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1.
China CDC Wkly ; 4(23): 504-508, 2022 Jun 10.
Article in English | MEDLINE | ID: covidwho-1893721

ABSTRACT

Introduction: Recently, a local cluster epidemic has occurred in Shijiazhuang City, Hebei Province. Failure to promptly identify patients with fever in rural areas was the major reason for this epidemic. Methods: We presented the field evaluation of a new real-time reverse transcription recombinase-aided amplification (RT-RAA) kit incorporating an endogenous internal control in a single-tube format, completed at the Hebei CDC from January 17, 2021 to January 27, 2021. Results: We evaluated the diagnostic performance of RT-RAA assay using automatic extracted RNA of 808 clinical samples. Compared with reverse transcriptase real-time quantitative PCR (qRT-PCR), RT-RAA kit achieved 92.41% sensitivity, 98.78% specificity and a 96.29% coincidence rate, demonstrating an excellent agreement between the RT-RAA assay and qRT-PCR assay. Furthermore, 58 samples were extracted using a manual extraction method within 5 minutes, but only samples with high nucleic acid concentration (cycle threshold value not higher than 32) could be stably detected. Discussion: The RT-RAA is more suitable to meet the needs of rapid, sensitive, and accurate detection in community-level medical institutions.

2.
Biosaf Health ; 2022 May 31.
Article in English | MEDLINE | ID: covidwho-1866924

ABSTRACT

The outbreak of COVID-19 in mainland China was declared controlled after April 2020. Since then, the origins of all local outbreaks were imported cases of COVID-19 infection or imported articles contaminated with SARS-CoV-2. Human contact with contaminated items may result in infection, which can lead to local COVID-19 outbreaks and even multiple transmission chains caused by different SARS-CoV-2 strains circulating within the same area. This study aimed to explore the possible causes of local outbreaks caused by articles contaminated with SARS-CoV-2 and to share "item-to-human" origin-tracing methods.

3.
Int J Infect Dis ; 114: 105-111, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1587637

ABSTRACT

OBJECTIVES: The democratization of diagnostics is one of the key challenges towards containing the transmission of coronavirus disease 2019 (COVID-19) around the globe. The operational complexities of existing PCR-based methods, including sample transfer to advanced central laboratories with expensive equipment, limit their use in resource-limited settings. However, with the advent of isothermal technologies, the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is possible at decentralized facilities. METHODS: In this study, two recombinase-based isothermal techniques, reverse transcription recombinase polymerase amplification (RT-RPA) and reverse transcription recombinase-aided amplification (RT-RAA), were evaluated for the detection of SARS-CoV-2 in clinical samples. A total of 76 real-time reverse transcription PCR (real-time RT-PCR) confirmed COVID-19 cases and 100 negative controls were evaluated to determine the diagnostic performance of the isothermal methods. RESULTS: This investigation revealed equally promising diagnostic accuracy of the two methods, with a sensitivity of 76.32% (95% confidence interval 65.18-85.32%) when the target genes were RdRP and ORF1ab for RT-RPA and RT-RAA, respectively; the combination of N and RdRP in RT-RPA augmented the accuracy of the assay at a sensitivity of 85.53% (95% confidence interval 75.58-92.55%). Furthermore, high specificity was observed for each of the methods, ranging from 94.00% to 98.00% (95% confidence interval 87.40-9.76%). CONCLUSIONS: Considering the diagnostic accuracies, both RT-RPA and RT-RAA appear to be suitable assays for point-of-need deployment for the detection of the pathogen, understanding its epidemiology, case management, and curbing transmission.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Nucleic Acid Amplification Techniques , RNA, Viral/genetics , Recombinases/metabolism , Reverse Transcription , Sensitivity and Specificity
4.
Biosaf Health ; 4(1): 1-5, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1588184

ABSTRACT

Although significant achievements have shown that the coronavirus disease 2019 (COVID-19) resurgence in Beijing, China, was initiated by contaminated frozen products and transported via cold chain transportation, international travelers with asymptomatic symptoms or false-negative nucleic acid may have another possible transmission mode that spread the virus to Beijing. One of the key differences between these two assumptions was whether the virus actively replicated since, so far, no reports showed viruses could stop evolution in alive hosts. We studied severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequences in this outbreak by a modified leaf-dating method with the Bayes factor. The numbers of single nucleotide variants (SNVs) found in SARS-CoV-2 sequences were significantly lower than those called from B.1.1 records collected at the matching time worldwide (P = 0.047). In addition, results of the leaf-dating method showed ages of viruses sampled from this outbreak were earlier than their recorded dates of collection (Bayes factors > 10), while control sequences (selected randomly with ten replicates) showed no differences in their collection dates (Bayes factors < 10). Our results which indicated that the re-emergence of SARS-CoV-2 in Beijing in June 2020 was caused by a virus that exhibited a lack of evolutionary changes compared to viruses collected at the corresponding time, provided evolutionary evidence to the contaminated imported frozen food should be responsible for the reappearance of COVID-19 cases in Beijing. The method developed here might also be helpful to provide the very first clues for potential sources of COVID-19 cases in the future.

5.
China CDC Wkly ; 3(46): 973-976, 2021 Nov 12.
Article in English | MEDLINE | ID: covidwho-1513533

ABSTRACT

INTRODUCTION: The best approach to preventing the importation of coronavirus disease 2019 (COVID-19) is enhancing the detection capacity at customs. The rapid detection is of utmost importance and therefore highly demanded. METHODS: We conducted a field validation study of a duplex real-time reverse transcription recombinase-aided amplification (RT-RAA) assay in Zhoushan and Hangzhou customs, in Zhejiang Province, China. The reverse transcriptase polymerase chain reaction (RT-PCR) assay kit routinely used at customs was used in parallel, and the duration the two methods took to complete a specific number of samples was compared. RESULTS: Among 506 samples collected, RT-RAA results were consistent with the RT-PCR results. The sensitivity and specificity were 100%, the total coincidence rate was 100%, and the Kappa value was 1 (P<0.05) for both methods. The RT-RAA kit took a significantly shorter time in testing the 20-200 samples than the RT-PCR kit. DISCUSSION: The RT-RAA detection method is more efficient and suitable for use at customs than RT-PCR assay to realize rapid customs clearance of 200 or fewer samples.

6.
China CDC Wkly ; 3(21): 448-453, 2021 May 21.
Article in English | MEDLINE | ID: covidwho-1237077

ABSTRACT

Background: COVID-19 infection is a major public health problem worldwide, and the D614G mutation enhances the infectivity of COVID-19.Methods: A probe-directed recombinase amplification (PDRA) assay was discussed to detect the D614G mutation at 39 ℃ for 30 min. The sensitivity, specificity, and reproducibility of the PDRA were evaluated by D614 and G614 recombinant plasmids. The clinical performance of PDRA assay was validated by testing of 53 previously confirmed COVID-19 positive RNAs and 10 negative samples. Direct sequencing was carried out in parallel for comparison.Result: With good reproducibility and specificity, the PDRA assay worked well with the concentration in the range of 103-107 copies/reaction. Compared with direct sequencing as a reference, the recombinase-aided amplification (RAA) assay obtained 100% sensitivity and 100% specificity using clinical samples.Conclusions: A rapid, convenient, sensitive, and specific method to detect D614G mutation was developed, which offers a useful tool to monitor mutations in COVID-19 virus RNA.

7.
Virol J ; 17(1): 197, 2020 12 28.
Article in English | MEDLINE | ID: covidwho-992505

ABSTRACT

BACKGROUND: Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. METHOD: In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. RESULT: The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23-1145.69) for ORF1ab and 528.1 (95% CI: 347.7-1248.7) for N, 401.8 (95% CI: 284.8-938.3) for ORF1ab and 336.8 (95% CI: 244.6-792.5) for N, and 194.74 (95% CI: 139.7-430.9) for ORF1ab and 189.1 (95% CI: 130.9-433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. CONCLUSION: In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.


Subject(s)
COVID-19/diagnosis , SARS-CoV-2/isolation & purification , COVID-19/virology , Humans , Limit of Detection , Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Sensitivity and Specificity
8.
J Cell Mol Med ; 25(2): 1274-1289, 2021 01.
Article in English | MEDLINE | ID: covidwho-978718

ABSTRACT

COVID-19 caused by SARS-CoV-2 is pandemic with a severe morbidity and mortality rate across the world. Despite the race for effective vaccine and drug against further expansion and fatality rate of this novel coronavirus, there is still lack of effective antiviral therapy. To this effect, we deemed it necessary to identify potential B and T cell epitopes from the envelope S protein. This can be used as potential targets to develop anti-SARS-CoV-2 vaccine preparations. In this study, we used immunoinformatics to identify conservative B and T cell epitopes for S proteins of SARS-CoV-2, which might play roles in the initiation of SARS-CoV-2 infection. We identified the B cell and T cell peptide epitopes of S protein and their antigenicity, as well as the interaction between the peptide epitopes and human leucocyte antigen (HLA). Among the B cell epitopes, 'EILDITPCSFGGVS' has the highest score of antigenicity and great immunogenicity. In T cell epitopes, MHC-I peptide 'KIADYNYKL' and MHC-II peptide 'LEILDITPC' were identified as high antigens. Besides, docking analysis showed that the predicted peptide 'KIADYNYKL' was closely bound to the HLA-A*0201. The results of molecular dynamics simulation through GROMACS software showed that 'HLA-A*0201~peptide' complex was very stable. And the peptide we selected could induce the T cell response similar to that of SARS-CoV-2 infection. Moreover, the predicted peptides were highly conserved in different isolates from different countries. The antigenic epitopes presumed in this study were effective new vaccine targets to prevent SARS-CoV-2 infection.


Subject(s)
COVID-19/immunology , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , COVID-19 Vaccines/immunology , HLA-A Antigens/immunology , Histocompatibility Antigens Class II/immunology , Humans , Molecular Dynamics Simulation , Pandemics/prevention & control , Viral Vaccines/immunology
9.
Reprod Biomed Online ; 42(3): 589-594, 2021 03.
Article in English | MEDLINE | ID: covidwho-955942

ABSTRACT

RESEARCH QUESTION: What are the risks associated with cryopreserved semen collected during and after the coronavirus disease 2019 (COVID-19) pandemic wave in Wuhan, China? DESIGN: Retrospective cohort study involving young adult men who were qualified sperm donors at the Hunan Province Human Sperm Bank (China) during the pandemic wave (1 January 2020 to 30 January 2020) and after the wave and return to work (7 April 2020 to 30 May 30 2020). One hundred paired semen and blood specimens from 100 donors were included. One-step single-tube nested quantitative real-time polymerase chain reaction (OSN-qRT-PCR) was used to detect SARS-CoV-2. Moreover, to control the unacceptable risk of false-negative results, a second round of screening was performed with pooled RNA from negative semen samples using crystal digital PCR (cd-PCR). RESULTS: For individual blood and semen samples, the target genes, namely the nucleocapsid protein (N) and open reading frame (ORF-1ab) genes, tested negative in all of the 100 paired samples. Further, as per cd-PCR results, there were >20,000 droplets per well in the RNA for each combined sample and no positive droplets were present for either of the aforementioned target genes. A total of 100 paired semen and blood samples from these two groups tested negative for SARS-CoV-2. CONCLUSIONS: Cryopreserved semen at the Hunan Province Human Sperm Bank during and after the COVID-19 pandemic wave was free of SARS-CoV-2 and was judged safe for external use in the future.


Subject(s)
COVID-19 , Pandemics , China/epidemiology , Humans , Male , Real-Time Polymerase Chain Reaction , Retrospective Studies , SARS-CoV-2 , Semen , Sperm Banks , Spermatozoa , Young Adult
10.
Anal Chem ; 92(13): 9399-9404, 2020 07 07.
Article in English | MEDLINE | ID: covidwho-342738

ABSTRACT

Coronavirus disease 2019 (COVID-19) has become a public health emergency. The reverse transcriptase real-time quantitative PCR (qRT-PCR) test is currently considered as the gold standard in the laboratory for the etiological detection of COVID-19. However, qRT-PCR results could be false-negative due to the inadequate sensitivity of qRT-PCR. In this study, we have developed and evaluated a novel one-step single-tube nested quantitative real-time PCR (OSN-qRT-PCR) assay for the highly sensitive detection of SARS-CoV-2 targeting the ORF1ab and N genes. The sensitivity of the OSN-qRT-PCR assay was 1 copy/reaction and 10-fold higher than that of the commercial qRT-PCR kit (10 copies/reaction). The clinical performance of the OSN-qRT-PCR assay was evaluated using 181 clinical samples. Among them, 14 qRT-PCR-negative samples (7 had no repetitive results and 7 had no cycle threshold (CT) values) were detected by OSN-qRT-PCR. Moreover, the 7 qRT-PCR-positives in the qRT-PCR gray zone (CT values of ORF1ab ranged from 37.48 to 39.07, and CT values of N ranged from 37.34 to 38.75) were out of the gray zone and thus were deemed to be positive by OSN-qRT-PCR, indicating that the positivity of these samples is confirmative. Compared to the qRT-PCR kit, the OSN-qRT-PCR assay revealed higher sensitivity and specificity, showing better suitability to clinical applications for the detection of SARS-CoV-2 in patients with low viral load.


Subject(s)
Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Real-Time Polymerase Chain Reaction/methods , Adult , Aged , Aged, 80 and over , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , COVID-19 , Coronavirus Infections/virology , Coronavirus Nucleocapsid Proteins , Female , Humans , Male , Middle Aged , Nucleocapsid Proteins/genetics , Pandemics , Phosphoproteins , Pneumonia, Viral/virology , Polyproteins , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2 , Sensitivity and Specificity , Viral Proteins/genetics
11.
Lancet ; 395(10224): 565-574, 2020 02 22.
Article in English | MEDLINE | ID: covidwho-80

ABSTRACT

BACKGROUND: In late December, 2019, patients presenting with viral pneumonia due to an unidentified microbial agent were reported in Wuhan, China. A novel coronavirus was subsequently identified as the causative pathogen, provisionally named 2019 novel coronavirus (2019-nCoV). As of Jan 26, 2020, more than 2000 cases of 2019-nCoV infection have been confirmed, most of which involved people living in or visiting Wuhan, and human-to-human transmission has been confirmed. METHODS: We did next-generation sequencing of samples from bronchoalveolar lavage fluid and cultured isolates from nine inpatients, eight of whom had visited the Huanan seafood market in Wuhan. Complete and partial 2019-nCoV genome sequences were obtained from these individuals. Viral contigs were connected using Sanger sequencing to obtain the full-length genomes, with the terminal regions determined by rapid amplification of cDNA ends. Phylogenetic analysis of these 2019-nCoV genomes and those of other coronaviruses was used to determine the evolutionary history of the virus and help infer its likely origin. Homology modelling was done to explore the likely receptor-binding properties of the virus. FINDINGS: The ten genome sequences of 2019-nCoV obtained from the nine patients were extremely similar, exhibiting more than 99·98% sequence identity. Notably, 2019-nCoV was closely related (with 88% identity) to two bat-derived severe acute respiratory syndrome (SARS)-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, collected in 2018 in Zhoushan, eastern China, but were more distant from SARS-CoV (about 79%) and MERS-CoV (about 50%). Phylogenetic analysis revealed that 2019-nCoV fell within the subgenus Sarbecovirus of the genus Betacoronavirus, with a relatively long branch length to its closest relatives bat-SL-CoVZC45 and bat-SL-CoVZXC21, and was genetically distinct from SARS-CoV. Notably, homology modelling revealed that 2019-nCoV had a similar receptor-binding domain structure to that of SARS-CoV, despite amino acid variation at some key residues. INTERPRETATION: 2019-nCoV is sufficiently divergent from SARS-CoV to be considered a new human-infecting betacoronavirus. Although our phylogenetic analysis suggests that bats might be the original host of this virus, an animal sold at the seafood market in Wuhan might represent an intermediate host facilitating the emergence of the virus in humans. Importantly, structural analysis suggests that 2019-nCoV might be able to bind to the angiotensin-converting enzyme 2 receptor in humans. The future evolution, adaptation, and spread of this virus warrant urgent investigation. FUNDING: National Key Research and Development Program of China, National Major Project for Control and Prevention of Infectious Disease in China, Chinese Academy of Sciences, Shandong First Medical University.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Genome, Viral , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , Receptors, Virus/metabolism , Betacoronavirus/metabolism , Bronchoalveolar Lavage Fluid/virology , COVID-19 , China/epidemiology , Coronavirus Infections/diagnosis , Coronavirus Infections/transmission , DNA, Viral/genetics , Disease Reservoirs/virology , Genomics/methods , High-Throughput Nucleotide Sequencing/methods , Humans , Phylogeny , Pneumonia, Viral/diagnosis , Pneumonia, Viral/transmission , SARS-CoV-2 , Sequence Alignment
12.
N Engl J Med ; 382(8): 727-733, 2020 02 20.
Article in English | MEDLINE | ID: covidwho-8

ABSTRACT

In December 2019, a cluster of patients with pneumonia of unknown cause was linked to a seafood wholesale market in Wuhan, China. A previously unknown betacoronavirus was discovered through the use of unbiased sequencing in samples from patients with pneumonia. Human airway epithelial cells were used to isolate a novel coronavirus, named 2019-nCoV, which formed a clade within the subgenus sarbecovirus, Orthocoronavirinae subfamily. Different from both MERS-CoV and SARS-CoV, 2019-nCoV is the seventh member of the family of coronaviruses that infect humans. Enhanced surveillance and further investigation are ongoing. (Funded by the National Key Research and Development Program of China and the National Major Project for Control and Prevention of Infectious Disease in China.).


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/virology , Lung/diagnostic imaging , Pneumonia, Viral/virology , Adult , Betacoronavirus/genetics , Betacoronavirus/ultrastructure , Bronchoalveolar Lavage Fluid/virology , COVID-19 , Cells, Cultured , China , Coronavirus Infections/diagnostic imaging , Coronavirus Infections/pathology , Epithelial Cells/pathology , Epithelial Cells/virology , Female , Genome, Viral , Humans , Lung/pathology , Lung/virology , Male , Microscopy, Electron, Transmission , Middle Aged , Phylogeny , Pneumonia, Viral/diagnostic imaging , Pneumonia, Viral/pathology , Radiography, Thoracic , Respiratory System/pathology , Respiratory System/virology , SARS-CoV-2
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