Comparison of CRISPR/Cas and Argonaute for nucleic acid tests.

Guided, programmable, and target-activated nucleases, exemplified by Cas in the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system and Argonaute (Ago), are emerging as a new generation of nucleic acid tests (NATs). A specific approach for comparison of these two nucleases side by side in terms of similarities, differences, and complementarities is instrumental for the sensible design of novel NATs.

SARS-CoV-2 Detection and COVID-19 Diagnosis: A Bird's Eye View.

The battle against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) associated coronavirus disease 2019 (COVID-19) is continued worldwide by administering firsttime emergency authorized novel mRNA-based and conventional vector-antigen-based antiCOVID-19 vaccines to prevent further transmission of the virus as well as to reduce the severe respiratory complications of the infection in infected individuals. However; the emergence of numerous SARS-CoV-2 variants is of concern, and the identification of certain breakthrough and reinfection cases in vaccinated individuals as well as new cases soaring in some low-to-middle income countries (LMICs) and even in some resource-replete nations have raised concerns that only vaccine jabs would not be sufficient to control and vanquishing the pandemic. Lack of screening for asymptomatic COVID-19-infected subjects and inefficient management of diagnosed COVID-19 infections also pose some concerns and the need to fill the gaps among policies and strategies to reduce the pandemic in hospitals, healthcare services, and the general community. For this purpose, the development and deployment of rapid screening and diagnostic procedures are prerequisites in premises with high infection rates as well as to screen mass unaffected COVID-19 populations. Novel methods of variant identification and genome surveillance studies would be an asset to minimize virus transmission and infection severity. The proposition of this pragmatic review explores current paradigms for the screening of SARS-CoV-2 variants, identification, and diagnosis of COVID-19 infection, and insights into the late-stage development of new methods to better understand virus super spread variants and genome surveillance studies to predict pandemic trajectories.

Design and Optimize of Campus Nucleic Acid Tests Queuing System

Covid-19 is more likely to spread in campus than it in other places because students live together without masks. In this case, it is necessary to take nucleic acid tests in a unified time regularly. To make nucleic acid tests efficient and convenient to manage students and the testing time, this article would apply queuing theory to design a nucleic acid tests queuing system by using the data from Sanda University in April 2022. According to the special conditions on campus, such as course schedule, students' daily activities, and campus management, students would be grouped by several management styles. The system would calculate the start time and waiting time for each group and would strive to take nucleic acid tests in an orderly manner with minimal waiting time. © 2022 IEEE.

Age and Sex Differences Among Mildly Symptomatic and Asymptomatic Patients with Omicron Infection in 2022 in Shanghai, China.

Background: An epidemic of the Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began in March 2022, and over 600,000 cases were confirmed until early May 2022 in Shanghai, China. Data on Omicron infections are available in other countries, but the clinical features of patients in the Chinese population, especially in Shanghai, are still lacking. We collected data from a subset of asymptomatic and mildly ill patients to learn about the age and sex disparity of Omicron infection based on changes in cycle threshold values. Methods: The basic information of 325 patients who were consecutively admitted to the Shanghai Geriatrics Center was collected through medical records, and patients were tested for viral nucleic acid carriage using nasal swab samples during hospitalization. SAS 9.4 was used for data analysis, and a p value < 0.05% was considered statistically significant. Results: Among the 325 included patients, 58.8% were males, with a mean age of 47.2 years and 13.6 days of hospitalization on average. The average number of nucleic acid tests among female patients was 4.7, which was higher than that among male patients (4.1). The median value of the slope for cycle threshold (Ct) changes in the nucleic acid detection (NAD) test was 1.4. Logistic regression indicated that the proportion of slope for Ct changes >1.5 was slightly higher among male patients than among female patients (odds ratio (OR) = 1.06, 95% confidence interval (CI): 0.68-1.66), and patients aged <45 years and 45-59 years had a higher proportion of slope for Ct changes >1.5 than patients aged ≥60 years. Ct values were more variable in the early stages of infection and stabilized in the later stages of infection. Conclusion: Among patients with mild illness or asymptomatic infection, the Ct value is a good, timely, and cost-effective method to reflect the recovery progress of patients. The slope of Ct changes was steeper among younger patients and male patients, which indicates faster disease recovery.

CRISPR-Cas13a cascade-based viral RNA assay for detecting SARS-CoV-2 and its mutations in clinical samples.

SARS-CoV-2 is one of the greatest threats to global human health. Point-of-care diagnostic tools for SARS-CoV-2 could facilitate rapid therapeutic intervention and mitigate transmission. In this work, we report CRISPR-Cas13a cascade-based viral RNA (Cas13C) assay for label-free and isothermal determination of SARS-CoV-2 and its mutations in clinical samples. Cas13a/crRNA was utilized to directly recognize the target of SARS-CoV-2 RNA, and the recognition events sequentially initiate the transcription amplification to produce light-up RNA aptamers for output fluorescence signal. The recognition of viral RNA via Cas13a-guide RNA ensures a high specificity to distinguish SARS-CoV-2 from MERS-CoV and SARS-CoV, as well as viral mutations. A post transcription amplification strategy was triggered after CRISPR-Cas13a recognition contributes to an amplification cascade that achieves high sensitivity for detecting SARS-CoV-2 RNA, with a limit of detection of 0.216 fM. In addition, the Cas13C assay could be able to discriminate single-nucleotide mutation, which was proven with N501Y in SARS-Cov-2 variant. This method was validated by a 100% agreement with RT-qPCR results from 12 clinical throat swab specimens. The Cas13C assay has the potential to be used as a routine nucleic acid test of SARS-CoV-2 virus in resource-limited regions.

Rapid Detection of SARS-CoV-2 Using Duplex Reverse Transcription-Multienzyme Isothermal Rapid Amplification in a Point-of-Care Testing.

In December 2019, a severe acute respiratory syndrome caused by SARS-CoV-2 spread rapidly worldwide. Portable nucleic acid tests of SARS-CoV-2 are critically important for diagnostics. In this study, we used an isothermal amplification method-Multienzyme Isothermal Rapid Amplification (MIRA)-for rapid detection of SARS-CoV-2. We designed the primers and probes in ORF1ab and N gene of SARS-CoV-2. The amplicons could be monitored by lateral flow dipsticks (LFDs). The reaction temperature, time, concentrations of primers and probes, and working volume were optimized. Four commercial swab collection buffers were used to test the amplification efficacy of our assay without RNA extraction. Our assay was able to amplify duplex targets of SARS-CoV-2 in one single reaction using one-step RT-MIRA. The assay worked well in a low volume of 10 µl at 38°C for 20 min. Using three collection buffers without guanidinium, our assay was able to amplify efficaciously without RNA extraction. The 95% limit of detection (LoD) of the RT-MIRA assay was 49.5 (95% CI, 46.8-52.7) copies/ml for ORF1ab gene and 48.8 (95% CI, 46.5-52.6) copies/ml for N gene. There is no cross-reaction with other human respiratory pathogens, such as SARS-CoV, MERS-CoV, influenza A virus, influenza B virus, human adenovirus, respiratory syncytial virus, human parainfluenza virus, and coronavirus 229E in our assay. The precision evaluation revealed that the C50-20% to C50+20% range bounds the C5-C95 interval. This assay also showed high anti-interference ability. The extraction-free RT-MIRA and qPCR detection results of 243 nucleic acid specimens from suspected patients or national references showed a 100.0% (95% confidence interval, 94.2%-100.0%) positive predictive value and a 100.0% (95% confidence interval, 92.7%-100.0%) negative predictive value. Compared with qPCR, the kappa value of the two assays was 1.00 (P < 0.0001). In conclusion, we provide a portable and visualized method for detection of SARS-CoV-2 without RNA extraction, allowing its application in SARS-CoV-2 on-site detection.

Evaluation of six different rapid methods for nucleic acid detection of SARS-COV-2 virus.

In the current coronavirus disease 2019 (COVID-19) pandemic there is a mass screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) happening around the world due to the extensive spread of the infections. There is a high demand for rapid diagnostic tests to expedite the identification of cases and to facilitate early isolation and control spread. Hence this study evaluates six different rapid nucleic acid detection assays that are commercially available for SARS-CoV-2 virus detection. Nasopharyngeal samples were collected from 4981 participants and were tested for the SARS-CoV-2 virus by the gold standard real-time reverse-transcription polymerase chain reaction (RT-PCR) method and with one of these six rapid methods of detection. Evaluation of the rapid nucleic acid detection assays was done by comparing the results of these rapid methods with the gold standard RT-qPCR results for SARS-COV-2 detection. AQ-TOP had the highest sensitivity (98%) and a strong kappa value of 0.943 followed by Genechecker and Abbot ID NOW. The POCKIT (ii RT-PCR) assay had the highest test accuracy of 99.29% followed by Genechecker and Cobas Liat. Atila iAMP showed the highest percentage of invalid reports (35.5%) followed by AQ-TOP with 6% and POCKIT with 3.7% of invalid reports. Genechecker system, Abbott ID NOW, and Cobas Liat were found to have the best performance and agreement when compared with the standard RT-PCR for COVID-19 detection. With further research, these rapid tests have the potential to be employed in large-scale screening of COVID-19.

Testing for COVID-19.

Accurate diagnostic tests that provide results in a timely manner are essential for the clinical and public health management of COVID-19 disease The choice as to which test to use will depend on the clinical presentation and the stage of the illness Nucleic acid tests, using real-time reverse transcriptase-polymerase chain reaction, are the most appropriate for diagnosing acute infection. Combined deep nasal (or nasopharyngeal) and throat swabs are the preferred sample Serology can be used to diagnose previous infection, more than 14 days after the onset of symptoms Antigen tests are in development and their role is not yet defined Interpretation of results must take into account the pre-test probability of the patient having the disease. This is based on their clinical presentation and epidemiological risk

COVID-19: Mechanisms of Vaccination and Immunity.

Vaccines are needed to protect from SARS-CoV-2, the virus causing COVID-19. Vaccines that induce large quantities of high affinity virus-neutralizing antibodies may optimally prevent infection and avoid unfavorable effects. Vaccination trials require precise clinical management, complemented with detailed evaluation of safety and immune responses. Here, we review the pros and cons of available vaccine platforms and options to accelerate vaccine development towards the safe immunization of the world's population against SARS-CoV-2. Favorable vaccines, used in well-designed vaccination strategies, may be critical for limiting harm and promoting trust and a long-term return to normal public life and economy.