DNA Hybridization Research Articles

Hereditary Colorectal Cancer Diagnosis by Next-Generation Sequencing.

Pathogenic germline variants causally contribute to the etiology of colorectal cancer (CRC) and polyposis. The era of massively parallel sequencing, also known as next-generation sequencing (NGS), make it highly possible, effective, and efficient to offer rapid and cost-effective diagnosis for CRC. To aid clinical laboratories in testing the most clinically significant genes, along with the published ACMG CRC technical standard guidelines, this protocol aims to provide a step-by-step technical workflow for carrying out the NGS-panel based CRC molecular diagnosis focusing on the wet lab portion of library preparation and massively parallel sequencing. Using the most popular pull-down-based target enrichment, the chapter particularly encompasses genomic DNA (gDNA) fragmentation, adapter ligation, indexing, hybridization, and capture, which is the most variable and technically challenging part of NGS testing involving at least 3 quality control (QC) checkpoints plus the pre- and post-capture PCR. The gDNA extraction and sequencing is less covered because they are relatively standard technologies with little variations and choices. Although this protocol also introduces pertinent testing algorithms and a brief guideline for pre- and post-testing genetic counselling, the audiences are required to refer to National Comprehensive Cancer Network (NCCN) clinical practice guidelines to determine the most appropriate testing strategies. Since NGS panel-based testing is a highly complex and dynamic platform with multiple choices from different technology and commercial resources, this technical benchtop-based protocol also aims to cover some of the key ramification points for decision-making by each laboratory at the discretion of the directors. © 2023 Wiley Periodicals LLC. Basic Protocol: Hereditary colorectal cancer (CRC) diagnosis by next-generation sequencing.

Plasmonic Biosensor for DNA Hybridization Using Integrated Graphene-Porous Silicon Waveguide

Plasmonic Biosensor for DNA Hybridization Using Integrated Graphene-Porous Silicon Waveguide

Genome Wide siRNA Screen Identifies CGAS-Sting Pathway As a Pharmacological Target That Promotes Survival of Hematopoietic Stem Cells Deficient in Fanconi Genes

Introduction Fanconi anemia (FA) is a homozygous recessive genetic disorder due to mutation in the Fanconi family of genes. The disease is characterized by early onset bone marrow failure and a predisposition to cancer. The FA genes are involved in DNA damage sensing and repair. Mutation in these genes leads to heightened DNA damage and extreme sensitivity to cross-linking agents. Hematopoietic stem cells of FA patients are defective in differentiation and self-renewal, leading to stem cell exhaustion and bone marrow failure early in life. The current treatment for bone marrow failure is allogeneic stem cell transplant from healthy donors. Due to difficulties in finding matching donors and the inherent problems associated with allogeneic transplants, pharmacological therapies that delay or prevent bone marrow failure are of clinical importance. Other proposed therapies including TGF-b, metformin, and resveratrol have met with limited clinical success. Methodology: To identify genes that promote survival of FA cells we performed a genome wide siRNA screen using isogenic pair of cell line with FANCD2 deletions and its corrected counterpart using transfection of siRNA libraries targeting 18,119 genes from Human Drug Targets, Human Druggable Genome, and Human Genome libraries at 2000 cells/well in 384-well plates followed by cell viability measurement after 72 hrs. We measured IRF3 phosphorylation and expression of immune related genes to analyze the CGAS-STING pathway. Using peripheral blood and bone marrow derived CD34+ cells transduced with lentivirus expressing shRNA against FANCD2 we tested the effect of CGAS inhibitor in promoting CFU-GM and CFU-E. Results: A genome wide siRNA screen identified IRF7 as one of the top 5 hits whose diminished expression resulted in increased viability of F-D2 mutant PD20 cells. IRF7 along with IRF3 serve as downstream transcription factors including various Toll-like receptors, CGAS-STING pathways, and other pattern recognition innate immune receptors. Using pIRF3 as an activation readout for CGAS-STING pathway we identified that both FANCD2 and FANCA mutant cells have elevated pIRF3 levels. The elevated pIRF3 levels were further increased upon addition of mitomycin C (MMC) in FA mutant cells as compared to gene corrected cells. The pIRF3 levels decreased upon both genetic KD and small molecule inhibition of both CGAS and STING. In line with increased pIRF3 FA mutant cells showed increased gene expression of various interferon-regulated genes, which were then also reduced by CGAS inhibitor. Overexpression of RNAseH1 partially reduced pIRF3 levels in FA mutant cells indicating that the RNA:DNA hybrids whose levels are elevated in FA mutant cells is one of the ligands of CGAS. Most importantly 10µM of CGAS inhibitor RU.51 was able to rescue FA-A mutant cell line 6914 cells from low dose MMC-induced cell death showing that the sensitivity of FA mutant cells to DNA damaging agents was at least partially driven by CGAS-STING pathway. We then tested if the protective effect of CGAS inhibitor extended to human CD34+ cells deficient in FA genes. 20µM of RU.51 was able to rescue the CFU-GM and CFU-E defects of CD34+ cells carrying lentiviral integrated shRNA against FANCD2 to a level to that in cells expressing non-silencing shRNAs. Interferon-regulated gene expression was also elevated in the FANCD2-deficient CD34+ cells, which was reversed by treatment with cGAS inhibitor. Conclusions: It has previously been shown that FA patients have elevated TNF-alpha, IFN-gamma levels, and NFkbactivation, and interferon alpha administration hastens the onset of bone marrow failure in FA patients. Our results show that FA mutant cells have elevated CGAS-STING signaling resulting in heightened interferon gene expression. Elevated R-loop levels in FA cells act as trigger for the CGAS-STING pathway. CGAS inhibitor is able to rescue low dose MMC-induced cell death in FA mutant fibroblasts and also restores the CFU-GM and CFU-E forming ability of CD34+ cells. These results indicate that CGAS-STING pathway is a pharmacologically tractable target to reverse the hematopoietic stem cell exhaustion seen in FA patients.

Assessment of A- to B- DNA Transitions Utilizing the Drude Polarizable Force Field.

In addition to the well-characterized B-form of DNA, duplex DNA can adopt various conformations, such as A or Z-DNA. Though less common, these structures can be induced biologically through protein or ligand interactions or experimentally with niche environmental conditions, such as high salt concentrations or in mixed water-ethanol. Reproducing these alternate structures through molecular dynamics simulations in recent years has been quite challenging with the currently available force fields, simulation techniques, and time scales. In this study, the Drude polarizable force field is tested for its ability to facilitate transitions between A-DNA and B-DNA or maintain A-DNA. Though transitions away from B-DNA were observed in high concentrations of ethanol, the resulting structures had hybrid properties taken from both B-DNA and A-DNA structures. This was also true for A-DNA in ethanol, which lost some of the A-DNA properties that it was expected to maintain. When B-DNA was tested in high salt environments, the resulting B-DNA structures showed no distinguishable differences with the increasing salt concentrations tested. These results with the Drude FF and recent results with additive force fields suggest that at present the current additive and polarizable force fields do not facilitate a complete transition between B- to A-DNA conformations under the conditions simulated. At present, the Drude FF favors A-B DNA hybrid structures when simulated in nonphysiological conditions.

DNA-Based Nanomaterials for Analysis of Extracellular Vesicles.

Extracellular vesicles (EVs) are cell-derived nanovesicles comprising a myriad of molecular cargo such as proteins and nucleic acids, playing essential roles in intercellular communication and physiological and pathological processes. EVs have received substantial attention as noninvasive biomarkers for disease diagnosis and prognosis. Owing to their ability to recognize protein and nucleic acid targets, DNA-based nanomaterials with excellent programmability and modifiability provide a promising tool for the sensitive and accurate detection of molecular cargo carried by EVs. In this perspective, recent advancements in EV analysis using a variety of DNA-based nanomaterials are summarized, which can be broadly classified into three categories: linear DNA probes, DNA nanostructures, and hybrid DNA nanomaterials. The design, construction, advantages, and disadvantages of different types of DNA nanomaterials, as well as their performance for detecting EVs are reviewed. The challenges and opportunities in the field of EV analysis by DNA nanomaterials are also discussed.

A target-triggered fluorescence-SERS dual-signal nano-system for real-time imaging of intracellular telomerase activity

Telomerase (TE) is a promising diagnostic and prognostic biomarker for many cancers. Quantification of TE activity in living cells is of great significance in biomedical and clinical research. Conventional fluorescence-based sensors for quantification of intracellular TE may suffer from problems of fast photobleaching and auto-fluorescence of some endogenous molecules, and hence are liable to produce false negative or positive results. To address this issue, a fluorescence-SERS dual-signal nano-system for real-time imaging of intracellular TE was designed by functionalizing a bimetallic Au@Ag nanostructure with 4-p-mercaptobenzoic acid (internal standard SERS tag) and a DNA hybrid complex consisted of a telomerase primer strand and its partially complimentary strand modified with Rhodamine 6G. The bimetallic Au@Ag nanostructure serves as an excellent SERS-enhancing and fluorescence-quenching substrate. Intracellular TE will trigger the extension of the primer strand and cause the shedding of Rhodamine 6G-modified complimentary strand from the nano-system through intramolecular DNA strand displacement, resulting in the recovery of the fluorescence of Rhodamine 6G and decrease in its SERS signal. Both the fluorescence of R6G and the ratio between the SERS signals of 4-p-mercaptobenzoic acid and Rhodamine 6G can be used for in situ imaging of intracellular TE. Experimental results showed that the proposed nano-system was featured with low background, excellent cell internalization efficiency, good biocompatibility, high sensitivity, good selectivity, and robustness to false positive results. It can be used to distinguish cancer cells from normal ones, identify different types of cancer cells, as well as perform absolute quantification of intracellular TE, which endows it with great potential in clinical diagnosis, target therapy and prognosis of cancer patients.

Gordonia metallireducens sp. nov., a tellurite- and selenite-resistant bacterium isolated from the sediment of an acid mine drainage stream.

A polyphasic taxonomic study was carried out on strain TSed Te1T, isolated from sediment of a stream contaminated with acid drainage from a coal mine. The bacterium forms pink-pigmented colonies and has a rod-coccus growth cycle, which also includes some coryneform arrangements. This bacterium is capable of growing in the presence of up to 750 μg ml-1 tellurite and 5000 μg ml-1 selenite, reducing each to elemental form. Nearly complete 16S rRNA gene sequence analysis associated the strain with Gordonia, with 99.5 and 99.3 % similarity to Gordonia namibiensis and Gordonia rubripertincta, respectively. Computation of the average nucleotide identity and digital DNA-DNA hybridization comparisons with the closest phylogenetic neighbour of TSed Te1T revealed genetic differences at the species level, which were further substantiated by differences in several physiological characteristics. The dominant fatty acids were C16 : 0, C18 : 1, C16 : 1 and tuberculostearic acid. The DNA G+C content was 67.6 mol%. Major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside, while MK-9(H2) was the only menaquinone found. Mycolic acids of C56-C60 were present. Whole-cell hydrolysates contained meso-diaminopimelic acid along with arabinose and galactose as the major cell-wall sugars. On the basis of the results obtained in this study, the bacterium was assigned to the genus Gordonia and represents a new species with the name Gordonia metallireducens sp. nov. The type strain is TSed Te1T (=NRRL B-65678T=DSM 114093T).

Tessaracoccus caeni sp. nov., a novel member of Propionibacteriaceae isolated from activated sludge in Hefei, PR China.

A floc-forming bacterial strain, designated HF-7T, was isolated from the activated sludge of an industrial wastewater treatment plant in Hefei, PR China. Cells of this strain were Gram-stain-positive, catalase- and oxidase-negative, facultatively anaerobic, and rod-shaped. Growth occurred at 20-42 °C (optimum, 28 °C), at pH 5.5-10.5 (optimum, pH 7.5) and with 0-8.0 % (w/v) NaCl (optimum, 1 %). The major fatty acid was anteiso-C15 : 0. The polar lipid profile contained phosphatidylglycerol, diphosphatidylglycerol and phosphatidylinositol. The DNA G+C content was 67 mol% from whole genomic sequence analysis. Based on the results of 16S rRNA gene sequence analysis, this strain should be assigned to the genus Tessaracoccus and is closely related to Tessaracoccus arenae CAU 1319T (95.87 % similarity), Tessaracoccus lapidicaptus IPBSL-7T (95.19 %) and Tessaracoccus bendigoensis Ben 106T (94.63 %) but separated from them by large distances in different phylogenetic trees. Based on whole genome analysis, the orthologous average nucleotide identity and in silico DNA-DNA hybridization values against two of the closest relatives were 75.21-76.50 % and 14.2-24.4 %, respectively. The phylogenetic, genotypic, phenotypic and chemotaxonomic data demonstrated that strain HF-7T could be distinguished from its phylogenetically related species and represents a novel species within the genus Tessaracoccus, for which the name Tessaracoccus caeni sp. nov. is proposed. The type strain is HF-7T (=KCTC 49959T=CCTCC AB 2023019T).

Mangrovimonas aestuarii sp. nov., isolated from tidal flat sediment.

A Gram-stain-negative, rod-shaped and light yellow-pigmented strain designated MBT5T was isolated from tidal flat sediment of an oyster farming area in Quanzhou Bay, PR China. Catalase activity and oxidase activity were positive. Flexirubin-type pigment was absent. Growth was observed at 10-40 °C (optimum, 35 °C), pH 6-9 (optimum, pH 7), and with 1-7 % NaCl (optimum, 2 %, w/v). The 16S rRNA gene of strain MBT5T had maximum sequence similarity values with Meridianimaribacter flavus NH57NT, Mangrovimonas yunxiaonensis LYYY01T and Mangrovimonas futianensis AS18T of 95.6, 95.4 and 94.9 %, respectively. Phylogenetic analysis based on 16S rRNA gene sequences and 120 conserved concatenated proteins indicated that strain MBT5T was affiliated to the genus Mangrovimonas and formed a distinct monophyletic branch. The digital DNA-DNA hybridization, average nucleotide identity and average amino acid identity values between strain MBT5T and the type strains of Mangrovimonas were estimated to be 17.3-18.7 %, 70.9-71.5 % and 66.4-68.2 %, respectively. The respiratory quinone was menaquinone-6. The major fatty acids were iso-C15 : 0 and iso-C15 : 1 G. The draft genome size was 2 952 053 bp with a DNA G+C content of 36.5 %. Based on phenotypic, physiological, phylogenetic and genomic data, together with chemotaxonomic characteristics, strain MBT5T represents a novel species, for which the name Mangrovimonas aestuarii sp. nov. is proposed. The type strain is MBT5T (=MCCC 1K06186T=KCTC 92888T=GDMCC 1.3851T).

Direct visualization of replication and R-loop collision using single-molecule imaging.

R-loops are three-stranded nucleic acid structures that can cause replication stress by blocking replication fork progression. However, the detailed mechanism underlying the collision of DNA replication forks and R-loops remains elusive. To investigate how R-loops induce replication stress, we use single-molecule fluorescence imaging to directly visualize the collision of replicating Phi29 DNA polymerase (Phi29DNAp), the simplest replication system, and R-loops. We demonstrate that a single R-loop can block replication, and the blockage is more pronounced when an RNA-DNA hybrid is on the non-template strand. We show that this asymmetry results from secondary structure formation on the non-template strand, which impedes the progression of Phi29 DNAp. We also show that G-quadruplex formation on the displaced single-stranded DNA in an R-loop enhances the replication stalling. Moreover, we observe the collision between Phi29 DNAp and RNA transcripts synthesized by T7 RNA polymerase (T7 RNAp). RNA transcripts cause more stalling because of the presence of T7 RNAp. Our work provides insights into how R-loops impede DNA replication at single-molecule resolution.

Characterization of Haloarcula terrestris sp. nov. and reclassification of a Haloarcula species based on a taxogenomic approach.

An extremely halophilic archaeon, strain S1AR25-5AT, was isolated from a hypersaline soil sampled in Odiel Saltmarshes Natural Area (Huelva, Spain). The cells were Gram-stain-negative, motile, pleomorphic rods. Cell growth was observed in the presence of 15-30 % (w/v) NaCl [optimum, 25 % (w/v) NaCl], at pH 6.0-9.0 (optimum, pH 6.5-7.5) and at 25-50 °C (optimum, 37 °C). Based on the 16S rRNA and rpoB' gene sequence comparisons, strain S1AR25-5AT was affiliated to the genus Haloarcula. Taxogenomic analysis, including comparison of the genomes and the phylogenomic tree based on the core-orthologous proteins, together with the genomic indices, i.e., orthologous average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity, confirmed that strain S1AR25-5AT (=CCM 9249T=CECT 30619T) represents a new species of the genus Haloarcula, for which we propose the name Haloarcula terrestris sp. nov. The major polar lipids were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulphate and an unidentified glycolipid, which correlated with the lipid profile of species of the genus Haloarcula. In addition, based on the modern approach in description of species in taxonomy of prokaryotes, the above mentioned genomic indexes indicated that the species Haloarcula tradensis should be considered as a heterotypic synonym of Haloarcula argentinensis.

RAD51-mediated R-loop formation acts to repair transcription-associated DNA breaks driving antigenic variation in Trypanosoma brucei.

RNA-DNA hybrids are epigenetic features of all genomes that intersect with many processes, including transcription, telomere homeostasis, and centromere function. Increasing evidence suggests that RNA-DNA hybrids can provide two conflicting roles in the maintenance and transmission of genomes: They can be the triggers of DNA damage, leading to genome change, or can aid the DNA repair processes needed to respond to DNA lesions. Evasion of host immunity by African trypanosomes, such as Trypanosoma brucei, relies on targeted recombination of silent Variant Surface Glycoprotein (VSG) genes into a specialized telomeric locus that directs transcription of just one VSG from thousands. How such VSG recombination is targeted and initiated is unclear. Here, we show that a key enzyme of T. brucei homologous recombination, RAD51, interacts with RNA-DNA hybrids. In addition, we show that RNA-DNA hybrids display a genome-wide colocalization with DNA breaks and that this relationship is impaired by mutation of RAD51. Finally, we show that RAD51 acts to repair highly abundant, localised DNA breaks at the single transcribed VSG and that mutation of RAD51 alters RNA-DNA hybrid abundance at 70 bp repeats both around the transcribed VSG and across the silent VSG archive. This work reveals a widespread, generalised role for RNA-DNA hybrids in directing RAD51 activity during recombination and uncovers a specialised application of this interplay during targeted DNA break repair needed for the critical T. brucei immune evasion reaction of antigenic variation.

Muricauda myxillae sp. nov., isolated from a marine sponge (Myxilla rosacea), and reclassification of Flagellimonas hymeniacidonis as Muricauda symbiotica nom. nov.

A Gram-stain-negative, aerobic and non-motile rods strain, designated as strain 2012CJ39-3T, was isolated from a marine sponge, Myxilla rosacea, collected on Chuja Island, Republic of Korea. Optimal growth of strain 2012CJ39-3T was observed at 25 °C, pH 7.0 and in the presence of 2.0-3.0 % (w/v) NaCl. Strain 2012CJ39-3T contained menaquinone-6 as the respiratory quinone, iso-C15 : 1 G, iso-C15 : 0, and iso-C17 : 0 3-OH as the predominant fatty acids, and phosphatidylethanolamine, an unidentified phospholipid, an unidentified glycolipid, three unidentified aminolipids and nine unidentified lipids as major polar lipids. The genomic DNA G+C content was 38.4 mol%. Results of phylogenetic analyses based on the 16S rRNA gene and whole-genome sequences revealed that strain 2012CJ39-3T formed a distinct phyletic lineage in the genus Muricauda. Strain 2012CJ39-3T was most closely related to Flagellimonas hymeniacidonis 176CP5-101T, Muricauda spongiicola 2012CJ35-5T, Muricauda algicola AsT0115T, Muricauda flava DSM 22638T and Muricauda parva SW169T with 96.5, 96.4, 96.3, 95.8 and 95.6 % 16S rRNA gene sequence similarity, respectively. The average nucleotide identity and digital DNA-DNA hybridization values between strain 2012CJ39-3T and M. spongiicola 2012CJ35-5T, F. hymeniacidonis 176CP5-101T, M. algicola AsT0115T, M. flava DSM 22638T and M. parva SW169T were 75.6, 74.2, 78.6, 75.3 and 74.8 % and 27.4, 19.9, 36.3, 24.2 and 18.9 %, respectively. Based on these results, strain 2012CJ39-3T represents a novel species of the genus Muricauda, for which the name Muricauda myxillae sp. nov. is proposed. The type strain is 2012CJ39-3T (=KACC 22644T= LMG 32582T). In addition, Flagellimonas hymeniacidonis is reclassified as Muricauda symbiotica nom. nov.

Marinihelvus fidelis gen. nov., sp. nov., isolated from marine sediment, and genomic comparison with multiple genera.

A Gram-stain-negative, rod-shaped, non-gliding, non-flagellated, yellow, facultatively aerobic bacterial strain, designated as W260T, was isolated from marine sediment of Xiaoshi Island, Weihai, PR China. The cells of W260T were 0.3-0.5 µm wide and 1.5-2.0 µm long. Strain W260T grows optimally at a temperature of 33 °C (range, 15-37 °C), pH 8 (range, pH 6.5-9.5) and witha NaCl concentration of 3.0 % (w/v; range, 1-8 %). It has the highest sequence similarity to Thiohalobacter thiocyanaticus DSM 21152T (91.7 %), followed by Wenzhouxiangella marina MCCC 1K00261T (91.4 %) and Thiohalospira alkaliphila DSM 17116T (90.7 %). The similarity between strain W260T and the species Thiohalophilus thiocyanatoxydans DSM 16326T was 89.4 %. Genome sequencing revealed a genome size of 3 430 000 bp and a DNA G+C content of 64.5 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain W260T and W. marina MCCC 1K00261T were 69.6 and 16.1-20.6 %, respectively. The predominant quinone was ubiquitin-8, and the major fatty acids were iso-C14 : 0 and iso-C16 : 0. The polar lipids consisted of phosphatidylethanolamine, phospholipid, phosphatidylglycerol, diphosphatidylglycerol and four unidentified lipids. Based on phenotypic, phylogenetic and chemotaxonomic information, it was determined that strain W260T represents a novel genus and species and it was given the name Marinihelvus fidelis sp. nov. The type strain is W260T (=MCCC 1H00471T=KCTC 92639T).

Cost-Effectiveness of Liquid Biopsy for Colorectal Cancer Screening in Patients Who Are Unscreened

Despite recommendations for universal screening, adherence to colorectal cancer screening in the US is approximately 60%. Liquid biopsy tests are in development for cancer early detection, but it is unclear whether they are cost-effective for colorectal cancer screening. To estimate the cost-effectiveness of liquid biopsy for colorectal cancer screening in the US. In this economic evaluation, a Markov model was developed to compare no screening and 5 colorectal cancer screening strategies: colonoscopy, liquid biopsy, liquid biopsy following nonadherence to colonoscopy, stool DNA, and fecal immunochemical test. Adherence to first-line screening with colonoscopy, stool DNA, or fecal immunochemical test was assumed to be 60.6%, and adherence for liquid biopsy was assumed to be 100%. For colonoscopy, stool DNA, and fecal immunochemical test, patients who did not adhere to testing were not offered other screening. In colonoscopy-liquid biopsy hybrid, liquid biopsy was second-line screening for those who deferred colonoscopy. Scenario analyses were performed to include the possibility of polyp detection for liquid biopsy. No screening, colonoscopy, fecal immunochemical test, stool DNA, liquid biopsy, and colonoscopy-liquid biopsy hybrid screening. Model outcomes included life expectancy, total cost, and incremental cost-effectiveness ratios. A strategy was considered cost-effective if it had an incremental cost-effectiveness ratio less than the US willingness-to-pay threshold of $100 000 per life-year gained. This study used a simulated cohort of patients aged 45 years with average risk of colorectal cancer. In the base case, colonoscopy was the preferred, or cost-effective, strategy with an incremental cost-effectiveness ratio of $28 071 per life-year gained. Colonoscopy-liquid biopsy hybrid had the greatest gain in life-years gained but had an incremental cost-effectiveness ratio of $377 538. Colonoscopy-liquid biopsy hybrid had a greater gain in life-years if liquid biopsy could detect polyps but remained too costly. In this economic evaluation of liquid biopsy for colorectal cancer screening, colonoscopy was a cost-effective strategy for colorectal cancer screening in the general population, and the inclusion of liquid biopsy as a first- or second-line screening strategy was not cost-effective at its current cost and screening performance. Liquid biopsy tests for colorectal cancer screening may become cost-effective if their cost is substantially lowered.

Precise Detection of Viral RNA by Programming Multiplex Rolling Circle Amplification and Strand Displacement.

Detection of viral infections (e.g., SARS-CoV-2) with high precision is critical to disease control and treatment. There is an urgent need to develop point-of-care detection methods to complement the gold standard laboratory-based PCR assay with comparable sensitivity and specificity. Herein, we developed a method termed mCAD to achieve ultraspecific point-of-care detection of SARS-CoV-2 RNA while maintaining high sensitivity by programming multiplex rolling circle amplification and toehold-mediated strand displacement reactions. RCA offers sufficient amplification of RNA targets for subsequent detection. Most importantly, a multilayer of detection specificity is implemented into mCAD via sequence-specific hybridization of nucleic acids across serial steps of this protocol to fully eliminate potential false-positive detections. Using mCAD, we demonstrated a highly specific, sensitive, and convenient visual detection of SARS-CoV-2 RNA from both synthetic and clinical samples, exhibiting performance comparable to qPCR. We envision that mCAD will find its broad applications in clinical prospects for nucleic acid detections readily beyond SARS-CoV-2 RNA.

Lab on a single fiber: A three-parameter sensor based on triple-SPR for one-to-one detection of DNA, pH and temperature

In order to realize the one-to-one correspondence between outputs and inputs in the multiparameter detection field, a multiparameter DNA hybridization sensor with temperature and pH detection channels based on a fiber-based triple surface plasmon resonance (Triple-SPR) effect is demonstrated in this paper. Compared with previous works, the significant innovation of this paper is the independent temperature testing and pH testing channels implemented based on a single fiber optic structure, and the high-sensitivity DNA hybridization detection channel. Polydimethylsiloxane and the self-assembled materials formed by polyacrylic acid (PAA) and chitosan (CS) are used for the real-time detection of temperature and pH. With the help of these two materials, the instantaneous response of the Triple-SPR sensor to temperature and pH has been realized, and the response times (RT) are less than 1 s and 20 s, respectively. After the modification of probe DNA (pDNA), the selective detection of complementary DNA (cDNA) is achieved. The sensitivity and detection limit (DL) reaches 0.09 nm/(nmol/L) and 3.69 nmol/L, respectively. The experimental results demonstrate that an approximately one-to-one correspondence exists between inputs and outputs. The output signals can be used to provide feedback signals for achieving stable temperature and pH environments. The advantages of multi-parameter independent detection, high-sensitivity as well as fast response of the fiber-based Triple-SPR sensing technology are of great significance in the field of future multi-parameter sensing, compensated detection as well as real-time bio-monitoring.

Ligilactobacillus cholophilus sp. nov., isolated from pickled potherb mustard (Brassica juncea Coss.).

Strain BD7642T was isolated from Chinese pickled potherb mustard (Brassica juncea Coss.) purchased from a local market in Shanghai, PR China. A polyphasic approach, including 16S rRNA gene sequence, housekeeping gene, average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH), G+C content and phenotypic analyses, was employed to characterize strain BD7642T. Cells of the bacterium were short round rods, Gram-stain-positive, non-spore-forming and catalase-negative. The strain grew at 30-45 °C and pH 4.0-8.0. Optimum growth occurred at 35-40 °C and pH 6.0-7.0. The strain exhibited growth with salt (NaCl) concentrations of up to 5 % (w/v). The G+C content of the strain's genomic DNA was 31.37 mol%. The major fatty acids were C16 : 0, C18 : 1 c9 and summed feature 10 (C18 : 1 c11/t9/t6). 16S rRNA gene sequencing revealed that strain BD7642T represents a member of the genus Ligilactobacillus and it had high sequence similarity to Ligilactobacillus aviarius NBRC 102162T (96.73 %), Ligilactobacillus araffinosus LGM 23560 (96.66 %) and Ligilactobacillus salivarius JCM 1231T (95.82 %). The dDDH values between strain BD7642T and its phylogenetically related species within the genus Ligilactobacillus ranged from 12.6 to 25.4 %. The ANI values between strain BD7642T and its closely related taxa were far lower than the threshold (95 %-96 %) used for species differentiation. Results of phylogenetic, physiological and phenotypic characterization confirmed that strain BD7642T represents a novel species within the genus Ligilactobacillus, for which the name Ligilactobacillus cholophilus sp. nov. is proposed. The type strain is BD7642T (=CCTCC AB 2022398T=JCM 36074T).

The Application of Hybridization Chain Reaction in the Detection of Foodborne Pathogens.

Today, with the globalization of the food trade progressing, food safety continues to warrant widespread attention. Foodborne diseases caused by contaminated food, including foodborne pathogens, seriously threaten public health and the economy. This has led to the development of more sensitive and accurate methods for detecting pathogenic bacteria. Many signal amplification techniques have been used to improve the sensitivity of foodborne pathogen detection. Among them, hybridization chain reaction (HCR), an isothermal nucleic acid hybridization signal amplification technique, has received increasing attention due to its enzyme-free and isothermal characteristics, and pathogenic bacteria detection methods using HCR for signal amplification have experienced rapid development in the last five years. In this review, we first describe the development of detection technologies for food contaminants represented by pathogens and introduce the fundamental principles, classifications, and characteristics of HCR. Furthermore, we highlight the application of various biosensors based on HCR nucleic acid amplification technology in detecting foodborne pathogens. Lastly, we summarize and offer insights into the prospects of HCR technology and its application in pathogen detection.

Cellulomonas endometrii sp. nov.: a novel bacterium isolated from the endometrial microbiota

An isolate of a bacterium recovered from an endometrial biopsy failed to be identified by MALDI-TOF mass spectrometry and was subjected to 16S rRNA sequencing. The obtained sequence was compared by BLASTn against the NCBI database, which revealed that the most closely related species was Cellulomonas hominis and Cellulomonas pakistanensis, with 98.85% and 98.45% identity, respectively. Phenotypic characterisation and genome sequencing were performed. The isolate was facultative anaerobic, gram-positive, motile, non-spore forming, and rod-shaped. Cell wall fatty acid profiling revealed that 12-methyl-tetradecanoic acid was the most abundant fatty acid (36%). The genome size was 4.25 Mbp with a G + C content of 74.8 mol%. Genomic comparison of species closely related to this strain showed that all digital DNA–DNA hybridisation (dDDH) and mean orthologous nucleotide identity (OrthoANI) values were below published species thresholds (70% and 95–96%, respectively). Based on these data, we conclude that this isolate represents a new bacterial species belonging to the family Cellulomonadaceae and the phylum Actinomycetota. We propose the name Cellulomonas endometrii sp. nov. The type strain is Marseille-Q7820T (= CSUR Q7820 = CECT 30716).