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1.
Gene ; 2022.
Article in English | EuropePMC | ID: covidwho-1755706

ABSTRACT

Both feline coronavirus (FCoV) and SARS-CoV-2 are coronaviruses that infect cats and humans, respectively. However, cats have been shown to be susceptible to SARS-CoV-2, and FCoV also had been shown to infect human. To elucidate the relationship between FCoV and SARS-CoV-2, we highlight the main characteristics of the genome, the receptor usage, and the correlation of the receptor-binding domain (RBD) of spike proteins in FCoV and SARS-CoV-2. It is demonstrated that FCoV and SARS-CoV-2 are closely related to the main characteristics of the genome, receptor usage, and RBD of spike proteins with similar furin cleavage sites. In particular, the affinity of the conserved feline angiotensin-converting enzyme 2 (fACE2) receptor to the RBD of SARS-CoV-2 suggests that cats are susceptible to SARS-CoV-2. In addition, cross-species of coronaviruses between cats and humans or other domesticated animals are also discussed. This review sheds light on cats as potential intermediate hosts for SARS-CoV-2 transmission, and cross-species transmission or zoonotic infection of FCoV and SARS-CoV-2 between cats and humans was identified.

2.
Chin J Integr Med ; 2022 Mar 23.
Article in English | MEDLINE | ID: covidwho-1756894

ABSTRACT

Rhizoma phragmitis is a common Chinese herbal medicine whose effects are defined as 'clearing heat and fire, promoting fluid production to quench thirst, eliminating irritability, stopping vomiting, and disinhibiting urine'. During the Novel Coronavirus epidemic in 2020, the Weijing Decoction and Wuye Lugen Decoction, with Rhizoma phragmitis as the main herbal component, were included in The Pneumonia Treatment Protocol for Novel Coronavirus Infection (Trial Version 5) due to remarkable antiviral effects. Modern pharmacological studies have shown that Rhizoma phragmitis has antiviral, antioxidative, anti-inflammatory, analgesic, and hypoglycemic functions, lowers blood lipids and protects the liver and kidney. This review aims to provide a systematic summary of the botany, traditional applications, phytochemistry, pharmacology and toxicology of Rhizoma phragmitis.

3.
Front Microbiol ; 13: 782421, 2022.
Article in English | MEDLINE | ID: covidwho-1742229

ABSTRACT

While IgM and IgG response to SARS-CoV-2 has been extensively studied, relatively little is known about secretory IgA (sIgA) response in respiratory mucosa. Here we report IgA response to the SARS-CoV-2 in sputum, throat swabs, and serum with nucleocapsid protein (NP) enzyme-linked immunosorbent assays (ELISA) in a cohort of 28 COVID-19 patients and 55 vaccine recipients. The assays showed sIgA in respiratory mucosa could be detected on the first day after illness onset (AIO), and the median conversion time for sIgA in sputum, throat swabs, and serum was 3, 4, and 10 days, respectively. The positive rates of sIgA first week AIO were 100% (24/28) and 85.7% (24/28) in sputum and throat swabs, respectively, and were both 100% during the mid-onset (2-3 weeks AIO). During the recovery period, sIgA positive rates in sputum and throat swabs gradually decreased from 60.7% (17/28) and 57.1% (16/28) 1 month AIO and the sIgA antibodies were all undetectable 6 months AIO. However, serum IgA positive rate was still 100% at 4 months and 53.6% (15/28) at 6 months. Throat swabs obtained from volunteers who received inactivated SARS-CoV-2 vaccines by intramuscular delivery all showed negative results in IgA ELISA. These findings will likely improve our understanding of respiratory mucosal immunity of this emerging disease and help in containing the pandemic and developing vaccines.

4.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-323573

ABSTRACT

Background: Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a significant threat to human health, but its clinical manifestations vary greatly among individuals. Early detection and treatment are important for severely ill patients to improve their prognosis and reduce the risk of death. Methods In the present study, serum markers were detected and analyzed in moderately ill and severely ill patients. Results The results found that there were statistically significant differences in age, serum Krebs von den Lungen-6 (KL-6) and Immunoglobulin A (IgA) levels between severely ill patients and moderately ill patients ( P  < 0.05). The cut-off of using KL-6 alone for the diagnosis of severely ill patients was 298.91 U/mL, with an AUC of 0.737, a sensitivity of 100%, and a specificity of 43%. When the diagnosis was performed using KL-6 in combination with Interleukin-6 (IL-6), an indicator of infection, the AUC was 0.776, with a sensitivity and specificity of 82% and 69%, respectively. When the three above were used in combination for diagnosis, the AUC was 0.785, and the sensitivity and specificity were 100% and 59%, respectively. After rehabilitation, the serum levels of KL-6, C-reactive protein (CRP), as well as antibodies, IgA, IgM and IgG, were significantly lower than those in the early stage of hospitalization. Conclusion In the present study, KL-6 and IgA were found to have some diagnostic efficacy for severely ill patients with COVID-19, but larger cohort studies are still needed for further confirmation, which in turn improves the diagnostic and therapeutic efficiency of severely ill patients.

5.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-318389

ABSTRACT

This protocol presents the Variant Nucleotide Guard (VaNGuard) assay, which is robust towards viral mutations and can be performed on purified RNA or directly on nasopharyngeal (NP) swab samples. The procedure typically comprises three parts, namely sample preparation, RT-LAMP reaction, and Cas12a-based detection via fluorescence or lateral flow assay. Sample preparation from NP swabs involves Proteinase K digestion followed by heat inactivation. Purified RNA or digested NP swab samples are then added as templates into RT-LAMP reactions and incubated at 65ºC for 22 minutes. Next, enAsCas12a and ssDNA-probes are added and the reactions are incubated at 60ºC for another 5 minutes. End-point fluorescence can be detected by a plate reader or a real-time PCR machine. Alternatively, a lateral flow strip can be inserted into each reaction tube for equipment-free read-out. The VaNGuard assay is a rapid and convenient point-of-care test for SARS-CoV-2 and is applicable to resource poor settings.

6.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-315290

ABSTRACT

Coronavirus disease (COVID-19) accompanies severe immune injury as well as a decrease and overactivation of T lymphocytes. We observed that vMIP-Ⅱ, a broad-spectrum chemokine receptor inhibitor, could improve the lymphocyte decrease of COVID-19. Comparisons of T cell populations in PBMCs showed that the effects of vMIP-II on the subsets of T cells and cytokine secretion stimulated by SARS-CoV-2 S protein were the same as those in the asymptomatic infection group: the proportion of CD8 + T CM cells in the vMIP-II treatment and asymptomatic groups was significantly higher than that in the symptomatic control group. Differential gene expression of effector CD8 + T cells suggested that vMIP-II inhibits multiple chemokine receptors and related signal pathway and strengthens their stem proliferating capacity. Thus, vMIP-II reconstitutes cellular immunity lost due to acute infection of SARS-CoV-2 by modulating effector CD8 + T cells to produce more T CM cells.

7.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-311943

ABSTRACT

The magnitude of SARS-CoV-2 infection, the dynamic changes of immune parameters in patients with the novel coronavirus disease (COVID-19) and their correlation with the disease severity remain unclear. The clinical and laboratory results from 154 confirmed COVID-19 patients were collected. The SARS-CoV-2 RNA levels in patients were estimated using the Ct values of specific RT-PCR tests. The lymphocyte subsets and cytokines profiles in the peripheral blood were analyzed by flow cytometry and specific immunoassays. 154 confirmed COVID-19 patients were clinically examined up to 4 weeks after admission. The initial SARS-CoV-2 RNA Ct values at admission varied but were comparable in the patient groups classified according to the age, gender, underlying diseases, and disease severity. Three days after admission significant higher Ct values were found in severe cases. Significantly reduced counts of T cells and T cell subsets were found in patients with old age and underlying diseases at admission and were characteristic for the development of severe COVID-19. Severe COVID-19 developed preferentially in patients with underlying compromised immunity and was not associated with initial virus levels. Higher SARS-CoV-2 RNA levels in severe cases were apparently a result of impaired immune control associated with dysregulation of inflammation.

8.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-308053

ABSTRACT

Background: Novel coronavirus pneumonia (NCP) is an emerging, highly contagious community acquired pneumonia (CAP) caused by severe acute SARS-CoV-2. Nucleic acid test currently played a crucial role in diagnosis of suspected COVID-19 patients. However, a high false-negative rate of this “gold standard” test has been reported and posed a major setback in blocking the spread of the virus. We here aim to describe an optimized laboratory detection strategy to reduce the false negative rate. Methods: : Suspected NCP patients were asked to collect both coughed up specimen and pharyngeal swab. Samples from the same patient were mixed and tested at a single pool. SARS-CoV-2 was then detected by real-time RT-PCR using two different detection kits. Only if both results were negative was the test reported as negative. The patients will be excluded after two consecutive negative tests at 24 hour intervals. We also used multiplex PCR to detect 13 common respiratory tract pathogens (RTP). Results: : Using this strategy, we confirmed 85 SARS-CoV-2 infections from 181 suspected patients, and 94.12% of patients were positive in the first test. The 96 excluded patients were followed up, and no additional NCP was found. We also found that 31.25% patients in 96 non-NCP patients were infected with at least one RTP that may cause CAP. Conclusion: Our studies suggest that dual reagents screening with pooled coughed up specimen and pharyngeal swab samples reduced the false negative rate of nucleic acid testing. During the epidemic of NCP in Anhui province, there was a certain proportion of infection and co-infection of other common pathogens of CAP. In comparison with SARS-CoV-2 detection alone, combining multiple pathogen detection reduces the rate of miss diagnosis.

9.
Evid Based Complement Alternat Med ; 2022: 8733598, 2022.
Article in English | MEDLINE | ID: covidwho-1685764

ABSTRACT

Background: Lianhua Qingke (LH) tablets is an effective traditional Chinese medicine against various viral infections, especially in relieving coughing. However, its effects on COVID-19 are unknown. Methods: To examine the therapeutic effectiveness of LH tablets in COVID-19 patients with mild and common types, a randomized, multicenter, controlled study was carried out. COVID-19 cases were randomized to undergo routine treatment with or without LH tablets (4 tablets, three times a day) for 14 days. The primary endpoints were the rate of achieving clinical symptom resolution and the corresponding time. Results: There were 144 participants in the full analysis set (72 each in the LH and control groups). The LH group participants had elevated symptom alleviation rate at 14 days compared with control cases (FAS: 98.61% vs. 84.72%, p = 0.0026). In comparison with control group participants, the LH group participants had reduced median time to clinical symptom alleviation (median: 4 vs. 7 days, p < 0.0001). Higher resolution rates of coughing (98.44% vs. 84.51%, p = 0.0045) and expectoration (100% vs. 82.35%, p = 0.0268) were observed in the LH group. Times to recovery of fever (median: 2 vs. 3 days, p = 0.0007), coughing (median: 4 vs. 7 days, p < 0.0001), and expectoration (median: 3 vs. 6 days, p < 0.0001) were also notably shorter in the LH group. Moreover, the LH group had elevated improvement rates in chest computed tomography signs (FAS: 86.11% vs. 72.22%, p = 0.0402) and clinical cure at day 28 (FAS: 83.33% vs. 68.06%, p = 0.0326). However, no differences were found in the laboratory test and viral assay. Serious adverse events were not detected. Conclusion: These preliminary findings indicate LH tablets may be effective in symptomatic COVID-19, especially in relieving coughing. This trial was registered in Chinese Clinical Trial Registry (ChiCTR2100042069).

10.
Matter ; 5(2): 694-709, 2022 Feb 02.
Article in English | MEDLINE | ID: covidwho-1670871

ABSTRACT

The current COVID-19 pandemic urges us to develop ultra-sensitive surface-enhanced Raman scattering (SERS) substrates to identify the infectiousness of SARS-CoV-2 virions in actual environments. Here, a micrometer-sized spherical SnS2 structure with the hierarchical nanostructure of "nano-canyon" morphology was developed as semiconductor-based SERS substrate, and it exhibited an extremely low limit of detection of 10-13 M for methylene blue, which is one of the highest sensitivities among the reported pure semiconductor-based SERS substrates. Such ultra-high SERS sensitivity originated from the synergistic enhancements of the molecular enrichment caused by capillary effect and the charge transfer chemical enhancement boosted by the lattice strain and sulfur vacancies. The novel two-step SERS diagnostic route based on the ultra-sensitive SnS2 substrate was presented to diagnose the infectiousness of SARS-CoV-2 through the identification standard of SERS signals for SARS-CoV-2 S protein and RNA, which could accurately identify non-infectious lysed SARS-CoV-2 virions in actual environments, whereas the current PCR methods cannot.

11.
Front Immunol ; 12: 715464, 2021.
Article in English | MEDLINE | ID: covidwho-1430698

ABSTRACT

The mutants resulted from the ongoing SARS-CoV-2 epidemic have showed resistance to antibody neutralization and vaccine-induced immune response. The present study isolated and identified two novel SARS-CoV-2 neutralizing antibodies (nAbs) from convalescent COVID-19 patients. These two nAbs (XG81 and XG83) were then systemically compared with nine nAbs that were reconstructed by using published data, and revealed that, even though these two nAbs shared targeting epitopes on spike protein, they were different from any of the nine nAbs. Compared with XG81, XG83 exhibited a higher RBD binding affinity and neutralization potency against wild-typed pseudovirus, variant pseudoviruses with mutated spike proteins, such as D614G, E484Q, and A475V, as well as the authentic SARS-CoV-2 virus. To explore potential broadly neutralizing antibodies, heavy and light chains from all 18 nAbs (16 published nAbs, XG81 and XG83) were cross-recombined, and some of the functional antibodies were screened and studied for RBD binding affinity, and neutralizing activity against pseudovirus and the authentic SARS-CoV-2 virus. The results demonstrated that several recombined antibodies had a more potent neutralization activity against variant pseudoviruses compared with the originally paired Abs. Taken together, the novel neutralizing antibodies identified in this study are a likely valuable addition to candidate antibody drugs for the development of clinical therapeutic agents against SARS-CoV-2 to minimize mutational escape.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Broadly Neutralizing Antibodies/immunology , Broadly Neutralizing Antibodies/therapeutic use , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/genetics , Antibodies, Viral/therapeutic use , Antibody Affinity/immunology , B-Lymphocytes/immunology , Broadly Neutralizing Antibodies/genetics , COVID-19/immunology , COVID-19/therapy , Cell Line , Epitopes/immunology , Humans , Immunotherapy/methods , Neutralization Tests , SARS-CoV-2/drug effects
12.
Front Immunol ; 12: 693775, 2021.
Article in English | MEDLINE | ID: covidwho-1394758

ABSTRACT

Small number of SARS-CoV-2 epidemic lineages did not efficiently exhibit a neutralization profile, while single amino acid mutation in the spike protein has not been confirmed in altering viral antigenicity resulting in immune escape. To identify crucial mutations in spike protein that escape humoral immune response, we evaluated the cross-neutralization of convalescent plasmas and RBD-specific monoclonal antibodies (mAbs) against various spike protein-based pseudoviruses. Three of 24 SARS-CoV-2 pseudoviruses containing different mutations in spike protein, including D614G, A475V, and E484Q, consistently showed an altered sensitivity to neutralization by convalescent plasmas. A475V and E484Q mutants are highly resistant to neutralization by mAb B38 and 2-4, suggesting that some crucial mutations in spike protein might evolve SARS-CoV-2 variants capable of escaping humoral immune response.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/blood , Antibodies, Viral/immunology , Mutation , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Substitution/genetics , Amino Acid Substitution/immunology , Antibodies, Neutralizing/immunology , Convalescence , Humans , Immune Evasion , Immunity, Humoral , Neutralization Tests , Protein Binding
15.
Microbiol Spectr ; 9(1): e0016921, 2021 09 03.
Article in English | MEDLINE | ID: covidwho-1270881

ABSTRACT

Nonstructural protein 1 (Nsp1) of severe acute respiratory syndrome coronaviruses (SARS-CoVs) is an important pathogenic factor that inhibits host protein translation by means of its C terminus. However, its N-terminal function remains elusive. Here, we determined the crystal structure of the N terminus (amino acids [aa] 11 to 125) of SARS-CoV-2 Nsp1 at a 1.25-Å resolution. Further functional assays showed that the N terminus of SARS-CoVs Nsp1 alone loses the ability to colocalize with ribosomes and inhibit protein translation. The C terminus of Nsp1 can colocalize with ribosomes, but its protein translation inhibition ability is significantly weakened. Interestingly, fusing the C terminus of Nsp1 with enhanced green fluorescent protein (EGFP) or other proteins in place of its N terminus restored the protein translation inhibitory ability to a level equivalent to that of full-length Nsp1. Thus, our results suggest that the N terminus of Nsp1 is able to stabilize the binding of the Nsp1 C terminus to ribosomes and act as a nonspecific barrier to block the mRNA channel, thus abrogating host mRNA translation.


Subject(s)
SARS-CoV-2/genetics , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , COVID-19 , Crystallography, X-Ray , HEK293 Cells , Humans , Protein Biosynthesis , Protein Conformation , Protein Domains , RNA, Messenger , Sequence Analysis, Protein , Viral Nonstructural Proteins/metabolism
16.
Journal of Modern Laboratory Medicine ; 35(3):90-93, 2020.
Article in Chinese | GIM | ID: covidwho-1264596

ABSTRACT

Objective: The kits produced by the two manufacturers were used to detect samples of patients with pneumonia infected by new coronavirus in parallel, and their detection effects were evaluated for limited application.

17.
J Virol Methods ; 295: 114185, 2021 09.
Article in English | MEDLINE | ID: covidwho-1243068

ABSTRACT

OBJECTIVE: Viral nucleic acid detection by real-time reverse transcription polymerase chain reaction (qPCR) is the current standard method for diagnosis of SARS-CoV-2 infection. However, due to low viral load in some COVID-19 patients, false negative results from this method have been repeatedly reported. METHOD: In this study, we compared the sensitivity and specificity of digital PCR (dPCR) in simulated samples and clinical samples with qPCR assay through a series of vigorous tests. RESULTS: The results showed that dPCR was more sensitive than qPCR especially for samples with low viral load (≤3 copies). In addition, dPCR had similar specificity as qPCR and could effectively distinguish other human coronaviruses and influenza virus from SARS-CoV-2. More importantly, dPCR was more sensitive than qPCR in detecting the virus in the "negative" samples from recurrent COVID-19 patients. CONCLUSIONS: In summary, dPCR could serve as a powerful complement to the current qPCR method for SARS-CoV-2 detection, especially for the samples with extremely low viral load, such as recurrent COVID-19 patients.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/isolation & purification , Viral Load , COVID-19/virology , Humans , RNA, Viral/genetics , Recurrence , SARS-CoV-2/genetics , Sensitivity and Specificity
18.
Virol Sin ; 35(6): 758-767, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-1217478

ABSTRACT

Reverse transcription-polymerase chain reaction (RT-PCR) is an essential method for specific diagnosis of SARS-CoV-2 infection. Unfortunately, false negative test results are often reported. In this study, we attempted to determine the principal causes leading to false negative results of RT-PCR detection of SARS-CoV-2 RNAs in respiratory tract specimens. Multiple sputum and throat swab specimens from 161 confirmed COVID-19 patients were tested with a commercial fluorescent RT-PCR kit targeting the ORF1ab and N regions of SARS-CoV-2 genome. The RNA level of a cellular housekeeping gene ribonuclease P/MRP subunit p30 (RPP30) in these specimens was also assessed by RT-PCR. Data for a total of 1052 samples were retrospectively re-analyzed and a strong association between positive results in SARS-CoV-2 RNA tests and high level of RPP30 RNA in respiratory tract specimens was revealed. By using the ROC-AUC analysis, we identified Ct cutoff values for RPP30 RT-PCR which predicted false negative results for SARS-CoV-2 RT-PCR with high sensitivity (95.03%-95.26%) and specificity (83.72%-98.55%) for respective combination of specimen type and amplification reaction. Using these Ct cutoff values, false negative results could be reliably identified. Therefore, the presence of cellular materials, likely infected host cells, are essential for correct SARS-CoV-2 RNA detection by RT-PCR in patient specimens. RPP30 could serve as an indicator for cellular content, or a surrogate indicator for specimen quality. In addition, our results demonstrated that false negativity accounted for a vast majority of contradicting results in SARS-CoV-2 RNA test by RT-PCR.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , RNA, Viral/genetics , SARS-CoV-2/genetics , Autoantigens/genetics , COVID-19/epidemiology , COVID-19/virology , China/epidemiology , Humans , Negative Results , Polyproteins/genetics , RNA, Viral/isolation & purification , Reference Standards , Retrospective Studies , Reverse Transcriptase Polymerase Chain Reaction/methods , Ribonuclease P/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Viral Proteins/genetics
19.
Aging (Albany NY) ; 13(7): 9265-9276, 2021 03 29.
Article in English | MEDLINE | ID: covidwho-1156226

ABSTRACT

BACKGROUND: Dysregulated immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are thought to underlie the progression of coronavirus disease 2019 (COVID-19). We sought to further characterize host antiviral and cytokine gene expression in COVID-19 patients based on illness severity. METHODS: In this case-control study, we retrospectively analyzed 46 recovered COVID-19 patients and 24 healthy subjects (no history of COVID-19) recruited from the Second People's Hospital of Fuyang City. Blood samples were collected from each study participant for RNA extraction and PCR. We assessed changes in antiviral gene expression between healthy controls and patients with mild/moderate (MM) and severe/critical (SC) disease. RESULTS: We found that type I interferon signaling (IFNA2, TLR8, IFNA1, IFNAR1, TLR9, IRF7, ISG15, APOBEC3G, and MX1) and genes encoding proinflammatory cytokines (IL12B, IL15, IL6, IL12A and IL1B) and chemokines (CXCL9, CXCL11 and CXCL10) were upregulated in patients with MM and SC disease. Moreover, we found that IFNA1, apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (APOBEC3G), and Fas-associated protein with death domain (FADD) were significantly downregulated (P < 0.05) in the SC group compared to the MM group. We also observed that microRNA (miR)-155 and miR-130a levels were markedly higher in the MM group compared to the SC group. CONCLUSION: COVID-19 is associated with the activation of host antiviral genes. Induction of the IFN system appears to be particularly important in controlling SARS-CoV-2 infection, as decreased expression of IFNA1, APOBEC3G and FADD genes in SC patients, relative to MM patients, may be associated with disease progression.


Subject(s)
COVID-19/genetics , COVID-19/immunology , Immunity, Innate , SARS-CoV-2/immunology , APOBEC-3G Deaminase/genetics , APOBEC-3G Deaminase/immunology , Adult , Aged , Case-Control Studies , Cytokines/genetics , Cytokines/immunology , Female , Humans , Interferon Type I/genetics , Interferon Type I/immunology , Male , Middle Aged , Retrospective Studies , Severity of Illness Index , Transcriptome , Up-Regulation
20.
Nat Commun ; 12(1): 1739, 2021 03 19.
Article in English | MEDLINE | ID: covidwho-1142438

ABSTRACT

Extensive testing is essential to break the transmission of SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Here, we present a CRISPR-based diagnostic assay that is robust to viral genome mutations and temperature, produces results fast, can be applied directly on nasopharyngeal (NP) specimens without RNA purification, and incorporates a human internal control within the same reaction. Specifically, we show that the use of an engineered AsCas12a enzyme enables detection of wildtype and mutated SARS-CoV-2 and allows us to perform the detection step with loop-mediated isothermal amplification (LAMP) at 60-65 °C. We also find that the use of hybrid DNA-RNA guides increases the rate of reaction, enabling our test to be completed within 30 minutes. Utilizing clinical samples from 72 patients with COVID-19 infection and 57 healthy individuals, we demonstrate that our test exhibits a specificity and positive predictive value of 100% with a sensitivity of 50 and 1000 copies per reaction (or 2 and 40 copies per microliter) for purified RNA samples and unpurified NP specimens respectively.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , RNA, Guide , SARS-CoV-2/genetics , Bacterial Proteins/genetics , COVID-19/virology , CRISPR-Associated Proteins/genetics , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Endodeoxyribonucleases/genetics , Humans , Molecular Diagnostic Techniques/methods , Mutation , Nasopharynx/virology , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics , Sensitivity and Specificity
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