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1.
Sensors (Basel) ; 21(21)2021 Oct 20.
Article in English | MEDLINE | ID: covidwho-1512554

ABSTRACT

The lack of portability and high cost of multiplex real-time PCR systems limits the device to be used in POC. To overcome this issue, this paper proposes a compact and cost-effective fluorescence detection system that can be integrated to a multiplex real-time PCR equipment. An open platform camera with embedded lens was used instead of photodiodes or an industrial camera. A compact filter wheel using a sliding tape is integrated, and the excitation LEDs are fixed at a 45° angle near the PCR chip, eliminating the need of additional filter wheels. The results show precise positioning of the filter wheel with an error less than 20 µm. Fluorescence detection results using a reference dye and standard DNA amplification showed comparable performance to that of the photodiode system.


Subject(s)
Nucleic Acid Amplification Techniques , Cost-Benefit Analysis , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction
2.
Sci Rep ; 11(1): 21872, 2021 11 08.
Article in English | MEDLINE | ID: covidwho-1506466

ABSTRACT

Severe acute respiratory syndrome (SARS) is a highly contagious viral respiratory illness. This illness is spurred on by a coronavirus known as SARS-associated coronavirus (SARS-CoV). SARS was first detected in Asia in late February 2003. The genome of this virus is very similar to the SARS-CoV-2. Therefore, the study of SARS-CoV disease and the identification of effective drugs to treat this disease can be new clues for the treatment of SARS-Cov-2. This study aimed to discover novel potential drugs for SARS-CoV disease in order to treating SARS-Cov-2 disease based on a novel systems biology approach. To this end, gene co-expression network analysis was applied. First, the gene co-expression network was reconstructed for 1441 genes, and then two gene modules were discovered as significant modules. Next, a list of miRNAs and transcription factors that target gene co-expression modules' genes were gathered from the valid databases, and two sub-networks formed of transcription factors and miRNAs were established. Afterward, the list of the drugs targeting obtained sub-networks' genes was retrieved from the DGIDb database, and two drug-gene and drug-TF interaction networks were reconstructed. Finally, after conducting different network analyses, we proposed five drugs, including FLUOROURACIL, CISPLATIN, SIROLIMUS, CYCLOPHOSPHAMIDE, and METHYLDOPA, as candidate drugs for SARS-CoV-2 coronavirus treatment. Moreover, ten miRNAs including miR-193b, miR-192, miR-215, miR-34a, miR-16, miR-16, miR-92a, miR-30a, miR-7, and miR-26b were found to be significant miRNAs in treating SARS-CoV-2 coronavirus.


Subject(s)
COVID-19/drug therapy , COVID-19/immunology , COVID-19/virology , Drug Repositioning , Gene Expression Profiling , Gene Expression Regulation, Viral , SARS-CoV-2 , Computational Biology , Gene Regulatory Networks , Genes, Viral , Genetic Techniques , Humans , MicroRNAs/metabolism , Oligonucleotide Array Sequence Analysis , Systems Biology , Transcription Factors
3.
Eur Rev Med Pharmacol Sci ; 25(20): 6411-6424, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-1503073

ABSTRACT

OBJECTIVE: The heterogeneity of clinical manifestations and mortality rates in Coronavirus disease 2019 (COVID-19) patients may be related to the existence of molecular subtypes in COVID-19. To improve current management, it is essential to find the hub genes and pathways associated with different COVID-19 subtypes. MATERIALS AND METHODS: The whole-genome sequencing information (GSE156063, GSE163151) of nasopharyngeal swabs from normal subjects and COVID-19 patients were downloaded from the Gene Expression Omnibus (GEO) database. The molecular subtypes of patients with COVID-19 were classified using the "consistent clustering" method, and the specific genes associated with each subtype were found. Differentially expressed genes (DEGs) were screened between normal subjects and COVID-19 patients; the Weighted gene co-expression network analysis (WGCNA) method was used to find the key module genes of COVID-19 patients. Subtype-specific, differentially expressed and module-related genes were collected and intersected. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were carried out and protein-protein interaction (PPI) networks were generated. The pathways enriched in COVID-19 subtypes were analyzed by gene set variation analysis (GSVA). RESULTS: Patients with COVID-19 were divided into three subtypes, and there was no significant difference in gender and age distribution between subtypes. 82 differential gene pathways were screened between Subtypes I and II, 131 differential gene pathways were screened between Subtypes I and III, and 107 differential gene pathways were screened between Subtypes II and III. Finally, 44 differentially expressed key genes were screened, including 11 hub genes (RSAD2, IFIT1, MX1, OAS1, OAS2, BST2, IFI27, IFI35, IFI6, IFITM3, STAT2). CONCLUSIONS: There are significant differences in gene activation and pathway enrichment among different molecular subtypes of COVID-19, which may account for the heterogeneity in clinical presentation and the prognosis of patients.


Subject(s)
Biomarkers, Tumor/genetics , COVID-19/genetics , Oligonucleotide Array Sequence Analysis , COVID-19/diagnosis , Genetic Variation/genetics , Humans
4.
Talanta ; 239: 122974, 2022 Mar 01.
Article in English | MEDLINE | ID: covidwho-1466916

ABSTRACT

Direct RNA detection is critical for providing the RNA insights into gene expression profiling, noncoding RNAs, RNA-associated diseases and pathogens, without reverse transcription. However, classical RNA analysis usually requires RT-PCR, which can cause bias amplification and quantitation errors. To address this challenge, herein we report a microfluidic RNA chip (the microchip prototype) for direct RNA detection, which is primarily based on RNA extension and labeling with DNA polymerase. This detection strategy is of high specificity (discriminating against single-nucleotide differences), rapidity, accuracy, nuclease resistance, and reusability. Further, we have successfully detected disease-associated RNAs in clinical samples, demonstrating its great potentials in biomedical research and clinical diagnosis.


Subject(s)
Microfluidic Analytical Techniques , RNA , Microfluidics , Nucleotides , Oligonucleotide Array Sequence Analysis , RNA/genetics
5.
Lab Chip ; 21(19): 3742-3747, 2021 09 28.
Article in English | MEDLINE | ID: covidwho-1351985

ABSTRACT

Droplet digital polymerase chain reaction (ddPCR) is an effective technique, with unparalleled sensitivity, for the absolute quantification of target nucleic acids. However, current commercial ddPCR devices for detecting the gene chip are time consuming due to complex image stitching. To address this issue, we propose a universal concentration determination system and realize one-time gene chip imaging with high resolution. All the functional units are controlled by self-developed software using the PyQt5 module in Python. Without stitching technology, images of the ddPCR chip (28 mm × 18 mm) containing 20 000 independent 0.81 nL micro chambers can be obtained in less than 15 seconds, which saves about 165 seconds. A white laser light source (2 mW cm-2) was employed as a substitute for the mercury lamp. Its wavelength matches well with typical fluorescent dyes (e.g., HEX, ROX and Cy5), and thus it can strengthen the fluorescence intensity for weak signals. The results also demonstrated that the correlation coefficient for the measured concentration and theoretical value was above 99%, by testing the ddPCR products with COVID-19 virus. Such a system can greatly reduce the time required for image acquisition and DNA concentration determination, and thus is able to speed up the lab-to-application process for ddPCR technology.


Subject(s)
COVID-19 , Nucleic Acids , Humans , Microfluidics , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , SARS-CoV-2
6.
Front Immunol ; 11: 575074, 2020.
Article in English | MEDLINE | ID: covidwho-1256374

ABSTRACT

Combined cellular and humoral host immune response determine the clinical course of a viral infection and effectiveness of vaccination, but currently the cellular immune response cannot be measured on simple blood samples. As functional activity of immune cells is determined by coordinated activity of signaling pathways, we developed mRNA-based JAK-STAT signaling pathway activity assays to quantitatively measure the cellular immune response on Affymetrix expression microarray data of various types of blood samples from virally infected patients (influenza, RSV, dengue, yellow fever, rotavirus) or vaccinated individuals, and to determine vaccine immunogenicity. JAK-STAT1/2 pathway activity was increased in blood samples of patients with viral, but not bacterial, infection and was higher in influenza compared to RSV-infected patients, reflecting known differences in immunogenicity. High JAK-STAT3 pathway activity was associated with more severe RSV infection. In contrast to inactivated influenza virus vaccine, live yellow fever vaccine did induce JAK-STAT1/2 pathway activity in blood samples, indicating superior immunogenicity. Normal (healthy) JAK-STAT1/2 pathway activity was established, enabling assay interpretation without the need for a reference sample. The JAK-STAT pathway assays enable measurement of cellular immune response for prognosis, therapy stratification, vaccine development, and clinical testing.


Subject(s)
Dengue Virus/immunology , Immunity, Cellular , Orthomyxoviridae/immunology , Respiratory Syncytial Virus, Human/immunology , Rotavirus/immunology , Viral Vaccines/therapeutic use , Virus Diseases/immunology , Yellow fever virus/immunology , Biomarkers/blood , Dengue/blood , Dengue/immunology , Dengue/prevention & control , Dengue/virology , Dengue Vaccines/therapeutic use , Dengue Virus/pathogenicity , Diagnosis, Differential , Host-Pathogen Interactions , Humans , Immunogenicity, Vaccine , Influenza Vaccines/therapeutic use , Influenza, Human/blood , Influenza, Human/immunology , Influenza, Human/prevention & control , Influenza, Human/virology , Oligonucleotide Array Sequence Analysis , Orthomyxoviridae/pathogenicity , Predictive Value of Tests , RNA, Messenger/blood , RNA, Messenger/genetics , Respiratory Syncytial Virus Infections/blood , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus Infections/prevention & control , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Human/pathogenicity , Rotavirus/pathogenicity , Rotavirus Infections/blood , Rotavirus Infections/immunology , Rotavirus Infections/prevention & control , Rotavirus Infections/virology , Rotavirus Vaccines , Signal Transduction/genetics , Virus Diseases/blood , Virus Diseases/prevention & control , Virus Diseases/virology , Yellow Fever/blood , Yellow Fever/immunology , Yellow Fever/prevention & control , Yellow Fever/virology , Yellow Fever Vaccine/therapeutic use , Yellow fever virus/pathogenicity
7.
Sci Rep ; 11(1): 7991, 2021 04 12.
Article in English | MEDLINE | ID: covidwho-1180274

ABSTRACT

To conquer the worldwide outbreak of COVID-19 virus, a large number of studies have been carried out on COVID-19 infection, transmission and treatment. However, few studies have been conducted from the perspectives of circRNA and lncRNA, which are known to be involved in regulating many life activities, such as immune tolerance and immune escapes, and hence may provide invaluable information in the emerging COVID-19 infection and recurrence. Moreover, exosomes has been reported to play an important role in COVID-19 recurrence, and thus may interact with the expression of circRNA and lncRNA. In this work, we sequenced circRNA, lncRNA and mRNA from recurrent COVID-19 patients and healthy people, and compared the differences. GO and KEGG enrichment analysis show that differentially expressed circRNA and lncRNA are mainly involved in the regulation of host cell cycle, apoptosis, immune inflammation, signaling pathway and other processes. The comparison to exosomes related databases shows that there are 114 differentially expressed circRNA, and 10 differentially expressed lncRNA related to exosomes. These studies provide reference for exploring circRNA and lncRNA to study the infection mechanism of COVID-19, their diagnostic and therapeutic values, as well as the possibility to employ them as biomarkers.


Subject(s)
COVID-19/blood , COVID-19/virology , RNA, Circular/blood , RNA, Long Noncoding/blood , Apoptosis , Biomarkers , COVID-19 Nucleic Acid Testing , Computational Biology , Exosomes/metabolism , Gene Expression Profiling , Gene Expression Regulation , Humans , Models, Statistical , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Recurrence , Signal Transduction
8.
Langmuir ; 37(16): 4763-4771, 2021 04 27.
Article in English | MEDLINE | ID: covidwho-1180210

ABSTRACT

SARS-CoV-2 has infected over 128 million people worldwide, and until a vaccine is developed and widely disseminated, vigilant testing and contact tracing are the most effective ways to slow the spread of COVID-19. Typical clinical testing only confirms the presence or absence of the virus, but rather, a simple and rapid testing procedure that sequences the entire genome would be impactful and allow for tracing the spread of the virus and variants, as well as the appearance of new variants. However, traditional short read sequencing methods are time consuming and expensive. Herein, we describe a tiled genome array that we developed for rapid and inexpensive full viral genome resequencing, and we have applied our SARS-CoV-2-specific genome tiling array to rapidly and accurately resequence the viral genome from eight clinical samples. We have resequenced eight samples acquired from patients in Wyoming that tested positive for SARS-CoV-2. We were ultimately able to sequence over 95% of the genome of each sample with greater than 99.9% average accuracy.


Subject(s)
COVID-19 , SARS-CoV-2 , Genome, Viral , Humans , Oligonucleotide Array Sequence Analysis
9.
Zhonghua Yi Xue Za Zhi ; 101(8): 573-578, 2021 Mar 02.
Article in Chinese | MEDLINE | ID: covidwho-1119574

ABSTRACT

Objective: To explore the difference in the expression profile of circular RNA in peripheral blood mononuclear cells between patients with mild and severe influenza pneumonia. Methods: From December 2018 to March 2019, 10 inpatients with mild and 10 inpatients with severe influenza pneumonia admitted to the Department of Infection and Clinical Microbiology of Beijing Chaoyang Hospital were included. Clariom™ D gene chip was used to explore the circRNA expression profiles of peripheral blood mononuclear cells (PBMC) isolated from the patients. The absolute value of the fold change (FC value)>2 and P<0.05 were used as the criteria to screen the differentially expressed circRNA, and the gene ontology (GO) enrichment analysis and the Kyoto Encyclopedia of Gene and Genome database (Kyoto Encyclopedia of Genes and Genomes, KEGG) signal pathway enrichment analysis were also performed. Results: The age of mild patients [M (P25, P75)] was 62.0 (34.5, 69.8) years old, including 4 males; the age of severe patients [M (P25, P75)] was 50.0 (37.0, 60.0) years old, all were males. A total of 137 differentially expressed circRNAs in PBMCs of mild and severe patients were screened. The numbers of up-regulated and down-regulated circRNAs in mild patients were 101 and 36, respectively. Among them, hsa_circ_0091073 (FC value=160.898, P<0.05) was the most significantly up-regulated circRNA and hsa_circ_0092219 (FC value =-17.630, P<0.05) was the most significantly down-regulated circRNA. GO enrichment analysis showed that a total of 111 secondary GO items were significantly associated with related differential expression of circRNA (P<0.05). The GO terms associated with upregulated circRNAs included DNA-templated transcription, regulation of DNA-templated transcription, regulation of transcription from RNA polymerase Ⅱ promoter, etc.; The GO terms associated with downregulated circRNAs included neutrophil degranulation, killing of cells of other organism, defense response to fungus, etc. KEGG signaling pathway analysis showed that there were 37 metabolic pathways related to differentially expressed circRNAs (P<0.05). Signaling pathways related to up-regulated circRNAs included nuclear factor-κB (NF-κB) signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, tumor necrosis factor (TNF) signaling pathway, etc. Signaling pathways related to down-regulation of circRNAs included cancer transcription disorders, folate carbon pool, and other types of O-glycan biosynthesis. Conclusion: The expression of circRNA in PBMC of mild and severe influenza pneumonia patients is significantly different, and it may play a role in the pathogenic mechanism of influenza pneumonia through multiple signal pathways.


Subject(s)
Influenza, Human , Pneumonia , Aged , Humans , Influenza, Human/genetics , Leukocytes, Mononuclear , Male , Middle Aged , Oligonucleotide Array Sequence Analysis , RNA, Circular
10.
Anal Biochem ; 626: 114124, 2021 08 01.
Article in English | MEDLINE | ID: covidwho-1085603

ABSTRACT

We report proof-of-principle experiments regarding a dynamic microarray protocol enabling accurate and semi-quantitative DNA analysis for re-sequencing, fingerprinting and genotyping. Single-stranded target molecules hybridise to surface-bound probes during initial gradual cooling with high-fidelity. Real-time tracking of target denaturation (via fluorescence) during a 'dynamic' gradual heating phase permits 'melt-curve' analysis. The probe most closely matching the target sequence is identified based on the highest melting temperature. We demonstrated a >99% re-sequencing accuracy and a potential detection rate of 1% for SNPs. Experiments employing Hypericum ribosomal ITS regions and HIV genomes illustrated a reliable detection level of 5% plus simultaneous re-sequencing and genotyping. Such performance suggests a range of potential real-world applications involving rapid sequence interrogation, for example, in the Covid-19 pandemic. Guidance is offered towards the development of a commercial platform and dedicated software required to bring this technique into mainstream science.


Subject(s)
COVID-19/genetics , Genome, Plant , Genome, Viral , Genotyping Techniques , HIV-1/genetics , Hypericum/genetics , Oligonucleotide Array Sequence Analysis , Software , COVID-19/epidemiology , Humans
11.
Nat Commun ; 11(1): 6176, 2020 12 02.
Article in English | MEDLINE | ID: covidwho-957574

ABSTRACT

Rapid screening and low-cost diagnosis play a crucial role in choosing the correct course of intervention when dealing with highly infectious pathogens. This is especially important if the disease-causing agent has no effective treatment, such as the novel coronavirus SARS-CoV-2, and shows no or similar symptoms to other common infections. Here, we report a disposable silicon-based integrated Point-of-Need transducer (TriSilix) for real-time quantitative detection of pathogen-specific sequences of nucleic acids. TriSilix can be produced at wafer-scale in a standard laboratory (37 chips of 10 × 10 × 0.65 mm in size can be produced in 7 h, costing ~0.35 USD per device). We are able to quantitatively detect a 563 bp fragment of genomic DNA of Mycobacterium avium subspecies paratuberculosis through real-time PCR with a limit-of-detection of 20 fg, equivalent to a single bacterium, at the 35th cycle. Using TriSilix, we also detect the cDNA from SARS-CoV-2 (1 pg) with high specificity against SARS-CoV (2003).


Subject(s)
COVID-19/diagnosis , Nucleic Acid Amplification Techniques/methods , Animals , DNA, Bacterial/genetics , Humans , Mycobacterium avium subsp. paratuberculosis/genetics , Oligonucleotide Array Sequence Analysis/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity , Silicon
12.
Euro Surveill ; 25(9)2020 03.
Article in English | MEDLINE | ID: covidwho-4532

ABSTRACT

The need for timely establishment of diagnostic assays arose when Germany was confronted with the first travel-associated outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Europe. We describe our laboratory experiences during a large contact tracing investigation, comparing previously published real-time RT-PCR assays in different PCR systems and a commercial kit. We found that assay performance using the same primers and probes with different PCR systems varied and the commercial kit performed well.


Subject(s)
Betacoronavirus , Clinical Laboratory Techniques , Coronavirus Infections , Pneumonia, Viral , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , COVID-19 , COVID-19 Testing , COVID-19 Vaccines , Coronavirus Infections/diagnosis , Coronavirus Infections/genetics , Germany , Humans , Oligonucleotide Array Sequence Analysis , Pneumonia, Viral/diagnosis , Pneumonia, Viral/genetics , SARS-CoV-2 , Sensitivity and Specificity , Time Factors , Viral Envelope Proteins/analysis , Viral Envelope Proteins/genetics , Workflow
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