A novel, ultrafast, ultrasensitive diagnosis platform for the detection of SARS-COV-2 using restriction endonuclease-mediated reverse transcription multiple cross displacement amplification.

Coronavirus disease 2019 (COVID-19) is a highly infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-COV-2). Though many methods have been used for detecting SARS-COV-2, development of an ultrafast and highly sensitive detection strategy to screen and/or diagnose suspected cases in the population, especially early-stage patients with low viral load, is significant for the prevention and treatment of COVID-19. In this study, a novel restriction endonuclease-mediated reverse transcription multiple cross displacement amplification (MCDA) combined with real-time fluorescence analysis (rRT-MCDA) was successfully established and performed to diagnose COVID-19 infection (COVID-19 rRT-MCDA). Two sets of specific SARS-COV-2 rRT-MCDA primers targeting opening reading frame 1a/b (ORF1ab) and nucleoprotein (NP) genes were designed and modified according to the reaction mechanism. The SARS-COV-2 rRT-MCDA test was optimized and evaluated using various pathogens and clinical samples. The optimal reaction condition of SARS-COV-2 rRT-MCDA assay was 65°C for 36 min. The SARS-COV-2 rRT-MCDA limit of detection (LoD) was 6.8 copies per reaction. Meanwhile, the specificity of SARS-COV-2 rRT-MCDA assay was 100%, and there was no cross-reaction with nucleic acids of other pathogens. In addition, the whole detection process of SARS-COV-2 rRT-MCDA, containing the RNA template processing (15 min) and real-time amplification (36 min), can be accomplished within 1 h. The SARS-COV-2 rRT-MCDA test established in the current report is a novel, ultrafast, ultrasensitive, and highly specific detection method, which can be performed as a valuable screening and/or diagnostic tool for COVID-19 in clinical application.

Response and Assessment of the Effectiveness of the Countermeasures for a COVID-19 Outbreak - Guizhou Province, China, March 2022.

What is already known about this topic?: Many regions in China have recently reported outbreaks of the coronavirus disease 2019 (COVID-19) caused by the Omicron variant. What is added by this report?: Wuchuan County, Guizhou Province reacted quickly and implemented accurate intervention measures to effectively control the outbreak. The susceptible-exposed-infectious-asymptomatic-removed (SEIAR) model was applied to evaluate the effectiveness of intervention measures. What are the implications for public health practice?: Fast response measures should be taken to prevent the spread of outbreaks caused by the Omicron variant.

Single-cell transcriptomic atlas reveals distinct immunological responses between COVID-19 vaccine and natural SARS-CoV-2 infection.

To control the ongoing coronavirus disease-2019 (COVID-19) pandemic, CoronaVac (Sinovac), an inactivated vaccine, has been granted emergency use authorization by many countries. However, the underlying mechanisms of the inactivated COVID-19 vaccine-induced immune response remain unclear, and little is known about its features compared to (Severe acute respiratory syndrome coronavirus 2) SARS-CoV-2 infection. Here, we implemented single-cell RNA sequencing (scRNA-seq) to profile longitudinally collected PBMCs (peripheral blood mononuclear cells) in six individuals immunized with CoronaVac and compared these to the profiles of COVID-19 infected patients from a Single Cell Consortium. Both inactivated vaccines and SARS-CoV-2 infection altered the proportion of different immune cell types, caused B cell activation and differentiation, and induced the expression of genes associated with antibody production in the plasma. The inactivated vaccine and SARS-COV-2 infection also caused alterations in peripheral immune activity such as interferon response, inflammatory cytokine expression, innate immune cell apoptosis and migration, effector T cell exhaustion and cytotoxicity, however, the magnitude of change was greater in COVID-19 patients, especially those with severe disease, than in immunized individuals. Further analyses revealed a distinct peripheral immune cell phenotype associated with CoronaVac immunization (HLA class II upregulation and IL21R upregulation in naïve B cells) versus SARS-CoV-2 infection (HLA class II downregulation and IL21R downregulation in naïve B cells from severe disease individuals). There were also differences in the expression of important genes associated with proinflammatory cytokines and thrombosis. In conclusion, this study provides a single-cell atlas of the systemic immune response to CoronaVac immunization and revealed distinct immune responses between inactivated vaccines and SARS-CoV-2 infection.

Initial Clinical Characteristics of 146 Patients with COVID-19 Reported in Guizhou Province, China: A Survival Analysis.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) has caused an outbreak around the world. Early detection of severe illness is crucial for patients' survival. We analysed initial clinical characteristics of 146 patients with COVID-19 reported in Guizhou province, China to explore risk factors for transforming mild illness to severe. METHODOLOGY: Data of 146 laboratory-confirmed cases were collected and evaluated by the survival analysis of univariate and multivariate Cox proportional hazards model. RESULTS: On initial presentation, patients had fever (51.05%), dry cough (45.45%), headache (16.08%), shortness of breath (7.75%) and gastrointestinal symptoms (13.99%). Among 146 laboratory-confirmed cases, 30 patients (20.55%) had severe illness and needed Intensive Care Unit care for supportive treatment. The remaining patients (116, 79.45%) were non-severe cases. Nineteen (19/146, 13.01%) of 30 patients in the Intensive Care Unit had comorbidities, including hypertension (12, 40.00%), diabetes (5, 16.67%), cardiovascular disease (5, 16.67%) and pulmonary disease (4, 13.33%). For survival analysis, patients who had fever (HR = 3.30, 95% CI = 1.31, 8.29) and comorbidities (HR = 9.76, 95% CI = 4.28, 22.23) at baseline were more likely to be admitted into the Intensive Care Unit. Few variables were not related to the survival time of discharge from baseline to discharge and from Intensive Care Unit care to discharge. CONCLUSIONS: Severe patients with COVID-19 should be paid more attention. On initial symptoms, many patients did not have fever, but those with fever were more likely to be admitted to the Intensive Care Unit. Comorbidities were likewise a risk factor of severe COVID-19.

Rapid and Visual Detection of SARS-CoV-2 Using Multiplex Reverse Transcription Loop-Mediated Isothermal Amplification Linked With Gold Nanoparticle-Based Lateral Flow Biosensor.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that has caused the outbreak of coronavirus disease 2019 (COVID-19) all over the world. In the absence of appropriate antiviral drugs or vaccines, developing a simple, rapid, and reliable assay for SARS-CoV-2 is necessary for the prevention and control of the COVID-19 transmission. Methods: A novel molecular diagnosis technique, named multiplex reverse transcription loop-mediated isothermal amplification, that has been linked to a nanoparticle-based lateral flow biosensor (mRT-LAMP-LFB) was applied to detect SARS-CoV-2 based on the SARS-CoV-2 RdRp and N genes, and the mRT-LAMP products were analyzed using nanoparticle-based lateral flow biosensor. The mRT-LAMP-LFB amplification conditions, including the target RNA concentration, amplification temperature, and time were optimized. The sensitivity and specificity of the mRT-LAMP-LFB method were tested in the current study, and the mRT-LAMP-LFB assay was applied to detect the SARS-CoV-2 virus from clinical samples and artificial sputum samples. Results: The SARS-CoV-2 specific primers based on the RdRp and N genes were valid for the establishment of mRT-LAMP-LFB assay to detect the SARS-CoV-2 virus. The multiple-RT-LAMP amplification condition was optimized at 63°C for 30 min. The full process, including reaction preparation, viral RNA extraction, RT-LAMP, and product identification, could be achieved in 80 min. The limit of detection (LoD) of the mRT-LAMP-LFB technology was 20 copies per reaction. The specificity of mRT-LAMP-LFB detection was 100%, and no cross-reactions to other respiratory pathogens were observed. Conclusion: The mRT-LAMP-LFB technique developed in the current study is a simple, rapid, and reliable method with great specificity and sensitivity when it comes to identifying SARS-CoV-2 virus for prevention and control of the COVID-19 disease, especially in resource-constrained regions of the world.

A one-step, one-pot CRISPR nucleic acid detection platform (CRISPR-top): Application for the diagnosis of COVID-19.

The existing CRISPR-mediated diagnostic tests require a two-step procedure (DNA or RNA amplification followed by CRISPR-mediated sequence-specific detection) for nucleic acid detection, which increases complexity and the risk of sample cross-contamination. Here, we report a new CRISPR-mediated test, called CRISPR-top (CRISPR-mediated testing in one-pot), which integrates simultaneous target pre-amplification with CRISPR/cas12b-mediated detection into a one-pot reaction mixture, performed at a constant temperature. The novel CRISPR-top assay was applied to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for COVID-19 (coronavirus disease 2019). COVID-19 CRISPR-top targets the ORF1ab (opening reading frame 1a/b) and NP (nucleoprotein) genes of SARS-CoV-2, and operates at 59 °C for 40 min with minimal instrument. The COVID-19 CRISPR-top assay can return results within 60-min and is easily interpreted by visual fluorescence or lateral flow readouts. The analytical limit of detection (LoD) for COVID-19 CRISPR-top is 10 copies (for each detection target) per reaction with no cross-reactivity observed from non-SARS-CoV-2 templates. Among clinically collected non-COVID-19 samples, the assay's specificity was 100% (80/80 oropharynx swab samples). Among 52 COVID-19 positive clinical samples collected, the COVID-19 CRISPR-top assay yielded 38 (73.1%) positive results using fluorescence readout and 35 (67.3%) positive results with lateral-flow readout. These diagnostic results were similar to those obtained using RT-PCR (34 positive (65.4%)). These data indicate that COVID-19 CRISPR-top is a simple, rapid, accurate and highly sensitive method for SARS-CoV-2 detection which can be used in the clinic, field laboratories and primary care facilities in resource-challenged settings.

Epidemiologic Characteristics of COVID-19 in Guizhou Province, China.

INTRODUCTION: At the end of 2019, the COVID-19 broke out, and spread to Guizhou province in January of 2020. METHODOLOGY: To acquire the epidemiologic characteristics of COVID-19 in Guizhou province, we collected data from 169 laboratory-confirmed COVID-19 related cases. We described the demographic characteristics of the cases and estimated the incubation period, serial interval and the effective reproduction number. We also presented two representative case studies in Guizhou province: Case Study 1 was an example of the asymptomatic carrier; while Case Study 2 was an example of a large and complex infection chain that involved four different regions, spanning three provinces and eight families. RESULTS: Two peaks in the incidence distribution associated with COVID-19 in Guizhou province were related to the 6.04 days (95% CI: 5.00 - 7.10) of incubation period and 6.14±2.21 days of serial interval. We also discussed the effectiveness of the control measures based on the instantaneous effective reproduction number that was a constantly declining curve. CONCLUSIONS: As of February 2, 2020, the estimated effective reproduction number was below 1, and no new cases were reported since February 26. These showed that Guizhou Province had achieved significant progress in preventing the spread of the epidemic. The medical isolation of close contacts was consequential. Meanwhile, the asymptomatic carriers and the super-spreaders must be isolated in time, who would cause a widespread infection.

Rapid, Ultrasensitive, and Highly Specific Diagnosis of COVID-19 by CRISPR-Based Detection.

Coronavirus Disease 2019 (COVID-19), which is caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), has rapidly spread leading to a global pandemic. Here, we combined multiple cross displacement amplification (MCDA) with CRISPR-Cas12a-based detection to develop a novel diagnostic test (MCCD) and applied for the diagnosis of COVID-19, called COVID-19 MCCD. The MCCD protocol conducts reverse transcription MCDA (RT-MCDA) reaction for RNA templates followed by CRISPR-Cas12a/CrRNA complex detection of predefined target sequences after which degradation of a single-strand DNA (ssDNA) molecule confirms detection of the target sequence. Two MCDA primer sets and two CrRNAs were designed targeting the opening reading frame 1a/b (ORF1ab) and nucleoprotein (N) of SARS-CoV-2. The optimal conditions include two RT-MCDA reactions at 63 °C for 35 min and a CRISPR-Cas12a/CrRNA detection reaction at 37 °C for 5 min. The COVID-19 MCCD assay can be visualized on a lateral flow biosensor (LFB) and completed within 1 h including RNA extraction (15 min), RT-MCDA reaction (35 min), CRISPR-Cas12a/CrRNA detection reaction (5 min), and reporting of result (within 2 min). The COVID-19 MCCD assay is very sensitive and detects the target gene with as low as seven copies per test and does not cross-react with non-SARS-CoV-2 templates. SARS-CoV-2 was detected in 37 of 37 COVID-19 patient samples, and nonpositive results were detected from 77 non-COVID-19 patients. Therefore, the COVID-19 MCCD assay is a useful tool for the reliable and quick diagnosis of SARS-CoV-2 infection.

Highly sensitive and specific diagnosis of COVID-19 by reverse transcription multiple cross-displacement amplification-labelled nanoparticles biosensor.

BACKGROUND: The ongoing outbreak of the novel human coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (also known as 2019-nCoV) has become a global health concern. Rapid and easy-to-use diagnostic techniques are urgently needed to diagnose SARS-CoV-2 infection. METHODS: We devised a reverse transcription multiple cross-displacement amplification (RT-MCDA) coupled with a nanoparticle-based biosensor assay (RT-MCDA-BS) for rapid, sensitive and specific diagnosis of coronavirus disease 2019 (COVID-19). Two primer sets were designed to target the open reading frame 1a/b and nucleoprotein gene of SARS-CoV-2. A total of 183 clinical samples, including 65 patients with COVID-19 infection and 118 patients with other pathogen infections were used to testify the assay's feasibility. Diagnosis results were reported visually using the biosensor. FINDINGS: The assay designed was performed using a simple instrument which could maintain the reaction in a constant temperature at 64°C for only 35 min. The total COVID-19 RT-MCDA-BS test procedure could be finished within 1 h. The COVID-19 RT-MCDA-BS could detect down to five copies of target sequences. Among 65 clinical samples from the COVID-19 patients, 22 (33.8%) positive results were obtained from faeces, nasal, pharyngeal and anal swabs via COVID-19 RT-MCDA-BS assay, while real-time reverse transcription-PCR assay only detected 20 (30.7%) positive results in these samples. No positive results were obtained from clinical samples with non-COVID-19 infections. INTERPRETATION: COVID-19 RT-MCDA-BS was a rapid, reliable, low-cost and easy-to-use assay, which could provide an attractive laboratory tool to diagnose COVID-19 in multiple clinical specimens, especially for field, clinic laboratories and primary care facilities in resource-poor settings.

Detection and analysis of nucleic acid in various biological samples of COVID-19 patients.

COVID-19 (corona virus disease 2019) is a kind of acute severe pneumonia caused by 2019-nCoV (2019-nCoV) infection. Since December 2019, it has been found in Wuhan, Hubei Province, and then spread to the whole country. Some parts of the world also showed an outbreak trend [1-3]. Real-time fluorescence quantitative reverse transcriptase polymerase chain reaction (reverse transcriptase-polymerase chain reaction,RT-PCR) and viral gene sequencing are the gold standard for the diagnosis of COVID-19. At present, upper respiratory tract nasopharyngeal swabs are mostly used as nucleic acid detection samples in China, but the positive rate is low. However, there are few reports on clinical application of 2019-nCoV nucleic acid detection in other biological samples. Methods: | The East Section of Renmin Hospital of Wuhan University is a designated COVID-19 hospital in Wuhan City, Hubei Province, China. This observation study included 132 patients diagnosed with COVID-19 in the infectious disease areas of the East Section of Renmin Hospital of Wuhan University from 2020.1.31 to 2020.2.29. COVID-19 diagnostic criteria: according to China's ⟪pneumonia diagnosis and treatment Program of novel coronavirus infection (trial version 7) ⟫, in accordance with the relevant epidemiological and clinical manifestations, nasopharyngeal swabs real-time fluorescence RT-PCR detection of 2019-nCoV nucleic acid positive, COVID-19 cases were divided into mild, ordinary, severe and severe [1]. The nasopharyngeal swabs of 132 cases of COVID-19 were positive for 2019-nCoV nucleic acid on admission, including 72 males and 60 females, with an average age of 66.7 ± 9.1 years, including 80 cases of common type, 44 cases of severe type and 8 cases of critical type. During the period of admission, under the condition of tertiary protection, nasopharyngeal swabs, sputum, blood, feces and anal swabs of COVID-19 cases were collected many times in the isolation ward for 2019-nCoV nucleic acid detection. All biological samples are sealed and transferred to the laboratory in strict accordance with the standard process. The RT-PCR test kits (BioGerm) were recommended by the Chinese Center for Disease Control and Prevention. The same technician and brand of test kit was used for all RT-PCR testing reported; both internal controls and negative controls were routinely performed with each batch of tests. Results: | 132 the results of 2019-nCoV nucleic acid test of various biological samples during the treatment of confirmed COVID-19 cases are as follows: the positive rate of 2019-nCoV nucleic acid test of nasopharyngeal swab is 38.13% (180/472 times), the positive rate of 2019-nCoV nucleic acid test of sputum is 48.68% (148/304 times), the positive rate of blood 2019-nCoV nucleic acid test is 3.03% (4/132 times), and the positive rate of 2019-nCoV nucleic acid test of feces is 9.83% (24/244 times). The positive rate of 2019-nCoV nucleic acid detection in anal swabs is 10.00% (12/120 times). Discussion|: In this study, it was found that the positive rate of 2019-nCoV nucleic acid in sputum of 132 patients with COVID-19 was higher than that of nasopharyngeal swabs, and viral nucleic acids were also detected in blood and digestive tract (fecal/anal swabs). Simple detection of nasopharyngeal swab 2019-nCoV nucleic acid detection positive rate is not high, multi-sample 2019-nCoV nucleic acid detection can improve the accuracy, reduce the false negative rate, better guide clinical treatment and evaluate the therapeutic effect.