Search Results (17)

Aptamers are powerful tools for detecting and analyzing biomolecules that consist of proteins or nucleic acids. However, their application to aptamers against viroids—highly structured self-replicating RNAs—has not yet been explored. In this study, a magnetic bead- and high-throughput sequencing-based SELEX (MB-HTS-SELEX) protocol for selecting potential aptamers against potato spindle tuber viroid (PSTVd) is presented. Full-length biotinylated-PSTVd RNA was transcribed in vitro, immobilized on streptavidin-coated magnetic beads, and incubated with a library of ~3.32 × 10¹⁴ molecules of random single-stranded oligo-DNAs (oligo-ssDNAs) of 20, 30, or 40 nucleotides (L20, L30, or L40, respectively) flanked by primer binding sites for downstream PCR amplification. Simultaneous biotin labeling of the anti-aptamer strand of the resulting double-stranded DNA (dsDNA) amplicons facilitated strand separation using streptavidin-coated magnetic beads. After 10 selection rounds, high-throughput sequencing, followed by bioinformatics analysis of the generated sequences, allowed for the detection of several enriched sequences, representing putative PSTVd-binding aptamers. Subsequent pull-down assays showed that the most abundant oligo-ssDNA in L30 was docked on PSTVd molecules. This combination method may ameliorate the selection of high-affinity aptamers against PSTVd, reduce the number of selection cycles, time, and other costs of aptamer production, thereby promoting future massive and cost-effective viroid detection and characterization. Full article

The removal of nitrogen compounds in wastewater has been successfully developed with various activated sludge-based processes. Microorganisms immobilized in media would enhance biological efficiency by the increase in biomass concentration; however, the microbial community composition in media has not been revealed. Attached microbial communities on immobilization media were analyzed after the operation of the wastewater treatment process, comparing aerobic and anoxic reactors. A modified Ludzack–Ettinger (MLE) process was operated with immobilized media with polyvinyl alcohol and polyethylene glycol. The mixed liquor suspended solid (MLSS) concentration in an aerobic reactor was maintained at 50,000 mg/L and 40,000 mg/L in an anoxic reactor by the media. A maximum of 99% of ammonium nitrogen from the influent was calculated to be oxidized; however, the organic nitrogen produced from microbial growth reduced the overall oxidation rate. The denitrification rate increased with the addition of glucose to adjust the carbon-to-nitrogen (C/N) ratio. Based on the total nitrogen concentration, the nitrogen removal efficiency was calculated to be 48.2% following the adjustment of the C/N ratio. A phylogenetic analysis of the microbial community in immobilized media using next-generation sequencing (NGS) revealed the dominance of nitrifying and denitrifying microorganisms in the aerobic and anoxic reactors, respectively. Sequences amplified using V3–V4 region primers of the 16S rRNA gene yielded 531,188 base pairs (bp) and 396,844 bp reads from the aerobic and anoxic reactors, respectively. Operational taxonomic units (OTUs) were identified at both the phylum and genus levels, with a total of 594 from the aerobic reactor and 375 from the anoxic reactor. Proteobacteria was the dominant phylum in both the aerobic and anoxic reactors, comprising 39.7% of the aerobic reactor and 65.9% of the anoxic reactor. The dominant genera in the aerobic reactor were Nitrospira and Povalibacter. Forty-five percent of the total number of OTUs consisted of known nitrification-related genera in the aerobic reactor. In contrast, the dominant genera in the anoxic reactor were Desulfomicrobium, Desulfobulbus, and Methyloversatilis. A total of 63% of the genera associated with denitrification, including Dechloromonas and Flavobacterium, were found in the anoxic reactor. The population of microorganisms in each reactor was compared in terms of diversity by the QIIME 2 algorithm. The Chao1 index values of α-diversity were 606.05 for the aerobic reactor and 415.53 for the anoxic reactor, indicating greater population diversity in the aerobic reactor compared to the anoxic one. The widespread distribution of nitrification activities among various groups has led to diverse population characteristics in the aerobic environment, particularly within the attached community. The microbiological community present in immobilized aerobic and anoxic media will contribute to future microbial studies on wastewater treatment processes. Full article

The thermal stability of DNA immobilized on a solid surface is one of the factors that affects the efficiency of solid-phase amplification (SP-PCR). Although variable temperature amplification ensures high specificity of the reaction by precisely controlling temperature changes, excessively high temperatures during denaturation can negatively affect DNA stability. Formamide (FA) enables DNA denaturation at lower temperatures, showing potential for SP-PCR. Research on FA’s impacts on DNA microarrays is still limited, necessitating further optimization in exploring the characteristics of FA in SP-PCR according to particular application needs. We immobilized DNA on a chip using a crosslinker and generated DNA microarrays through bridge amplification based on FA denaturation on our automated reaction device. We optimized the denaturation and hybridization parameters of FA, achieving a maximum cluster density of 2.83 × 10⁴ colonies/mm². Compared to high-temperature denaturation, FA denaturation required a lower template concentration and milder reaction conditions and produced higher cluster density, demonstrating that FA effectively improves hybridization rates on surfaces. Regarding the immobilized DNA stability, the FA group exhibited a 45% loss of DNA, resulting in a 15% higher DNA retention rate compared to the high-temperature group, indicating that FA can better maintain DNA stability. Our study suggests that using FA improves the immobilized DNA stability and amplification efficiency in SP-PCR. Full article

Nucleic acid amplification reactions such as polymerase chain reaction (PCR), which uses a DNA polymerase to amplify individual double-stranded DNA fragments, are a useful technique for visualizing the presence of specific genomes. Although the fluorescent labeling method is mainly used with DNA amplification, other detection methods should be considered for further improvements, such as miniaturization and cost reduction, of reaction-monitoring devices. In this study, the quartz-crystal microbalance (QCM) method, which can measure nanogram-order masses, was applied for the real-time detection of DNA fragments in a solution with nucleic acids. This was combined with an isothermal nucleic acid amplification reaction based on the recombinase polymerase amplification (RPA) method, which allowed DNA amplification at a constant temperature. When the DNA amplification reaction was initiated on a QCM sensor plate with an immobilized primer DNA strand, a significant increase in mass was observed compared to when the primer DNA was not immobilized. QCM was shown to be sufficiently sensitive for the in situ detection of amplified DNA fragments. Combining a portable QCM device and RPA offers a sensitive point-of-care method for detecting nucleic acids. Full article

Adenosine is a vital biological small molecule that regulates various physiological processes in the human body. A high expression of adenosine in cells can facilitate tumor growth. Therefore, detecting adenosine is crucial for early disease diagnosis. In this paper, we designed a fluorescent biosensor for the sensitive detection of adenosine based on the cationic comb-type copolymer PLL-g-Dex for assisted rapid hybridization of nucleic acids at room temperature. In this strategy, adenosine preferentially binds to the aptamer immobilized on the surface of magnetic nanobeads, releasing free aDNA in solution as the primer strand, which rapidly forms DNA nanowires with auxiliary probes of bDNA with the assistance of PLL-g-Dex. SYBR Green I is embedded in DNA duplexes to generate strong fluorescence. The experimental results showed that PLL-g-Dex promotes DNA hybridization reactions at room temperature to form ultra-long DNA nanowires, thus achieving signal amplification and shortening the detection time. In addition, magnetic nanobeads can reduce the background signal during the reaction. Compared with several previous studies on the fluorescence detection of adenosine, this strategy has a lower detection limit of 2.32 nM. Furthermore, this novel system exhibited a good detection performance even under complex environments, such as serum, providing some reference for the quantitative detection of adenosine in early disease diagnosis. Full article

Nucleic acid detection is widely used to identify infectious diseases and ensure food safety. However, conventional PCR-based techniques are time consuming. Thus, this study aims to combine recombinase polymerase amplification (RPA), which enables the rapid amplification of even trace amounts of nucleic acid fragments within 10–40 min at 37–42 °C, and solution-processed oxide thin-film transistor (TFT) technology, which exhibits high detection sensitivity, to detect Leishmania. A single-stranded anti-probe was incorporated into the RPA primer to facilitate effective hybridization between the RPA product and the immobilized probe on the solution-processed oxide TFT. The RPA-amplified product carrying an anti-probe enabled specific binding to the chip surface. Changes in current were monitored before and after sample incubation to identify the target nucleic acids in the samples accurately. The proposed method achieved a remarkable limit of detection of 10¹ copies/μL of the Leishmania HSP70 fragment within 30 min. The design of the probes on the solution-processed oxide TFT surface and the anti-probe simplified the detection of other target nucleic acids, eliminating the need to denature DNA double-strands for specific binding during nucleic acid detection. Thus, the novel method offers the advantage of requiring minimal reagent resources and eliminates the need for complex procedures. Full article

Since its discovery, polymerase chain reaction (PCR) has emerged as an important technology for the diagnosis and identification of infectious diseases. It is a highly sensitive and reliable nucleic acids (NA) detection tool for various sample types. However, stool, which carries the most abundant micro-organisms and physiological byproducts, remains to be the trickiest clinical specimen for molecular detection of pathogens. Herein, we demonstrate the novel application of hydrogel microparticles as carriers of viral RNA from stool samples without prior RNA purification for real-time polymerase chain reaction (qPCR). In each microparticle of primer-incorporated network (PIN) as a self-sufficient reaction compartment, immobilized reverse transcription (RT) primers capture the viral RNA by hybridization and directly initiate RT of RNA to generate a pool of complementary DNA (PIN-cDNA pool). Through a simple operation with a portable thermostat device, a PIN-cDNA pool for influenza A virus (IAV) was obtained in 20 min. The PIN-cDNA pools can be stored at room temperature, or directly used to deliver cDNA templates for qPCR. The viral cDNA templates were freely released in the subsequent qPCR to allow amplification efficiency of over 91%. The assay displayed good linearity, repeatability, and comparable limit of detection (LoD) with a commercialized viral RNA purification kit. As a proof of concept, this technology carries a huge potential for onsite application to improve human and animal infectious disease surveillance activities using stool samples without the need for a laboratory or centrifuge for sample preparation. Full article

The potato cyst nematode (PCN), Globodera pallida, has acquired significant importance throughout Europe due to its widespread prevalence and negative effects on potato production. Thus, rapid and reliable diagnosis of PCN is critical during surveillance programs and for the implementation of control measures. The development of innovative technologies to overcome the limitations of current methodologies in achieving early detection is needed. Lab-on-a-chip devices can swiftly and accurately detect the presence of certain nucleotide sequences with high sensitivity and convert the presence of biological components into an understandable electrical signal by combining biosensors with microfluidics-based biochemical analysis. In this study, a specific DNA-probe sequence and PCR primers were designed to be used in a magnetoresistive biosensing platform to amplify the internal transcribed spacer region of the ribosomal DNA of G. pallida. Magnetic nanoparticles were used as the labelling agents of asymmetric PCR product through biotin–streptavidin interaction. Upon target hybridization to sensor immobilized oligo probes, the fringe field created by the magnetic nanoparticles produces a variation in the sensor’s electrical resistance. The detection signal corresponds to the concentration of target molecules present in the sample. The results demonstrate the suitability of the magnetic biosensor to detect PCR target product and the specificity of the probe, which consistently distinguishes G. pallida (DV/V > 1%) from other cyst nematodes (DV/V < 1%), even when DNA mixtures were tested at different concentrations. This shows the magnetic biosensor’s potential as a bioanalytical device for field applications and border phytosanitary inspections. Full article

Acinetobacter baumannii (A. baumannii) are phenotypically indistinguishable from the Acinetobacter calcoaceticus–A. baumannii (ACB) complex members using routine laboratory methods. Early diagnosis plays an important role in controlling A. baumannii infections and this could be assisted by the development of a rapid, yet sensitive diagnostic test. In this study, we developed an enzyme-based electrochemical genosensor for asymmetric PCR (aPCR) amplicon detection of the bla_OXA-51-like gene in A. baumannii. A. baumanniibla_OXA-51-like gene PCR primers were designed, having the reverse primer modified at the 5′ end with FAM. A bla_OXA-51-like gene sequence-specific biotin labelled capture probe was designed and immobilized using a synthetic oligomer (FAM-labelled) deposited on the working electrode of a streptavidin-modified, screen-printed carbon electrode (SPCE). The zot gene was used as an internal control with biotin and FAM labelled as forward and reverse primers, respectively. The bla_OXA-51-like gene was amplified using asymmetric PCR (aPCR) to generate single-stranded amplicons that were detected using the designed SPCE. The amperometric current response was detected with a peroxidase-conjugated, anti-fluorescein antibody. The assay was tested using reference and clinical A. baumannii strains and other nosocomial bacteria. The analytical sensitivity of the assay at the genomic level and bacterial cell level was 0.5 pg/mL (1.443 µA) and 10³ CFU/mL, respectively. The assay was 100% specific and sensitive for A. baumannii. Based on accelerated stability performance, the developed genosensor was stable for 1.6 years when stored at 4 °C and up to 28 days at >25 °C. The developed electrochemical genosensor is specific and sensitive and could be useful for rapid, accurate diagnosis of A. baumannii infections even in temperate regions. Full article

Background: Infection that is related to implanted biomaterials is a serious issue in the clinic. Antimicrobial peptides (AMPs) have been considered as an ideal alternative to traditional antibiotic drugs, for the treatment of infections, while some problems, such as aggregation and protein hydrolysis, are still the dominant concerns that compromise their antimicrobial efficiency in vivo. Methods: In this study, antimicrobial peptides underwent self-assembly on gold substrates, forming good antibacterial surfaces, with stable antibacterial behavior. The antimicrobial ability of AMPs grafted on the surfaces, with or without glycine spaces or a primer layer, was evaluated. Results: Specifically, three Pac-525 derivatives, namely, Ac-CGn-KWRRWVRWI-NH₂ (n = 0, 2, or 6) were covalently grafted onto gold substrates via the self-assembling process for inhibiting the growth of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Furthermore, the alkanethiols HS(CH)₁₀SH were firstly self-assembled into monolayers, as a primer layer (SAM-SH) for the secondary self-assembly of Pac-525 derivatives, to effectively enhance the bactericidal performance of the grafted AMPs. The -(CH)₁₀-S-S-G₆Pac derivative was highly effective against S. aureus and E. coli, and reduced the viable amount of E. coli and S. aureus to 0.4% and 33.2%, respectively, after 24 h of contact. In addition, the immobilized AMPs showed good biocompatibility, promoting bone marrow stem cell proliferation. Conclusion: the self-assembled monolayers of the Pac-525 derivatives have great potential as a novel therapeutic method for the treatment of implanted biomaterial infections. Full article

The emergence of the novel coronavirus, SARS-CoV-2, has highlighted the need for rapid, accurate, and point-of-care diagnostic testing. Lab-on-a-Chip (LoC) devices offer the possibility to run such tests at a low cost, while at the same time permitting the multiplexed detection of several viruses when coupled with microarray detection of the amplified products. Herein, we report the development of a protocol for the qualitative detection of SARS-CoV-2, through the design of appropriate primers that target the evolutionary conserved regions of the virus. The proposed protocol relies on an improved version of asymmetric RT-PCR, the linear-after-the-exponential (LATE)-PCR that uses primers that are deliberately designed for use at unequal concentrations. As a result, LATE-PCR exhibits similar efficiency to symmetric PCR, while promoting accumulation of single-stranded products that can subsequently hybridize to a single-strand DNA probe-spotted microarray. The performance of the developed LATE-PCR protocol was compared to that of symmetric RT-PCR, and validated with the use of artificial viral RNA and nasopharyngeal swab samples from real patients. Furthermore, and in order to illustrate its potential for integration into a biosensor platform, the amplicons were allowed to hybridize with probes that were covalently immobilized onto commercially available functionalized glass, without the need for heat denaturation. Full article

The accurate diagnosis of bacterial infections is of critical importance for effective treatment decisions. Due to the multietiologic nature of most infectious diseases, multiplex assays are essential for diagnostics. However, multiplexability in nucleic acid amplification-based methods commonly resorts to multiple primers and/or multiple reaction chambers, which increases analysis cost and complexity. Herein, a polymerase chain reaction (PCR) offer method based on a universal pair of primers and an array of specific oligonucleotide probes was developed through the analysis of the bacterial 16S ribosomal RNA gene. The detection system consisted of DNA hybridization over an array of magnetoresistive sensors in a microfabricated biochip coupled to an electronic reader. Immobilized probes interrogated single-stranded biotinylated amplicons and were obtained using asymmetric PCR. Moreover, they were magnetically labelled with streptavidin-coated superparamagnetic nanoparticles. The benchmarking of the system was demonstrated to detect five major bovine mastitis-causing pathogens: Escherichia coli, Klebsiella sp., Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae. All selected probes proved to specifically detect their respective amplicon without significant cross reactivity. A calibration curve was performed for S. agalactiae, which demonstrates demonstrating a limit of detection below 30 fg/µL. Thus, a sensitive and specific multiplex detection assay was established, demonstrating its potential as a bioanalytical device for point-of-care applications. Full article

In this paper we describe an isothermal rolling-circle amplification (RCA) protocol to detect gene point mutations on chips. The method is based on an allele-specific oligonucleotide circularization mediated by a special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and HIV cDNA gene. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe) can be amplified by rolling circle amplification to generate multimeric singlestranded DNA (ssDNA) under isothermal conditions. There are four sequence regions to bind respectively with fluorescent probe, RCA primer, solid probe and HIV cDNA template in the C-probe which we designed. These ssDNA products are hybridized with fluorescent probes and solid probes which are immobilized on a glass slide composing a regular microarray pattern. The fluorescence signals can be monitored by a scanner in the presence of HIV cDNA templates, whereas the probes cannot be circularized and signal of fluorescence cannot be found. The RCA array has capability of high-throughput detection of the point mutation and the single-nucleotide polymorphism (SNP).The development of C-probe-based technologies offers a promising prospect for situ detection, microarray, molecular diagnosis, single nucleotide polymorphism, and whole genome amplification. Full article

In this paper we describe a novel method for detecting many DNA fragments through efficient amplification by using an emulsion system based on “on-chip” PCR instead of conventional multiplex polymerase chain reaction (PCR). During the preparation of on-chip PCR, a set of primers were immobilized on a slide and other sets were in an emulsion system. Different emulsion phase primers and other related PCR components were dispersed in different droplets of the emulsion system, and then, due to the thermal instability of emulsion droplets, they would be released onto the surface of the slide after preheating in the first PCR step. To test the above method, we used plasma DNAs from pregnant women who was carrying a male fetus for gender identification. Four different Y chromosome DNA fragments were selected. Results showed that different DNA fragments could be simultaneously amplified with satisfactory results. It is suggested that a simple, convenient and inexpensive on-chip PCR method has been developed. Full article

A visible sensor array system for simultaneous multiple SNP genotyping has been developed using a new plastic base with specific surface chemistry. Discrimination of SNP alleles is carried out by an allele-specific extension reaction using immobilized oligonucleotide primers. The 3’-ends of oligonucleotide primers are modified with a locked nucleic acid to enhance their efficiency in allelic discrimination. Biotin-dUTPs included in the reaction mixture are selectively incorporated into extending primer sequences and are utilized as tags for alkaline phosphatase-mediated precipitation of colored chemical substrates onto the surface of the plastic base. The visible precipitates allow immediate inspection of typing results by the naked eye and easy recording by a digital camera equipped on a commercial mobile phone. Up to four individuals can be analyzed on a single sensor array and multiple sensor arrays can be handled in a single operation. All of the reactions can be performed within one hour using conventional laboratory instruments. This visible genotype sensor array is suitable for “focused genomics” that follows “comprehensive genomics”. Full article