Target DNA Fragments Research Articles

Advanced Electrochemical Biosensing toward Staphylococcus aureus Based on the RPA-CRISPR/Cas12a System and Conductive Nanocomposite.

Staphylococcus aureus (S. aureus) is a prevalent foodborne pathogen that poses significant challenges to food safety. Herein, a sensitive and specific electrochemical biosensor based on RPA-CRISPR/Cas12a is developed for evaluating S. aureus. In the presence of S. aureus, the extracted target DNA fragments are efficiently amplified by recombinase polymerase amplification (RPA). The designed crRNA, binding to Cas12a, effectively recognizes the target fragment cleaving hpDNA. The signal molecule of hpDNA is cleaved from the sensing interface, resulting in a reduction of current response. Under optimal experimental conditions, the developed electrochemical biosensor exhibits remarkable sensitivity in detecting S. aureus. The linear range for quantifying S. aureus in pure culture is 1.04 × 101-1.04 × 108 CFU/mL, with a detection limit as low as 3 CFU/mL. In addition, the biosensor enables the accurate and sensitive detection of S. aureus in milk within a linear range of 1.07 × 101-1.07 × 107 CFU/mL. The electrochemical biosensor enhances anti-interference capability owing to the specific amplification of RPA primers and the single-base recognition ability of crRNA. The RPA-CRISPR/Cas12a biosensor exhibits exceptional anti-interference capability, precision, and sensitivity, thereby establishing a robust foundation for real-time monitoring of microbial contamination.

Development of a 45K pepper GBTS liquid-phase gene chip and its application in genome-wide association studies.

Pepper (Capsicum spp.) is a vegetable that is cultivated globally and has undergone extensive domestication, leading to a significant diversification in its agronomic traits. With the advancement of genomics in pepper and the reduction in sequencing costs, the high-throughput detection of single nucleotide polymorphisms (SNPs) and small insertions-deletions (indels) has become increasingly critical for analyzing pepper germplasms and improving breeding programs. As a result, there is a pressing need for a cost-effective, high-throughput, and versatile technique suitable for both foreground and background selection in pepper breeding. In the present study, Python-based web scraping scripts were utilized to systematically extract data from published literatures and relevant sequence databases focusing on pepper genomes. Subsequent to data extraction, SNPs and indels were meticulously identified and filtered. This process culminated in the delineation of core polymorphic sites, which were instrumental in the development of specific probes. Following this, comprehensive phenotypic and genotypic analyses were conducted on a diverse collection of 420 pepper germplasms. Concurrently, a genome-wide association study (GWAS) was conducted to elucidate the genetic determinants of helical fruit shape in peppers. In this study, a 45K pepper Genotyping-By-Target-Sequencing (GBTS) liquid-phase gene chip was developed on the GenoBaits platform. This chip is composed of 45,389 probes, of which 42,535 are derived from core polymorphic sites (CPS) in the background genetic landscape, while 2,854 are associated with foreground agronomic traits, spanning across 43 traits. The CPS probes are spaced at an average interval of 68 Kb. We have assessed the performance of this chip on 420 pepper germplasms, with successful capture of target DNA fragments by 45,387 probes. Furthermore, the probe capture ratio surpassed 70% in 410 of the 420 germplasms tested. Using this chip, we have efficiently genotyped 273 germplasms for spiciness levels and elucidated the genetic relationships among 410 pepper germplasms. Our results allowed for precise clustering of sister lines and C. chinense germplasms. In addition, through a GWAS for helical fruit shape, we identified three quantitative trait loci (QTLs): heli2.1, heli11.1, and heli11.2. Within the heli11.1 QTL, a gene encoding the tubulin alpha chain was identified, suggesting its potential role in the helical growth pattern of pepper fruits. In summary, the 45K pepper GBTS liquid-phase gene chip offers robust detection of polymorphic sites and is a promising tool for advancing research into pepper germplasm and the breeding of new pepper varieties.

Development of a Multiplex PCR Assay for Rapid Differentiation of Fowlpox and Pigeonpox Viruses.

The aim of this study was to develop a multiplex PCR assay capable of rapidly differentiating two major Avipoxvirus (APV) species, Fowlpox virus (FWPV) and Pigeonpox virus (PGPV), which cause disease in bird species. Despite the importance of a rapid differentiation assay, no such assay exists that can differentiate the APV species without sequencing. To achieve this, species-specific target DNA fragments were selected from the fpv122 gene of FWPV and the HM89_gp120 gene of PGPV, which are unique to each genome. Nine samples collected from unvaccinated chickens, pigeons, and a turkey with typical pox lesions were genetically identified as FWPV and PGPV. The designed primers and target DNA fragments were validated using in silico analyses with the nucleotide Basic Local Alignment Search Tool. The multiplex PCR assay consisted of species-specific primers and previously described PanAPV primers (genus-specific) and was able to differentiate FWPV and PGPV, consistent with the phylogenetic outputs. This study represents the first successful differentiation of FWPV and PGPV genomes using a conventional multiplex PCR test. This assay has the potential to facilitate the rapid diagnosis and control of APV infections.

A Test to Comprehensively Capture the Known Genetic Component of Familial Pulmonary Fibrosis.

The recent European Respiratory Society statement on familial pulmonary fibrosis supports the need for genetic testing in the care of patients and their relatives. However, no solution (i.e., a concrete test) was provided to implement genetic testing in daily practice. Herein, we tabulated and standardized the nomenclature of 128 genetic variants in 20 genes implicated in adult-onset pulmonary fibrosis. The objective was to develop a laboratory-developed test (LDT) on the basis of standard Sanger sequencing to capture all known familial pulmonary fibrosis-associated variants. Targeted DNA fragments were amplified using harmonized PCR conditions to perform the LDT in a single 96-well plate. The new genetic test was evaluated in 62 sporadic cases of idiopathic pulmonary fibrosis. As expected in this population, we observed a low yield of disease-causing mutations. More important, 100% of targeted variants by the LDT were successfully evaluated. Furthermore, four variants of uncertain significance with in silico-predicted deleterious scores were identified in three patients, suggesting novel pathogenic variants in genes known to cause idiopathic pulmonary fibrosis. Finally, the MUC5B promoter variant rs35705950 was strongly enriched in these patients with a minor allele frequency of 41.1% compared with 10.6% in a matched population-based cohort (n = 29,060), leading to an estimation that this variant may explain up to 35% of the population-attributable risk. This LDT provides a solution for rapid clinical translation. Technical laboratory details are provided so that specialized pulmonary centers can implement the LDT in house to expedite the clinical recommendations of expert panels.

Designing and validation of specific primers for the quantitative detection of bacteria in sugarcane inoculant.

Endophytic diazotrophic plant growth-promoting bacteria Herbaspirillum rubrisubalbicans (HCC103), Herbaspirillum seropedicae (HRC54), Paraburkholderia tropica (Ppe8T), Gluconacetobacter diazotrophicus (Pal5T), and Nitrospirillum amazonense (CBAmC) have been used as inoculants for sugarcane. The genome sequences of these strains were used to design a set of specific primers for the real-time PCR (qPCR) assay. Primer specificity was confirmed by conventional PCR using the genomic DNAs of 25 related bacterial species and the five target strains. The qPCR assays were conducted using root and shoot samples from two sugarcane varieties (RB867515 and RB92579). These samples were collected both with and without inoculation, using the target strains specified in this study. The sugarcane plants were grown in a greenhouse, utilizing a substrate composed of sterile sand and vermiculite in a 2:1 ratio, for a duration of 55days. The primers designed for this study successfully amplified target DNA fragments from each of the bacterial species, enabling their differentiation at the species level. The total bacterial population present in the sugarcane quantified using qPCR was on average 105.2 cells g-1 of fresh tissue. Across both evaluated varieties, it was observed that the population of inoculated bacteria tended to decrease over time and became more concentrated in the sugarcane roots compared to the aerial parts. The qPCR results suggest that both the host and the microbes influence the endophytic population and the bacterial number decreases with plant age.

Nanopore sequencing of DNA barcodes succeeds in unveilling the diversity of fungal mock communities

Background: The fungal component of the gut microbiome has been underrepresented in most gut microbiome studies. While metabarcoding approaches have been used to assess the diversity and role of the gut mycobiome, differences in experimental design and downstream analyses may induce bias and differential outcomes. This study assessed the capacity of nanopore sequencing to retrieve the microbial profile and relative abundance of a mock fungal community comprised of Candida glabrata, Meyerozyma guilliermondii, Pichia kudriavzevii, Clavispora lusitaniae and Candida parapsilosis. Methods: The approach was implemented using the MinION system and involved the analysis of sequencing libraries made from equimolar mixtures of the PCR-amplified nuclear ribosomal internal transcribed spacer (ITS) of the target species. Results: Nanopore sequencing successfully retrieved the composition of the fungal mock community in terms of the different taxa present. However, the approach was unable to correctly assess the expected relative abundances of each species in the same community, showing some yet undetermined bias that may be related to the size of the respective target DNA fragments. Clavispora lusitaniae was consistently overrepresented in the mixtures, while C. glabrata was underrepresented. The remaining three species showed relative abundances more aligned with the expected values of an equimolar mixture. Conclusions: Although not yielding the expected results for the relative abundances, the values obtained from independent sequencing runs were similar for all species, suggesting a good reliability but questionable accuracy in this sequencing approach.

Long-range Energy Transfer Between Dye-loaded Nanoparticles: Observation and Amplified Detection of Nucleic Acids.

Förster resonance energy transfer (FRET) is essential in optical materials for light-harvesting, photovoltaics and biosensing, but its operating range is fundamentally limited by the Förster radius of ∼5nm. Here, FRET between fluorescent organic nanoparticles (NPs) is studied for the first time in order to break this limit. The donor and acceptor NPs are built from charged hydrophobic polymers loaded with cationic dyes and bulky hydrophobic counterions. Their surface is functionalized with DNA in order to control surface-to-surface distance. It is found that the FRET efficiency does not follow the canonic Förster law, reaching 0.70 and 0.45 values for NP-NP distance of 15 and 20nm, respectively. This corresponds to the FRET efficiency decay as power four of the surface-to-surface NP-NP distance. Based on this long-distance FRET, a DNA nanoprobe is developed, where a target DNA fragment, encoding cancer marker survivin, brings together donor and acceptor NPs at ∼15nm distance. In this nanoprobe, a single molecular recognition results in unprecedented color switch for >5000 dyes, yielding a simple and fast assay with 18 attomoles limit of detection. Breaking the Förster distance limit for ultrabright NPs opens the route to advanced optical nanomaterials for amplified FRET-based sensing of biomolecules. This article is protected by copyright. All rights reserved.

Detection of chronic toxoplasmosis in the brain of mice using loop-mediated isothermal amplification (LAMP) and conventional PCR

BackgroundToxoplasma gondii is a common protozoan parasite that infects approximately one-third of the world's population. It is a disease with multiple manifestations. In immunocompetent individuals, symptoms are mild and flu-like, whereas, in immunocompromised patients, it often results in severe morbidity and mortality. Thus, studies for developing a simple, rapid diagnostic tool for early detection of Toxoplasma are emerging. Molecular diagnosis is highly accurate and helpful in congenitally infected and immunocompromised patients. The loop-mediated isothermal amplification (LAMP) technique was invented to improve nucleic acid amplification efficacy in terms of sensitivity and specificity. Aim of the studyThe study aimed to validate a LAMP protocol for detecting Toxoplasma DNA in the brain homogenates from mice experimentally infected with Toxoplasma's ME-49 (cyst-forming type II) strain in comparison to PCR. MethodsIn this study, the target DNA fragment was the Toxoplasma 529-bp, repeated 200–300 copies/genome. The sensitivity of both LAMP and conventional PCR techniques was estimated in brain homogenates in experimental mice at eight weeks post-infection and compared to the histopathology data. ResultsThe LAMP reaction showed positive results in 18 of the 26 examined samples of brain homogenates. PCR showed the characteristic 529-bp band in 15 of the 26 examined samples. ConclusionThe LAMP showed a higher sensitivity over PCR in detecting Toxoplasma infection in brain homogenates of infected mice.

Nanopore sequencing of DNA barcodes to unveil the diversity of fungal mock communities

Background: The fungal component of the gut microbiome has been underrepresented in most gut microbiome studies. While next-generation sequencing (NGS) approaches have been used to assess the diversity and role of the gut mycobiome, differences in experimental design and downstream analyses may induce bias and differential outcomes. This study assessed the capacity of nanopore sequencing to retrieve the microbial profile and relative abundance of a mock fungal community comprised of Candida glabrata, Meyerozyma guilliermondii, Pichia kudriavzevii, Clavispora lusitaniae and Candida parapsilosis. Methods: The approach was implemented using the MinION system and involved the analysis of sequencing libraries made from equimolar mixtures of the PCR-amplified internal transcriber spacer genomic regions of the target species. Results: Nanopore sequencing successfully retrieved the composition of the fungal mock community in terms of the different taxa present. However, the approach was unable to correctly assess the expected relative abundances of each species in the same community, showing some yet undetermined bias that may be related to the size of the respective target DNA fragments. Clavispora lusitaniae was consistently overrepresented in the mixtures, while C. glabrata was underrepresented. The remaining three species showed relative abundances more aligned with the expected values of an equimolar mixture. Conclusions: Although not yielding the expected results for the relative abundances, the values obtained from independent sequencing runs were similar for all species, suggesting a good reliability but questionable accuracy in this sequencing approach.

Zinc finger and SCAN domain containing 1, ZSCAN1, is a novel stemness-related tumor suppressor and transcriptional repressor in breast cancer targeting TAZ.

Cancer stem cells (CSCs) targeted therapy holds the potential for improving cancer management; identification of stemness-related genes in CSCs is necessary for its development. The Cancer Genome Atlas (TCGA) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) datasets were used for survival analysis. ZSCAN1 correlated genes was identified by Spearman correlation analysis. Breast cancer stem-like cells (BCSLCs) were isolated by sorting CD44+CD24- cells from suspension cultured breast cancer (BC) spheroids. The sphere-forming capacity and sphere- and tumor-initiating capacities were determined by sphere formation and limiting dilution assays. The relative gene expression was determined by qRT-PCR, western blot. Lentivirus system was used for gene manipulation. Nuclear run-on assay was employed to examine the levels of nascent mRNAs. DNA pull-down and Chromatin immunoprecipitation (ChIP) assays were used for determining the interaction between protein and target DNA fragments. Luciferase reporter assay was used for evaluating the activity of the promoter. ZSCAN1 is aberrantly suppressed in BC, and this suppression indicates a bad prognosis. Ectopic expression of ZSCAN1 inhibited the proliferation, clonogenicity, and tumorigenicity of BC cells. ZSCAN1-overexpressing BCSLCs exhibited weakened stemness properties. Normal human mammary epithelial (HMLE) cells with ZSCAN1 depletion exhibited enhanced stemness properties. Mechanistic studies showed that ZSCAN1 directly binds to -951 ~ -925bp region of WWTR1 (encodes TAZ) promoter, inhibits WWTR1 transcription, thereby inhibiting the stemness of BCSCs. Our work thus revealed ZSCAN1 as a novel stemness-related tumor suppressor and transcriptional repressor in BC.

A Magnetoelectrochemical Bioassay for Highly Sensitive Sensing of Point Mutations in Interleukin-6 Gene Using TMB as a Hybridization Intercalation Indicator.

Point mutations are common in the human DNA genome and are closely related to higher susceptibility to cancer diseases. Therefore, suitable methods for their sensing are of general interest. In this work, we report on a magnetic electrochemical bioassay using DNA probes tethered to streptavidin magnetic beads (strep-MBs) to detect T > G single nucleotide polymorphism (SNP) within the inteleukin-6 (IL6) gene in human genomic DNA. In the presence of the target DNA fragment and tetramethylbenzidine (TMB), the electrochemical signal related to the oxidation of TMB is observed, which is much higher than the one obtained in the absence of the target. The key parameters affecting the analytical signal, such as the concentration of the biotinylated probe, its incubation time with strep-MBs, DNA hybridization time, and TMB loading, were optimized using the electrochemical signal intensity and signal-to-blank (S/B) ratio as selection criteria. Using spiked buffer solutions, the bioassay can detect the mutated allele in a wide range of concentrations (over six decades) with a low detection limit (7.3 fM). Furthermore, the bioassay displays a high specificity with high concentrations of the major allele (one mismatched), and two mismatched and non-complementary DNA. More importantly, the bioassay can detect the variation in scarcely diluted human DNA, collected from 23 donors, and can reliably distinguish between heterozygous (TG genotype) and homozygous (GG genotype) in respect to the control subjects (TT genotype), where the differences are statistically highly significant (p-value < 0.001). Thus, the bioassay is useful for cohort studies targeting one or more mutations in human DNA.

High sensitivity and specificity rates of cobas® HPV test as a primary screening test for cervical intraepithelial lesions in a real-world setting.

Cervical carcinoma (CC) is the fourth most common malignancy among women. Screening with Papanicolau smear is linked to a reduction in CC incidence rates when screening programs have been developed. However, this technique has several limitations, including moderate sensitivity rates for detection of cervical preneoplastic HPV-related lesions. In this real-world study, we proposed to evaluate the sensitivity and specificity rates of cobas® test, which amplifies target DNA fragments by polymerase chain reaction and hybridization of nucleic acids for the detection of 14 HR-HPV types in a single analysis) used as primary screening test for CC and preneoplastic lesions in women aged 25-65 years in a large University Hospital in Buenos Aires. A total of 1044 patients were included in the sample (median age: 46 years); sensitivity and specificity rates for the HR-HPV test used as primary screening test were 98.66% (95% confidence interval [95CI]: 97.67-99.3%) and 87.15% (95CI: 84.93-89.15%), respectively. The positive predictive value was 88.47% (95CI: 86.54%-90.42%) and the negative predictive value was 98.48% (95CI: 97.75%-99.23%). The cobas® HR-HPV testing was highly sensitive and specific for the detection of CC and preneoplastic lesions in real practice.

Emulsion Polymerase Chain Reaction Coupled with Denaturing Gradient Gel Electrophoresis for Microbial Diversity Studies.

Emulsion PCR-DGGE is a molecular biology technique used to amplify and analyze DNA fragments. This technique combines two processes, emulsion PCR and denaturing gradient gel electrophoresis (DGGE), to enhance the specificity and yield of the amplification process and to separate the amplified fragments based on their melting behavior. In the emulsion PCR step, a high-quality DNA template is mixed with the PCR reagents and droplet generator oil to create an oil-in-water emulsion. The emulsion is then subjected to thermal cycling to amplify the target DNA fragments. The amplified fragments are recovered from the droplets and purified to remove any impurities that may interfere with downstream applications. In the DGGE step, the purified amplicon is loaded onto a DGGE apparatus, where the DNA fragments are separated and visualized based on their melting behavior. This method allows for the concurrent amplification and separation of multiple DNA fragments, thereby enhancing the resolution and sensitivity of the analysis. It is widely used in environmental and medical microbiology research, as well as in other fields that require the identification and characterization of microorganisms, such as the study of microbial diversity in soil, water, and other natural environments, as well as in the human gut microbiome and other medical samples.

A higher DNA damage rate in aqueous eDNA particles suggests intra‐cellular eDNA degradation in cellular environments

AbstractA higher DNA quality in an environmental DNA (eDNA) sample is crucial for the improved and precise assessment of biodiversity via eDNA analysis. However, the frequency of damage of DNA molecules released from macro‐organisms in aquatic environments remains unclear. As a proof of concept, the rate of DNA damage (λ; per base pair) in an eDNA sample was estimated by measuring the quantity of target DNA with different fragment lengths using quantitative real‐time PCR (qPCR). Rearing water was collected from tanks housing zebrafish (Danio rerio) using filters with different pore sizes ranging from 0.2 to 10 μm, multiple fragment lengths of their mitochondrial eDNA concentrations were quantified and estimated the DNA damage rate based on an exponential relationship between target DNA concentrations and DNA fragment lengths. The DNA damage rate in zebrafish eDNA particles was higher for larger pore size filters, implying that cell membranes do not necessarily protect intra‐cellular DNA from degradation and high activity of intra‐cellular DNases actively degrades intra‐cellular eDNA molecules. Moreover, this finding may partly account for the apparently shorter persistence of larger eDNA particles in aquatic environments and the initial rapid decay of eDNA fragments in a biphasic degradation process. Additional field studies are required to investigate the frequency of DNA damage in different eDNA size fractions and states to facilitate the efficient collection of population genetic information on aquatic macro‐organisms from environmental samples.

The Method of Screening and Identification of Transcription Factor in Klebsiella.

This protocol describes a method for verifying the specific transcription factor regulating glycerol dehydratase (GDH) expression in Klebsiella. DNA pull-down accompanied with mass spectrometry is used to screen and identify the transcription factor interacting with the promoter region of the key gene in Klebsiella. EMSA method is used to validate the specific binding of the transcription factor to the promoter region in vitro. In addition, the target DNA fragments are constructed by fusion PCR to prepare competent cells from Klebsiella for electrical transformation and further transformed to obtain key gene deletion strains to verify the transcription factor responsible for the target gene expression in Klebsiella.

Loop-mediated isothermal amplification of PBAN gene for molecular diagnosis of Bemisia tabaci biotype Q (Hemiptera: Aleyrodidae)

• LAMP assay for PBAN of the whitefly was developed. • The PBAN-based LAMP primer set showed specific amplification of the target region. • The PBAN-based LAMP assay can be applicable for monitoring B. tabaci biotype Q. The tobacco whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), is a serious pest that transmits tomato yellow leaf curl virus (TYLCV) in tomato. The tobacco whitefly exhibits morphological similarity with the greenhouse whitefly, Trialeurodes vaporariorum (Hemiptera: Aleyrodidae) which also damages various plants in the greenhouse. Therefore, it is very important to quickly and accurately diagnose whiteflies for applying pest management strategies. In this study, we used a loop-mediated isothermal amplification (LAMP) assay to quickly and effectively detect B. tabaci . Primer sets were investigated for the specificity of B. tabaci by visible detection of the target DNA fragment. Two primer sets for pheromone biosynthesis activating neuropeptide (PBAN) gene were obtained from transcriptomic analysis of B. tabaci biotype Q. The PBAN-based LAMP primer set showed specific amplification of the target region. The optimal conditions for B. tabaci detection were 60 °C for 60 min with four LAMP primers of PBAN. The minimum amount of genomic DNA required for visible detection was 100 ng. These results suggest that the PBAN-based LAMP assay can be applicable for field monitoring of B. tabaci .

Field investigation- and dietary metabarcoding-based screening of arthropods that prey on primary tea pests.

Predatory natural enemies play key functional roles in biological control. Abundant predatory arthropod species have been recorded in tea plantation ecosystems. However, few studies have comprehensively evaluated the control effect of predatory arthropods on tea pests in the field. We performed a 1‐year field investigation and collected predatory arthropods and pests in the tea canopy. A total of 7931 predatory arthropod individuals were collected, and Coleosoma blandum (Araneae, Theridiidae) was the most abundant species in the studied tea plantation. The population dynamics between C. blandum and four main tea pest species (Aleurocanthus spiniferus, Empoasca onukii, Ectropis grisescens, and Scopula subpunctaria) were established using the individual number of predators and pests in each month. The results showed that C. blandum appeared to co‐occur in the tea canopy with A. spiniferus, Em. onukii, and Ec. grisescens in a longer period. The prey spectrum of C. blandum was further analyzed using DNA metabarcoding. Among prey species, A. spiniferus, Em. onukii, and Ec. grisescens were included, and the relative abundance and positive rates of target DNA fragments of A. spiniferus were greater than that of other two pests. Combined with the high dominance index of C. blandum, co‐occurrence between C. blandum and A. spiniferus in time and space and high positive rate and relative abundance of target DNA fragments of A. spiniferus, C. blandum was identified to prey on A. spiniferus, and C. blandum may be an important predator of A. spiniferus. Thus, C. blandum has potential as a biological control agent of A. spiniferus in an integrated pest management strategy.

Development and evaluation of PCR primers for environmental DNA (eDNA) metabarcoding of Amphibia

Biodiversity monitoring is important for the conservation of natural ecosystems in general, but particularly for amphibians, whose populations are pronouncedly declining. However, amphibians’ ecological traits (e.g. nocturnal or aquatic) often prevent their precise monitoring. Environmental DNA (eDNA) metabarcoding – analysis of extra-organismal DNA released into the environment – allows the easy and effective monitoring of the biodiversity of aquatic organisms. Here, we developed and tested the utility of original PCR primer sets. First, we conducted in vitro PCR amplification tests with universal primer candidates using total DNA extracted from amphibian tissues. Five primer sets successfully amplified the target DNA fragments (partial 16S rRNA gene fragments of 160–311 bp) from all 16 taxa tested (from the three living amphibian orders Anura, Caudata and Gymnophiona). Next, we investigated the taxonomic resolution retrieved using each primer set. The results revealed that the universal primer set “Amph16S” had the highest resolution amongst the tested sets. Finally, we applied Amph16S to the water samples collected in the field and evaluated its detection capability by comparing the species detected using eDNA and physical survey (capture-based sampling and visual survey) in multiple agricultural ecosystems across Japan (160 sites in 10 areas). The eDNA metabarcoding with Amph16S detected twice as many species as the physical surveys (16 vs. 8 species, respectively), indicating the effectiveness of Amph16S in biodiversity monitoring and ecological research for amphibian communities.

LIFE-Seq: a universal Large Integrated DNA Fragment Enrichment Sequencing strategy for deciphering the transgene integration of genetically modified organisms.

SummaryMolecular characterization of genetically modified organisms (GMOs) yields basic information on exogenous DNA integration, including integration sites, entire inserted sequences and structures, flanking sequences and copy number, providing key data for biosafety assessment. However, there are few effective methods for deciphering transgene integration, especially for large DNA fragment integration with complex rearrangement, inversion and tandem repeats. Herein, we developed a universal Large Integrated DNA Fragments Enrichment strategy combined with PacBio Sequencing (LIFE‐Seq) for deciphering transgene integration in GMOs. Universal tilling DNA probes targeting transgenic elements and exogenous genes facilitate specific enrichment of large inserted DNA fragments associated with transgenes from plant genomes, followed by PacBio sequencing. LIFE‐Seq were evaluated using six GM events and four crop species. Target DNA fragments averaging ~6275 bp were enriched and sequenced, generating ~26 352 high fidelity reads for each sample. Transgene integration structures were determined with high repeatability and sensitivity. Compared with next‐generation whole‐genome sequencing, LIFE‐Seq achieved better data integrity and accuracy, greater universality and lower cost, especially for transgenic crops with complex inserted DNA structures. LIFE‐Seq could be applied in molecular characterization of transgenic crops and animals, and complex DNA structure analysis in genetics research.

Detection of small-sized DNA fragments in a glassy nanopore by utilization of CRISPR-Cas12a as a converter system.

The fabrication of nanopores with a matched pore size, and the existence of multiple interferents make the reproducible detection of small-sized molecules by means of solid-state nanopores still challenging. A useful method to solve these problems is based on the detection of large DNA nanostructures related to the existence of small-sized targets. In particular, a DNA tetrahedron with a well-defined 3D nanostructure is the ideal candidate for use as a signal transducer. Here, we demonstrate the detection of an L1-encoding gene of HPV18 as a test DNA target sequence in a reaction buffer solution, where long single-stranded DNA linking DNA tetrahedra onto the surface of the magnetic beads is cleaved by a target DNA-activated CRISPR-cas12 system. The DNA tetrahedra are subsequently released and can be detected by the current pulse in a glassy nanopore. This approach has several advantages: (1) one signal transducer can be used to detect different targets; (2) a glassy nanopore with a pore size much larger than the target DNA fragment can boost the tolerance of the contaminants and interferents which often degrade the performance of a nanopore sensor.