lys353 from hACE2 forms a hydrogen bond with the main chain of SARS-CoV-2 RBM while maintaining the salt bridge with Asp38 from hACE2 (Figure 5b). Both hotspots have adjusted to reduced support from nearby RBD residues, yet still, become well stabilized at the SARS-CoV-2/hACE2 interface. Figure 5 | (a) Structural analysis at the interface between SARS-CoV-2 RBM and human ACE2. (b) Structural analysis at the interface between SARS-CoV RBM and human ACE2. Reproduced with permission from ref 19. Copyright 2020 Springer Nature. Download figure Download PowerPoint Besides, based on sequence alignment of SARS-CoV-2 S with multiple related SARS-CoVs, Walls et al.20,26 revealed that SARS-CoV-2 S had a unique polybasic “Arginine-Arginine-Alanine-Arginine (RRAR)” furin protease cleavage site at the S1/S2 boundary (Figure 6). The furin cleavage site of SARS-CoV-2 was also confirmed by other research groups.27–30 In addition, it was reported that SARS-CoV-2 entry into host cells was dependent on the cleavage ability of cathepsins (e.g., cathepsin L and cathepsin B),31 which is involved with antigen processing during the viral infection, and transmembrane protease serine protease-2 (TMPRSS-2)32 that cleaves and activates the viral surface proteins. Therefore, the presence of the protease cleavage sites in S glycoprotein could facilitate the fusion between SARS-CoV-2 and host cells, and consequently, enhance viral entry in tissues. Based on numerous research studies, it can be deduced that the enhanced binding affinity to ACE2 and the presence of protease cleavage sites in S glycoprotein account for the rapid and virulent human-to-human transmission of SARS-CoV-2. Figure 6 | Sequence comparison of SARS-CoV-2 S glycoprotein with S glycoproteins from multiple SARS-CoVs and SARS-related CoVs (SARSr-CoVs) indicating that only SARS-CoV-2 spike contains a putative “RRAR” furin cleavage site at the S1/S2 boundary. Download figure Download PowerPoint Diagnosis of COVID-19 Compared with SARS-CoV, SARS-CoV-2 is characterized by more rapid spread and virulent human-to-human transmission. SARS-CoV-2 is airborne transmitted and causes infectious and deadly pneumonia, termed as COVID-19. Patients with COVID-19 usually have pneumonia-like symptoms, such as fever, dry cough, fatigue, sputum production, short breath, and sore throat. However, some patients with COVID-19 show mild or even no symptoms. These asymptomatic individuals are highly contagious. The World Health Organization (WHO) declared a COVID-19 as pandemic on March 11, 2020. As of July 29, 2020, the number of cases of COVID-19 infection had exceeded 16.5 million, with 655,112 deaths at an astonishing speed. To prevent the route of transmission and eliminate the source of infection, the best strategy for COVID-19 management is early detection, early isolation, and treatment of the infected. This leads us to a discussion of diagnostic methods and drug development. Computed tomography COVID-19 is an airborne respiratory disease, ultimately resulting in severe acute respiratory syndrome. Therefore, the clinical status of the lung of patients can provide important information for COVID-19 diagnosis. As a noninvasive imaging test based on the combination of X-ray, reconstruction mathematics, and computer technology, CT scan of the chest is expected to provide detailed pictures of lung abnormalities, particularly to a COVID-19 diagnosis.33 Up to now, numerous CT scans of COVID-19 patients have revealed the presence of ground-glass opacities (GGO), characterized by a peripheral and subpleural distribution, as the main CT feature of COVID-19 pneumonia (Figure 7).34–37 To explore the correlation between chest CT and reverse transcription-polymerase chain reaction (RT-PCR) testing in COVID-19, Ai et al.38 systematically analyzed their response to clinical manifestations in 1014 cases of suspected COVID-19. The results indicated that the favorable rates of RT-PCR assay and chest CT scans were 59% (601/1014) and 88% (888/1014), respectively. Given its sensitivity, a CT scan was adopted as a clinical criterion for the COVID-19 cases from Hubei Province of China in the revised fifth edition of the Guideline of Diagnosis and Treatment. Although the specificity of CT for COVID-19 is relatively low (∼25%) for overlapped imaging features with other viral pneumonia,38,39 chest CT scan undoubtedly plays a critical role in early identification, disease progression, and treatment monitoring of COVID-19 pneumonia, especially for the hospitals or communities lacking nucleic acid testing equipment and kits. Figure 7 | CT images from the survival group and mortality group. GGO with predominant peripheral distribution in middle and lower lung zones were found in the CT images of a 76-year-old woman from the survival group (a–c). Air bronchogram with extensive consolidations and GGO were observed in the CT images of a 72-year-old woman from the mortality group (d–f). Cited from ref 36. Copyright 2020 The Authors. Download figure Download PowerPoint Nucleic acid detection As the critical component of SARS-CoV-2, RNA represents a significant molecular marker for COVID-19 diagnosis. Advances in nucleic acid sequencing techniques make the identification and detection of viral RNA feasible.40 Early in January 2020, the full-length genome sequences of SARS-CoV-2 were identified from infected patients.41 Sequence information was uploaded in GenBank with the accession code of MN908947 on January 10, 2020. The next day, PCR diagnostic reagents were provided for SARS-CoV-2 testing. Such high efficiency in obtaining the sequence information and developing diagnostic reagents was critical for curbing the domestic spread of SARS-CoV-2. RT-PCR For nucleic acid testing of COVID-19, the predominant strategy is real-time RT-PCR, which combines the reverse transcription of RNA and amplification of specific cDNA regions. RT-PCR provides quantitative information about viral loads with high sensitivity and selectivity (Figure 8).42 For general workflow, respiratory samples, such as nasopharyngeal swabs, oropharyngeal swabs, bronchoalveolar lavage (BAL), or tracheal aspirates, are collected, followed by extraction of the viral RNAs.43 The extracted viral RNA is reverse transcribed into cDNA. Then a specific region of the cDNA is amplified with polymerase for fluorescence detection. Of note, although BAL and tracheal aspirates containing higher viral loads are more desirable samples for nucleic acid testing, the sampling processes place health care workers at a high risk of infection. As a result, in clinics, nasopharyngeal and oropharyngeal swabs are the more commonly used RNAs.44 To optimize the RT-PCR system, a key consideration in the design of primers and the fluorescent reporter probes to ensure the sensitivity and specificity of SARS-CoV-2 detection and prevention of false-negative results. Three regions of the SARS-CoV-2 genomes have been recommended, including RNA-dependent RNA polymerase (RdRp) gene, N gene, and E gene.45 Another key consideration is to optimize the conditions for the reverse transcription step and the amplification step. For conventional RT-PCR detection kits, the two steps are carried out separately, and the whole detection process requires 2–3 h. To simplify the operation and reduce the possibility of cross-contamination, a one-step RT-PCR has been developed by combining reverse transcription and PCR reaction in the same tube. Benefiting from less overall hands-on time, one-step RT-PCR is superior in high-throughput screening of COVID-19. On the other hand, its sensitivity and feasibility are, to some extent, compromised compared with the two-step strategy.46 Up to March 31, 2020, among the COVID-19 diagnostic kits approved by the National Medical Products Administration (NMPA) of China, 40% (10/25) was based on the RT-PCR technology. Figure 8 | Schematics of RT-PCR. cDNA is first to reverse transcribed from an RNA template. After denaturation and annealing, primers and probe hybridize with cDNA. The probe contains a phosphors dye at the 5′-end and a quencher at the 3′-end, in which the fluorescence signal is quenched. During the polymerization step, the 5′ nuclease activity of the Taq polymerase displaces and cleaves the probe, resulting in fluorescence recovery of the dye. The fluorescence signal increase is indirectly proportional to the number of mRNA template. Download figure Download PowerPoint On the other hand, it was reported that ∼70% of patients who showed typical CT manifestations were diagnosed as negative by RT-PCR testing.38 Such a high false-negative rate of RT-PCR detection might have resulted from an undisciplined sampling operation, ineffective RNA extraction, or low viral load of the sample. To avoid misdiagnosis of this deadly disease, multiple criteria have been recommended for determination, especially for those with an epidemiological link. Jiang et al.47 evaluated the performance of RT-PCR combined with chest CT in diagnosing COVID-19 from 87 confirmed cases and 481 excluded cases. They found that the highest sensitivity (91.9%, 79/86) was obtained in COVID-19 screening through the combination of RT-PCR test and CT scan, compared with that of RT-PCR test alone (78.2%, 68/87), CT scan alone (66.7%, 54/81), or even a combination of two RT-PCR analyses (86.2%, 75/87). Isothermal nucleic acid amplification Although RT-PCR is a gold standard strategy for the detection of viral RNA, long turnaround times, and complicated operation limits its applications for the high-throughput screening of COVID-19 patients. Isothermal nucleic acid amplification technologies could be utilized to rapidly amplify nucleic acids at a constant temperature and provide alternative strategies for virus testing.48 Currently, these technologies include loop-mediated isothermal amplification (LAMP),49 recombinase polymerase amplification (RPA)50 and clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection,51 and so on.52 Without the need to involve a series of alternating temperature cycles, these technologies are highly efficient and free from the requirement of sophisticated thermal cyclers, thus potentially suitable for the rapid and low-cost diagnosis of COVID-19. As a typical example, a general LAMP system usually contains 4–6 primers, which are designed to amplify six specific regions of the target gene, and polymerase (e.g., Bst polymerase), which has a high activity of both DNA replication and strand displacement (Figure 9).53 After DNA polymerase initiates synthesis, two of the primers can form loop structures to facilitate subsequent rounds of amplification. As a result, LAMP can achieve 109–101