SUMMARYIn recent decades, several new diseases have emerged in different geographical areas, with pathogens including Ebola virus, Zika virus, Nipah virus, and coronaviruses (CoVs). Recently, a new type of viral infection emerged in Wuhan City, China, and initial genomic sequencing data of this virus do not match with previously sequenced CoVs, suggesting a novel CoV strain (2019-nCoV), which has now been termed severe acute respiratory syndrome CoV-2 (SARS-CoV-2). Although coronavirus disease 2019 (COVID-19) is suspected to originate from an animal host (zoonotic origin) followed by human-to-human transmission, the possibility of other routes should not be ruled out. Compared to diseases caused by previously known human CoVs, COVID-19 shows less severe pathogenesis but higher transmission competence, as is evident from the continuously increasing number of confirmed cases globally. Compared to other emerging viruses, such as Ebola virus, avian H7N9, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2 has shown relatively low pathogenicity and moderate transmissibility. Codon usage studies suggest that this novel virus has been transferred from an animal source, such as bats. Early diagnosis by real-time PCR and next-generation sequencing has facilitated the identification of the pathogen at an early stage. Since no antiviral drug or vaccine exists to treat or prevent SARS-CoV-2, potential therapeutic strategies that are currently being evaluated predominantly stem from previous experience with treating SARS-CoV, MERS-CoV, and other emerging viral diseases. In this review, we address epidemiological, diagnostic, clinical, and therapeutic aspects, including perspectives of vaccines and preventive measures that have already been globally recommended to counter this pandemic virus.

Coronavirus Disease 2019–COVID-19

An ongoing theme of the COVID-19 pandemic is the need for widespread availability of accurate and efficient diagnostic testing for detection of SARS-CoV-2 and antiviral antibodies in infected indiv...

Assay Techniques and Test Development for COVID-19 Diagnosis

https://escholarship.org/content/qt1k5378ws/qt1k5378ws.pdf?t=qnu6z3

Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy.

In December 2019, an outbreak of pneumonia of unknown origin was reported in Wuhan, Hubei Province, China. Pneumonia cases were epidemiologically linked to the Huanan Seafood Wholesale Market. Inoculation of respiratory samples into human airway epithelial cells, Vero E6 and Huh7 cell lines, led to the isolation of a novel respiratory virus whose genome analysis showed it to be a novel coronavirus related to SARS-CoV, and therefore named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is a betacoronavirus belonging to the subgenus Sarbecovirus. The global spread of SARS-CoV-2 and the thousands of deaths caused by coronavirus disease (COVID-19) led the World Health Organization to declare a pandemic on 12 March 2020. To date, the world has paid a high toll in this pandemic in terms of human lives lost, economic repercussions and increased poverty. In this review, we provide information regarding the epidemiology, serological and molecular diagnosis, origin of SARS-CoV-2 and its ability to infect human cells, and safety issues. Then we focus on the available therapies to fight COVID-19, the development of vaccines, the role of artificial intelligence in the management of the pandemic and limiting the spread of the virus, the impact of the COVID-19 epidemic on our lifestyle, and preparation for a possible second wave.

The COVID-19 pandemic

The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus is a major public health challenge. Rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. Approaches to detect viral RNA based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) have potential as simple, scalable, and broadly applicable testing methods. Compared to RT quantitative polymerase chain reaction (RT-qPCR)-based methods, RT-LAMP assays require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. Here, we tested a two-color RT-LAMP assay protocol for detecting SARS-CoV-2 viral RNA using a primer set specific for the N gene. We tested our RT-LAMP assay on surplus RNA samples isolated from 768 pharyngeal swab specimens collected from individuals being tested for COVID-19. We determined the sensitivity and specificity of the RT-LAMP assay for detecting SARS-CoV-2 viral RNA. Compared to an RT-qPCR assay using a sensitive primer set, we found that the RT-LAMP assay reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a sensitivity of 97.5% and a specificity of 99.7%. We also developed a swab-to-RT-LAMP assay that did not require a prior RNA isolation step, which retained excellent specificity (99.5%) but showed lower sensitivity (86% for CT < 30) than the RT-LAMP assay. In addition, we developed a multiplexed sequencing protocol (LAMP-sequencing) as a diagnostic validation procedure to detect and record the outcome of RT-LAMP reactions.

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A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples.

An outbreak of betacoronavirus severe acute respiratory syndrome (SARS)-CoV-2 began in Wuhan, China in December 2019. COVID-19, the disease associated with SARS-CoV-2 infection, rapidly spread to produce a global pandemic. We report development of a rapid (<40 min), easy-to-implement and accurate CRISPR–Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA extracts. We validated our method using contrived reference samples and clinical samples from patients in the United States, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. Our CRISPR-based DETECTR assay provides a visual and faster alternative to the US Centers for Disease Control and Prevention SARS-CoV-2 real-time RT–PCR assay, with 95% positive predictive agreement and 100% negative predictive agreement. SARS-CoV-2 in patient samples is detected in under an hour using a CRISPR-based lateral flow assay.

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CRISPR-Cas12-based detection of SARS-CoV-2.

Described herein are devices, systems, fluidic devices, kits, and methods for detection of target nucleic acids associated with diseases, cancers, genetic disorders, a genotype, a phenotype, or ancestral origin. The devices, systems, fluidic devices, kits, and methods may comprise reagents of a guide nucleic acid targeting a target nucleic acid, a programmable nuclease, and a single stranded detector nucleic acid with a detection moiety. The target nucleic acid of interest may be indicative of a disease, and the disease may be communicable diseases, or of a cancer or genetic disorder. The target nucleic acid of interest may be indicative of a genotype, a phenotype, or ancestral origin.

Programmable nuclease compositions and methods of use thereof

The application of cancer organoids is of great value in individualized therapy as an embodiment of the tumor of a patient, a gastric cancer organoid model was established and its application in individualized drug screening was explored. The primary tumor tissues of 3 patients with gastric cancer who underwent primary surgery at the Fourth Department of General Surgery of the First Hospital of Lanzhou University (Lanzhou, China) between July and August 2021 were selected and digested with mixed enzymes to prepare cell suspensions. Of these, two were cultured by mixing with Matrigel, while the cells from the third patient were placed in a 24-well ultra-low adhesion plate for suspension organoid culture. After intensive organoid growth, they were digested, passaged, cryopreserved and thawed for further analyses. The formation of gastric cancer organoids was observed under an inverted microscope. One case was selected, and organoids were compared with the original tumor tissue via H&E and immunohistochemical staining to evaluate the consistency of the two. Finally, paclitaxel, oxaliplatin and fluorouracil were administered to the organoids to verify the value of screening individualized drugs. It was indicated that the passage and cryopreservation of gastric cancer organoids were successfully established in all three cases. The H&E and immunohistochemical staining results suggested that the structure and protein expression of the organoids were highly similar to those of the source tumor tissue. The use of established gastric cancer organoids for individualized chemotherapy drug screening is of high clinical value. Gastric cancer organoids with high similarity to the original tissue may be successfully constructed by the suspension growth culture method. The established organoids may serve as an effective model for individualized drug screening.

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Establishment of gastric cancer organoid and its application in individualized therapy

Described herein are devices, systems, fluidic devices, kits, and methods for detection of target nucleic acids.

Xin Miao

Papers

CRISPR-Cas12-based detection of SARS-CoV-2.

Programmable nuclease compositions and methods of use thereof

Establishment of gastric cancer organoid and its application in individualized therapy

Assays and methods for detection of nucleic acids