Fast Nucleic Acid Research Articles

The detection of Bursaphelenchus xylophilus via accelerated strand exchange amplification: An ultra‐rapid and accurate method

AbstractOne of the most damaging pathogens of pinewood is the pinewood nematode, Bursaphelenchus xylophilus, which could cause an adverse effect on the ecosystems of forests and the commerce of timber. Therefore, it is crucial to realize rapid and accurate B. xylophilus detection. In this work, an accelerated strand exchange amplification method (ASEA) was established to detect B. xylophilus for the first time. By integrating with fast nucleic acid extraction, the whole detection procedure could be finished within 30 min, dramatically shortened the detection time. The ASEA method exhibited high specificity towards B. xylophilus and the detection limit for B. xylophilus plasmid DNA was as low as 1.0 × 100 copies/μL. Furthermore, the ASEA approach also exhibited accurate detection for B. xylophilus when applied to actual pinewood samples, meeting the demand of B. xylophilus detection in realistic scenario. We believe the ASEA method has significant potential for B. xylophilus detection, and it will be helpful for controlling forest pest and quarantine regulations.

Meat Authenticity Made Easy: DNA Extraction-Free Rapid Onsite Detection of Duck and Pork Ingredients in Beef and Lamb Using Dual-Recombinase-Aided Amplification and Multiplex Lateral Flow Strips.

Meat adulteration is a major global concern that poses a threat to public health and consumer rights. However, current detection techniques, such as quantitative polymerase chain reaction (qPCR) and gas chromatography-mass spectrometry, are time-consuming and require sophisticated equipment. In this study, we developed a rapid onsite identification method for animal-derived ingredients by utilizing a fast nucleic acid lysis buffer to expedite the release of sample nucleic acids and combined it with dual-recombinase-aided amplification (dual-RAA) technology and visual multiplex lateral flow strips (MLFSs). Our method successfully detected duck- and bovine-derived, porcine- and bovine-derived, duck- and ovine-derived, and porcine- and ovine-derived meat in a rapid 20 min onsite detection assay, with a detection limit of 101 copies/50 μL reaction system for target genes. Moreover, our method accurately detected adulterated meat with proportions as low as 1:999. These findings have significant implications for food safety and the protection of consumer rights.

Implementation and clinical evaluation of an Mpox virus laboratory-developed test on a fully automated random-access platform.

While Mpox virus (MPXV) diagnostics were performed in specialized laboratories only, the global emergence of Mpox cases in 2022 revealed the need for a more readily available diagnostic. Automated random-access platforms with fast nucleic acid extraction and PCR have become established in many laboratories, providing faster and more accessible testing. In this study, we adapted a previously published generic MPXV-PCR as a lab-developed test (LDT) on a NeuMoDx Molecular System and isolated MPXV clones from patient materials. To reduce the handling of infectious material, we evaluated a viral lysis buffer (VLB) for sample pretreatment. We further compared the MPXV-LDT-PCR to conventional real-time PCR, determined its sensitivity and specificity using positive swabs, and assessed its performance using external quality assessment samples. Pretreatment of samples with 50% VLB reduced MPXV infectivity by approximately 200-fold while maintaining PCR sensitivity. The assay demonstrated a sensitivity and specificity of 100% with no cross-reactivity in the samples tested and performed with a limit of detection of 262 GE/mL. In summary, the assay had a turnaround time of fewer than 2 h and can easily be transferred to other automated PCR platforms, providing a basis for developing rapid assays for upcoming pandemics.

Efficient mRNA delivery using lipid nanoparticles modified with fusogenic coiled-coil peptides.

Gene delivery has great potential in modulating protein expression in specific cells to treat diseases. Such therapeutic gene delivery demands sufficient cellular internalization and endosomal escape. Of various nonviral nucleic acid delivery systems, lipid nanoparticles (LNPs) are the most advanced, but still, are very inefficient as the majority are unable to escape from endosomes/lysosomes. Here, we develop a highly efficient gene delivery system using fusogenic coiled-coil peptides. We modified LNPs, carrying EGFP-mRNA, and cells with complementary coiled-coil lipopeptides. Coiled-coil formation between these lipopeptides induced fast nucleic acid uptake and enhanced GFP expression. The cellular uptake of coiled-coil modified LNPs is likely driven by membrane fusion thereby omitting typical endocytosis pathways. This direct cytosolic delivery circumvents the problems commonly observed with the limited endosomal escape of mRNA. Therefore fusogenic coiled-coil peptide modification of existing LNP formulations to enhance nucleic acid delivery efficiency could be beneficial for several gene therapy applications.

Toward a CRISPR-Based Point-of-Care Test for Tomato Brown Rugose Fruit Virus Detection

Implementing effective monitoring strategies is fundamental to protecting crops from pathogens and ensuring sufficient food supply as the global population continues to grow. This is especially important for emergent plant pathogens such as tomato brown rugose fruit virus (ToBRFV), which overcomes the genetic resistance resources used in tomato breeding against tobamoviruses and has become a pandemic in less than a decade. Here, we report the development of a CRISPR/Cas12a-based test to detect ToBRFV in the laboratory and potentially in a field setting. Using different tobamoviruses to assess specificity, our test showed a clear positive signal for ToBRFV-infected samples, whereas no cross-reactivity was observed for closely related viruses. Next, we compared the limit of detection of our CRISPR-based test with a widely used reference real-time quantitative PCR test, revealing similar sensitivities for both tests. Finally, to reduce complexity and achieve field applicability, we used a fast nucleic acid purification step and compared its results side by side with those of a commonly used column-mediated protocol. The new protocol saved time and resources but at the expense of sensitivity. However, it could still be useful to confirm ToBRFV detection in samples with incipient symptoms of infection. Although there is room for improvement, to our knowledge, this is the first field-compatible CRISPR-based test to detect ToBRFV that combines isothermal amplification with a simplified nucleic acid extraction protocol. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Cas14a1-mediated nucleic acid detectifon platform for pathogens

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease (Cas) based biosensing system provides a novel genomic diagnostic tool for pathogenic detection. However, most of the discovered Cas effectors have poor single strand DNA (ssDNA) target recognition capability with the constraint of protospacer adjacent motif (PAM) sites, which are not suitable for universal pathogenic diagnosis. Herein, we developed a highly sensitive and specific fluorescence tool for bacterial detection by utilizing the unique collateral cleavage activity of a Cas14a1-mediated nucleic acid detection platform (CMP). We combine CMP with molecular amplification to build a CRISPR-Cas based bioanalysis technique, offering fast nucleic acid detection with high sensitivity and specificity. This technique can identify different species of pathogens in milk samples with excellent accuracy. The CMP technique is a promising platform for pathogenic genomic diagnostic in biomedicine and food safety field.

Quantifying and Adjusting Plasmon‐Driven Nano‐Localized Temperature Field around Gold Nanorods for Nucleic Acids Amplification

Fast nucleic acid (NA) amplification has found widespread biomedical applications, where high thermocycling rate is the key. The plasmon-driven nano-localized thermocycling around the gold nanorods (AuNRs) is a promising alternative, as the significantly reduced reaction volume enables a rapid temperature response. However, quantifying and adjusting the nano-localized temperature field remains challenging for now. Herein, a simple method is developed to quantify and adjust the nano-localized temperature field around AuNRs by combining experimental measurement and numerical simulation. An indirect method to measure the surface temperature of AuNRs is first developed by utilizing the temperature-dependent stability of Authiol bond. Meanwhile, the relationship of AuNRs' surface temperature with the AuNRs concentration and laser intensity, is also studied. In combination with thermal diffusion simulation, the nano-localized temperature field under the laser irradiation is obtained. The results show that the restricted reaction volume (≈aL level) enables ultrafast thermocycling rate (>104 °C s-1 ). At last, a duplex-specific nuclease (DSN)-mediated isothermal amplification is successfully demonstrated within the nano-localized temperature field. It is envisioned that the developed method for quantifying and adjusting the nano-localized temperature field around AuNRs is adaptive for various noble metal nanostructures and will facilitate the development of the biochemical reaction in the nano-localized environment.

Identification for adulteration of beef with chicken based on single primer-triggered isothermal amplification

Abstract Adulteration of beef with cheap chicken has become a growing problem worldwide. In this study, a quick, single primer-triggered isothermal amplification (SAMP) combined with a fast nucleic acid extraction method was employed to detect the chicken meat in adulterated beef. Chicken from adulterated beef was identified using the chicken species-specific primer designed according to the Gallus gallus mitochondrial conserved sequences. Our SAMP method displayed good specificity and sensitivity in detecting chicken and beef meat DNA–the limit of detection (LOD) of SAMP is 0.33 pg/μL of chicken and beef total DNA and 2% w/w chicken meat in beef. The whole work flow from DNA extraction to signal detection can be finished within 1 h, fulfilling the requirement of on-site meat species identification.

Evaluation of fast nucleic acid detection system in severe acute respiratory syndrome coronavirus 2

Objective: To evaluate the performance and application of a fast nucleic acid detection system for testing severe acute respiratory syndrome virus 2 (SARS-COV-2). Methods: Clinical samples were collected from February to July 2020 from Beijing Center for Diseases Prevention and Control and the Laboratory Department of China-Japan Friendship Hospital, to evaluate the sensitivity, specificity, anti-interference ability, precision and clinical sample coincidence rate of fast nucleic acid detection system for SARS-CoV-2. The analytical sensitivity was determined by a dilution series of 20 replications for each concentration. Analytical specificity study was performed by testing organisms whose infection produces symptoms similar to those observed at the onset of corona virus disease 2019 (COVID-19), and of the normal or pathogenic microflora that may be present in specimens collected. Potential interference substances were evaluated with different concentration in the interference study. Precision study was conducted by estimating intra-and inter-batch variability. Clinical evaluation was performed by testing 230 oropharyngeal swab specimens and 95 sputum specimens in fast nucleic acid detection system, comparing with conventional real-time fluorescent quantitative PCR (RT-qPCR) and clinical diagnostic results. Results: The analytical sensitivity of SARS-CoV-2 using fast nucleic acid detection system was 400 copies/ml. The result is negative for testing with the organisms that may likely in the circulating area or causing similar symptoms with SARS-CoV-2 and human nucleic acid, indicating that no cross reactivity with organisms. The results of precision test showed that the Coefficient of variation of Ct value of high, medium and low concentration samples was 1.90%-3.92%, and all of them were less than 5% in intra-and inter-batch testing. The results of the samples were still positive after adding the potential interfering substances, indicating that the possible interfering substances in the samples had no effect on the results. 98.46% and 97.85% diagnosis results of fast nucleic acid detection system were consistent with RT-qPCR and clinical diagnostic results, respectively. Conclusion: The fast nucleic acid detection system based on molecular parallel reaction can be used as a selection method for SARS-CoV-2 testing.

Complex Size and Surface Charge Determine Nucleic Acid Transfer by Fusogenic Liposomes.

Highly efficient, biocompatible, and fast nucleic acid delivery methods are essential for biomedical applications and research. At present, two main strategies are used to this end. In non-viral transfection liposome- or polymer-based formulations are used to transfer cargo into cells via endocytosis, whereas viral carriers enable direct nucleic acid delivery into the cell cytoplasm. Here, we introduce a new generation of liposomes for nucleic acid delivery, which immediately fuse with the cellular plasma membrane upon contact to transfer the functional nucleic acid directly into the cell cytoplasm. For maximum fusion efficiency combined with high cargo transfer, nucleic acids had to be complexed and partially neutralized before incorporation into fusogenic liposomes. Among the various neutralization agents tested, small, linear, and positively charged polymers yielded the best complex properties. Systematic variation of liposomal composition and nucleic acid complexation identified surface charge as well as particle size as essential parameters for cargo-liposome interaction and subsequent fusion induction. Optimized protocols were tested for the efficient transfer of different kinds of nucleic acids like plasmid DNA, messenger RNA, and short-interfering RNA into various mammalian cells in culture and into primary tissues.

Evaluation of GeneXpert MTB for detection of Mycobacterium tuberculosis in Fayoum Governorate

Background Despite being one of the most ancient diseases, tuberculosis (TB) is still a major health problem globally. It is one of the most important causes of death worldwide. GeneXpert MTB/RIF assay is a fast nucleic acid amplification test used for detection of TB as well as identification of rifampicin resistance. Design This was a cross-sectional study. Setting The study was conducted at Fayoum Chest Hospital, Egypt, between December 2017 and December 2018. Objectives To evaluate GeneXpert MTB/RIF in detection of Mycobacterium tuberculosis in Fayoum Chest Hospital. Patients and methods One hundred participants were enrolled in this study with clinical or radiological susceptibility of pulmonary or extrapulmonary TB. All patients underwent the following: full history taking, clinical examination, plain chest radiography, examination of specimen by Ziehl–Neelsen stain, cultivation on Lowenstein–Jensen media, and processed in GeneXpert MTB/RIF. Results GeneXpert assay is a good, fast, noninvasive, and accurate diagnostic tool for detection of M. tuberculosis, showing sensitivity of 100.0%, specificity of 85.0%, positive predictive value of 96.4%, negative predictive value of 100%, and accuracy of 97%. On the contrary, Ziehl–Neelsen had sensitivity of 83.75%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 60.61%. Conclusion GeneXpert is a fast, accurate, and promising technique with good sensitivity and specificity, but it cannot be used alone as a diagnostic tool for TB.

Role of Viral Agents in Childhood Central Nervous System Infections

AbstractRapid laboratory diagnosis in acute viral infections in the central nervous system (CNS) is important for the direction of treatment. In this study, we aimed to evaluate viral agents detected by real-time polymerase chain reaction (PCR) in cerebrospinal fluid (CSF) samples sent to the national virology reference laboratory for the diagnosis of acute CNS viral infection in the 0 to 18 age group. The results of 5,255 CSF samples submitted to the laboratory between January 1, 2009, and February 14, 2018, were retrospectively reviewed. Test results of CSF samples tested with the multiplex real-time PCR panel (Fast Track Diagnostics/Viral Meningitis commercial kit, Luxembourg) kit were evaluated. PCR positivity was detected in 216 (4.11%) of the 5,255 CSF samples tested. The most common agents were enteroviruses 92 (42.6%) and herpes simplex virus-1 (HSV-1) 87 (40.2%) while varicella-zoster virus (VZV) 24 (11.1%), mumps virus 10 (4.62%), parechovirus 2 (0.92%), and herpes simplex virus-2 (HSV-2) 1 (0.46%) were less often identified. Considering the possibility that a wide variety of microorganisms may be agents of CNS infections, the performing of routine laboratory tests with multiple, fast, and reliable nucleic acid tests such as multiplex PCR provides a practical and effective approach. In addition to the identification of agents, retrospective studies of the incidence of agents will shed light on the clinical approaches in terms of the agents to be considered in the preliminary diagnosis. We consider that our results are valuable in terms of the high number of samples examined by the national virology reference laboratory over a 9-year period, and will contribute to clinicians' understanding of the epidemiology of meningitis in our country, and assist in identifying when and how to investigate patients for possible meningitis.

A fast nucleic acid extraction system for point-of-care and integration of digital PCR

Digital PCR is a powerful amplification method for absolute quantification of nucleic acids. The systems that integrated the nucleic acid extraction and amplification can reduce detection time, improve accuracy, and reduce labor costs. However, current nucleic acid extraction systems cannot be integrated well with integrated fluidic circuit (IFC) dPCR or droplet digital PCR chips perfectly and limit the application of digital PCR. In this study, a polytetrafluoroethylene (PTFE)-based nucleic acid extraction (PNE) system, which was able to achieve fully closed extraction for micro samples and was able to be integrated with IFC dPCR or droplet digital dPCR (ddPCR) chips perfectly is proposed. For this system, PTFE tubing with an inner diameter of 1 mm was used to load the reagents and superparamagnetic particles (PMPs) were used to extract nucleic acids. The system can extract nucleic acids from cells and blood in 5 minutes. Meanwhile, when nucleic acid extraction was completed, PNE was able to be directly combined with IFC dPCR or ddPCR chips without any intermediate steps. Therefore, the PNE system can realize sample-in-digital-answer-out. It will be highly useful in point-of-care (POC) and promote the development and application of dPCR.

A programmable type of CRISPR system

Biotechnology CRISPR-Cas9 systems have been causing a revolution in biology. Harrington et al. describe the discovery and technological implementation of an additional type of CRISPR system based on an extracompact effector protein, Cas14. Metagenomics data, particularly from uncultivated samples, uncovered the CRISPR-Cas14 systems containing all the components necessary for adaptive immunity in prokaryotes. At half the size of class 2 CRISPR effectors, Cas14 appears to target single-stranded DNA without class 2 sequence restrictions. By leveraging this activity, a fast and high-fidelity nucleic acid detection system enabled detection of single-nucleotide polymorphisms. Science , this issue p. [839][1] [1]: /lookup/doi/10.1126/science.aav4294

QuantGenius: implementation of a decision support system for qPCR-based gene quantification

BackgroundQuantitative molecular biology remains a challenge for researchers due to inconsistent approaches for control of errors in the final results. Due to several factors that can influence the final result, quantitative analysis and interpretation of qPCR data are still not trivial. Together with the development of high-throughput qPCR platforms, there is a need for a tool allowing for robust, reliable and fast nucleic acid quantification.ResultsWe have developed “quantGenius” (http://quantgenius.nib.si), an open-access web application for a reliable qPCR-based quantification of nucleic acids. The quantGenius workflow interactively guides the user through data import, quality control (QC) and calculation steps. The input is machine- and chemistry–independent. Quantification is performed using the standard curve approach, with normalization to one or several reference genes. The special feature of the application is the implementation of user-guided QC-based decision support system, based on qPCR standards, that takes into account pipetting errors, assay amplification efficiencies, limits of detection and quantification of the assays as well as the control of PCR inhibition in individual samples. The intermediate calculations and final results are exportable in a data matrix suitable for further statistical analysis or visualization. We additionally compare the most important features of quantGenius with similar advanced software tools and illustrate the importance of proper QC system in the analysis of qPCR data in two use cases.ConclusionsTo our knowledge, quantGenius is the only qPCR data analysis tool that integrates QC-based decision support and will help scientists to obtain reliable results which are the basis for biologically meaningful data interpretation.

Fast therapeutic DNA internalization – A high potential transfection system based on a peptide mimicking cationic lipid

The delivery of nucleic acids into cells is a determining factor for successful gene therapy. In this study we investigate the uptake and time dependent processing of a lipid-based non-viral nucleic acid delivery system composed of a peptide-mimicking cationic lipid (N-{6-amino-1-[N-(9Z)-octadec-9-enylamino]-1-oxohexan-(2S)-2-yl}-N′-{2-[N,N-bis(2-aminoethyl)amino]ethyl}-2-hexadecylpropandiamide - OH4) and a phospholipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine - DOPE). Studies by confocal laser scanning microscopy (CLSM) indicate a rapid internalization of fluorescent labelled DNA within 1h. Furthermore, vesicular structures on the lipid surface were reported, which are associated with the application of the lipid-based non-viral vector. Time dependent investigations of the gene expression of a reporter gene encoding for enhanced green fluorescent protein (eGFP) or luciferase in 4 different cell lines demonstrate an initial gene expression soon after 4h followed by a boost in gene expression beginning from 12h to 24h. Investigations with selective blocking of endocytic pathways using low molecular weight inhibitors suggested clathrin-mediated endocytosis as main internalization route in 3 cell lines. Our research presents a new horizon in rapid gene therapy using non-viral vectors; due to the modifications of the lipid components, fast nucleic acid internalizations could be achieved using our delivery systems.

Digitale PCR in der Labordiagnostik

The need for new highly sensitive, cost-efficient, fast and robust nucleic acid detection and quantification technologies is a driving force. PCR, especially quantitative PCR (qPCR), is the method of choice in diagnostics and life-sciences. The digital PCR (dPCR) provides a new technology to measure absolute quantities of nucleic acids without the need for calibration curves. This review gives details on the dPCR technology and available platforms. We discuss the platform differences, common features as well as their advantages and disadvantages.

Fast nucleic acid amplification for integration in point‐of‐care applications

An ultrafast microfluidic PCR module (30 PCR cycles in 6 min) based on the oscillating fluid plug concept was developed. A robust amplification of native genomic DNA from whole blood samples could be achieved at operational conditions established from systematic investigations of key parameters including heat transfer and in particular flow velocities. Experimental data were augmented with results from computational fluid dynamics simulations. The reproducibility of the current system was substantially improved compared to previous concepts by integration of a closed reservoir instead of utilizing a vented channel end at ambient pressure rendering the devised module suitable for integration into complex sample-to-answer analysis platforms such as point-of-care applications.

Extraction of genomic DNA and detection of single nucleotide polymorphism genotyping utilizing an integrated magnetic bead-based microfluidic platform

This study presents a new magnetic bead-based microfluidic platform, which integrates three major modules for rapid leukocytes purification, genomic DNA (gDNA) extraction and fast analysis of genetic gene. By utilizing microfluidic technologies and magnetic beads conjugated with CD15/45 antibodies, leukocytes in a human whole blood sample can be first purified and concentrated, followed by extraction of gDNA utilizing surface-charge switchable, DNA-specific, magnetic beads in the lysis solution. Then, specific genes associated with genetic diseases can be amplified by an on-chip polymerase chain reaction (PCR) process automatically. The whole pretreatment process including the leukocytes purification and gDNA extraction can be performed in an automatic fashion with the incorporation of the built bio-separators consisting of microcoils array within less than 20 min. The detection of single nucleotide polymorphism (SNP) genotyping of methylenetetra-hydrofolate reductase (MTHFR) C677T region associated with an increased risk of genetic diseases was further performed to demonstrate the capability of the proposed system. The extracted gDNA can be transported into a micro PCR chamber for on-chip fast nucleic acid amplification of detection genes with minimum human intervention. Hence, the developed system may provide a powerful automated platform for pretreatment of human leukocytes, gDNA extraction and fast analysis of genetic gene.

Microfluidic Systems Integrated With a Sample Pretreatment Device for Fast Nucleic-Acid Amplification

This paper presents a new miniature reverse-transcription polymerase chain-reaction (RT-PCR) system integrating a sample pretreatment device for fast nucleic-acid amplification and diagnosis of viruses and bacteria. In the system, a two-way serpentine-shape (s-shape) pneumatic micropump and a magnetic bioseparator were developed for separation and enrichment of viruses and bacteria. This new bioseparator can also be used as microheating chambers to perform RT-PCR. Taking advantage of the specific interaction between the antibodies on the surface of magnetic beads and the surface antigens on viruses or bacteria, the target virus and bacteria were recognized and further separated and purified from the biosamples by a magnetic field generated by the bioseparator. The target purified virus/bacteria was then lysed to release ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) for the subsequent RT-PCR processes. Experimental results showed that the target virus/bacteria was successfully separated and enriched by the high specificity and selectivity of antibody-conjugated magnetic beads, and the subsequent amplification of RNA/DNA was automatically completed by utilizing the on-chip microheaters and the micro temperature sensor. The high mixing efficiency of the two-way s-shape pump and the rapid heating/cooling rate of the microheating chambers can significantly shorten the pretreatment and diagnosis processes. As a whole, the developed system may provide a powerful platform for sample pretreatment and fast disease diagnosis.