On-Chip Nucleic Acid Purification Followed by ddPCR for SARS-CoV-2 Detection.

We developed a microfluidic chip integrated with nucleic acid purification and droplet-based digital polymerase chain reaction (ddPCR) modules to realize a 'sample-in, result-out' infectious virus diagnosis. The whole process involved pulling magnetic beads through drops in an oil-enclosed environment. The purified nucleic acids were dispensed into microdroplets by a concentric-ring, oil-water-mixing, flow-focusing droplets generator driven under negative pressure conditions. Microdroplets were generated with good uniformity (CV = 5.8%), adjustable diameters (50-200 µm), and controllable flow rates (0-0.3 µL/s). Further verification was provided by quantitative detection of plasmids. We observed a linear correlation of R2 = 0.9998 in the concentration range from 10 to 105 copies/µL. Finally, this chip was applied to quantify the nucleic acid concentrations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The measured nucleic acid recovery rate of 75 ± 8.8% and detection limit of 10 copies/µL proved its on-chip purification and accurate detection abilities. This chip can potentially be a valuable tool in point-of-care testing.

Recent Progress in Development and Application of DNA, Protein, Peptide, Glycan, Antibody, and Aptamer Microarrays.

Microarrays are one of the trailblazing technologies of the last two decades and have displayed their importance in all the associated fields of biology. They are widely explored to screen, identify, and gain insights on the characteristics traits of biomolecules (individually or in complex solutions). A wide variety of biomolecule-based microarrays (DNA microarrays, protein microarrays, glycan microarrays, antibody microarrays, peptide microarrays, and aptamer microarrays) are either commercially available or fabricated in-house by researchers to explore diverse substrates, surface coating, immobilization techniques, and detection strategies. The aim of this review is to explore the development of biomolecule-based microarray applications since 2018 onwards. Here, we have covered a different array of printing strategies, substrate surface modification, biomolecule immobilization strategies, detection techniques, and biomolecule-based microarray applications. The period of 2018-2022 focused on using biomolecule-based microarrays for the identification of biomarkers, detection of viruses, differentiation of multiple pathogens, etc. A few potential future applications of microarrays could be for personalized medicine, vaccine candidate screening, toxin screening, pathogen identification, and posttranslational modifications.

Identification of Potential Key Genes and Prognostic Biomarkers of Lung Cancer Based on Bioinformatics.

Objective: To analyze and identify the core genes related to the expression and prognosis of lung cancer including lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) by bioinformatics technology, with the aim of providing a reference for clinical treatment. Methods: Five sets of gene chips, GSE7670, GSE151102, GSE33532, GSE43458, and GSE19804, were obtained from the Gene Expression Omnibus (GEO) database. After using GEO2R to analyze the differentially expressed genes (DEGs) between lung cancer and normal tissues online, the common DEGs of the five sets of chips were obtained using a Venn online tool and imported into the Database for Annotation, Visualization, and Integrated Discovery (DAVID) database for Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The protein-protein interaction (PPI) network was constructed by STRING online software for further study, and the core genes were determined by Cytoscape software and KEGG pathway enrichment analysis. The clustering heat map was drawn by Excel software to verify its accuracy. In addition, we used the University of Alabama at Birmingham Cancer (UALCAN) website to analyze the expression of core genes in P53 mutation status, confirmed the expression of crucial core genes in lung cancer tissues with Gene Expression Profiling Interactive Analysis (GEPIA) and GEPIA2 online software, and evaluated their prognostic value in lung cancer patients with the Kaplan-Meier online plotter tool. Results: CHEK1, CCNB1, CCNB2, and CDK1 were selected. The expression levels of these four genes in lung cancer tissues were significantly higher than those in normal tissues. Their increased expression was negatively correlated with lung cancer patients (including LUAD and LUSC) prognosis and survival rate. Conclusion: CHEK1, CCNB1, CCNB2, and CDK1 are the critical core genes of lung cancer and are highly expressed in lung cancer. They are negatively correlated with the prognosis of lung cancer patients (including LUAD and LUSC) and closely related to the formation and prediction of lung cancer. They are valuable predictors and may be predictive biomarkers of lung cancer.

A Miniaturized Silicon Lab-on-Chip for Integrated PCR and Hybridization Microarray for High Multiplexing Nucleic Acids Analysis.

A silicon lab-on-chip, for the detection of nucleic acids through the integrated PCR and hybridization microarray, was developed. The silicon lab-on-chip manufactured through bio-MEMS technology is composed of two PCR microreactors (each volume 11.2 µL) and a microarray-hybridization microchamber (volume 30 µL), fluidically connected by buried bypass. It contains heaters and temperature sensors for the management and control of the temperature cycles during the PCR amplification and hybridization processes. A post-silicon process based on (i) plasmo-O2 cleaning/activation, (ii) vapor phase epoxy silanization, (iii) microarray fabrication and (iv) a protein-based passivation step was developed and fully characterized. The ssDNA microarray (4 rows × 10 columns) composed of 400 spots (spot size-70 ± 12 µm; spot-to-spot distance-130 ± 13 µm) was manufactured by piezo-dispense technology. A DNA microarray probe density in the range of 1310 to 2070 probe µm-2 was observed, together with a limit of detection of about 19 target µm-2. The performances of the silicon lab-on-chip were validated by the detection of the beta-globin gene directly from human blood. Remarkable sensitivity, multiplexing analysis and specificity were demonstrated for the detection of beta-globin and mycobacterium tuberculosis sequences.

Cost-Effective Multiplex Fluorescence Detection System for PCR Chip.

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.

Drug repurposing for coronavirus (SARS-CoV-2) based on gene co-expression network analysis.

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.

Identification of hub genes and molecular subtypes in COVID-19 based on WGCNA.

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.

A novel microfluidic RNA chip for direct, single-nucleotide specific, rapid and partially-degraded RNA detection.

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.

A rapid nucleic acid concentration measurement system with large field of view for a droplet digital PCR microfluidic chip.

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.

Measurement of Cellular Immune Response to Viral Infection and Vaccination.

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.

A study of differential circRNA and lncRNA expressions in COVID-19-infected peripheral blood.

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.

Highly Accurate Chip-Based Resequencing of SARS-CoV-2 Clinical Samples.

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.

[Differential circRNA expression profiles in peripheral blood mononuclear cells among mild and severe influenza-associated pneumonia patients].

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.

Array-based dynamic allele specific hybridization (Array-DASH): Optimization-free microarray processing for multiple simultaneous genomic assays.

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.

Disposable silicon-based all-in-one micro-qPCR for rapid on-site detection of pathogens.

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).

Rapid establishment of laboratory diagnostics for the novel coronavirus SARS-CoV-2 in Bavaria, Germany, February 2020.

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.