Integrating CRISPR/Cas within isothermal amplification for point-of-Care Assay of nucleic acid.

Nucleic acid detection technology is now widely used in scientific research and clinical testing, such as infectious and genetic diseases screening, molecular diagnosis of tumors and pharmacogenomic research, which is also an important part of in vitro diagnostics (IVD). However, with the increasing requirements of diagnosis and treatment, existing nucleic acid detection technologies are facing challenges in dealing with the current problems (especially since the outbreak of coronavirus disease in 2019 (Covid-19)). Recently, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (CRISPR/Cas)-based diagnostics have become a hot spot of attention. CRISPR/Cas has been developed as a molecular detection tool besides scientific research in biology and medicine fields, and some CRISPR-based products have already been translated. It is known as the "next-generation molecular diagnostic technology" because of its advantages such as easy design and accurate identification. CRISPR/Cas relies on pre-amplification of target sequences and subsequent detection of Cas proteins. Combining the CRISPR/Cas system with various isothermal nucleic acid amplification strategies can generate amplified detection signals, enrich low abundance molecular targets, improve the specificity and sensitivity of analysis, and develop point-of-care (POC) diagnostic techniques. In this review, we analyze the current status of CRISPR/Cas systems and isothermal amplification, report the advantages of combining the two and summarize the recent progress with the integration of both technologies with POC sensors in the nucleic acid field. In addition, the challenges and future prospects of CRISPR technology combined with isothermal amplification strategies in biosensing and clinical applications are discussed.

Analysis of respiratory virus infection in 114 suspected COVID-19 patients

To explore the value of simultaneous detection of multiple viruses in epidemic prevention and control of COVID-19. To analyze respiratory virus infection of 114 suspected COVID-19 patients, real-time RT-PCR was used to detect SARS-CoV-2 nucleic acid. At the same time, the thermostatic amplification was used to detect other 18 respiratory virus. As results, the nucleic acid of 114 suspected COVID-19 patients was negative, and 21 of them were infected with non-other respiratory viruses, with an infection rate of 18.42%. A total of 10 respiratory viruses were detected in 21 cases, including coronavirus NL63/229E, respiratory syncytial virus, human coxsackie virus A16, influenza B virus, human parainfluenza virus type 1, human parainfluenza virus type 3, human metapneumovirus, influenza A virus, seasonal influenza a virus subtype H3, and enterovirus/rhinovirus. There were 6 cases of influenza B virus infection and 5 cases of respiratory syncytial virus. Three patients were co-infected with two viruses: respiratory syncytial virus mixed with coxsackie virus A16, coronavirus NL63/229E mixed with human parainfluenza virus 1, and influenza A virus mixed with influenza A virus seasonal H3 subtype. In conclusion, in response to the SARS-CoV-2 epidemic, attention should be paid to the identification of SARS-CoV-2 and other respiratory viruses in suspected COVID-19 patients, so as to effectively exclude suspected cases.

Distinguishable Immunologic Characteristics of COVID-19 Patients with Comorbid Type 2 Diabetes Compared with Nondiabetic Individuals.

BACKGROUND: COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has threatened every civilian as a global pandemic. The immune system poses the critical interactive chain between the human body and the virus. Here, we make efforts to examine whether comorbidity with type 2 diabetes (T2D) affects the immunological response in COVID-19 patients. METHODS: We conducted a retrospective pilot study investigating immunological characteristics of confirmed cases of COVID-19 with or without comorbid T2D. Two subcohorts of sex- and age-matched participants were eligible for data analysis, of which 33 participants were with T2D and the remaining 37 were nondiabetic (NDM). Cellular immunity was assessed by flow cytometric determination of surface markers including CD3, CD4, CD8, CD19, CD16, and CD56 in peripheral blood. Levels of C reactive protein, immunoglobulin (IgG, IgM, IgA, and IgE), and complements (C3, C4) were detected by rate nephelometry immunoassay. And Th1/Th2 cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ) were detected by Cytometric Bead Array. RESULTS: Neutrophil counts were found to be significantly higher in the T2D group than in the NDM group and had a significant relevance with clinical severity. Lymphocyte frequencies showed no significant differences in the two groups. However, the proportions and absolute counts of T, Tc, Th, and NK cells decreased in both groups to different degrees. An abnormal increase in neutrophil count and a decrease in lymphocyte subpopulations may represent risk factors of COVID-19 severity. The level of IgG, IgM, IgA, C3, and C4 showed no significant difference between the two groups, while the IgE levels were higher in the T2D group than in the NDM group (p < 0.05). Th1 cytokines including IFN-γ, TNF-α, and IL-6, as well as CRP, appeared significantly higher in the T2D group. CONCLUSIONS: The COVID-19 patients comorbid with T2D demonstrated distinguishable immunological parameters, which represented clinical relevancies with the predisposed disease severity in T2D.

Gastrointestinal involvement of COVID-19 and potential faecal transmission of SARS-CoV-2.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was found initially in Wuhan, China in early December 2019. The pandemic has spread to 216 countries and regions, infecting more than 23310 000 people and causing over 800 000 deaths globally by Aug. 24, 2020, according to World Health Organization (https://www.who.int/emergencies/diseases/ novel-coronavirus-2019). Fever, cough, and dyspnea are the three common symptoms of the condition, whereas the conventional transmission route for SARS-CoV-2 is through droplets entering the respiratory tract. To date, infection control measures for COVID-19 have been focusing on the involvement of the respiratory system. However, ignoring potential faecal transmission and the gastrointestinal involvement of SARS-CoV-2 may result in mistakes in attempts to control the pandemic.