Rapid DNA Research Articles

A rapid, efficient and cost-effective DNA isolation protocol for various herbal products which is appropriate for different downstream genomics applications

A rapid, efficient and cost-effective DNA isolation protocol for various herbal products which is appropriate for different downstream genomics applications

A simple and rapid DNA extraction method from meat by microneedle patch for detection of adulterated components

ABSTRACT The simple and rapid identification of meat via DNA-based methods is still subject to the conditions of complex and time-consuming preprocessing. It usually takes 3–4 h to obtain DNA sample using conventionally available DNA extraction kits. The existing approaches are cumbersome, require large equipment, and are not suitable for field operation. This study developed a swelling microneedle patch, constituted by micron-sized copolymers of methyl vinyl ether and maleic acid (PMVE/MA). The microneedle patch was applied to the meat sample for 1 min before being pulled out and rinsed with Tris-EDTA (TE) buffer to obtain DNA, which could be used for polymerase chain reaction (PCR) without purification. The DNA extracted by the microneedle patch could meet the needs for amplification and identification of meat samples. The developed approach which combines the patch-based extraction method and conventional PCR amplification is quick, simple, and reliable for DNA extraction and identification of meat samples.

Rapid DNA extraction and colorimetric amplicon visualisation speed up LAMP-based detection of soybean allergen in foods

AbstractDetection of allergens in foods, including soybean, is relevant for food labelling requirements. Moreover, allergen-specific methods may allow standardisation of allergens in food matrices for use in food challenges as allergy diagnostic approaches. Rapid methods are preferred for screening and along the manufacturing line. Previously, we demonstrated sensitive and specific detection of soybean DNA by combining loop-mediated isothermal amplification (LAMP) and lateral flow device (LFD)-like visualisation. However, lengthy DNA extraction and potential contamination of subsequent by previous LAMP reactions from unclosed LFD may impact its use as a rapid and robust method. Here, we developed a rapid protocol for DNA extraction. Moreover, we identified phenol red for distinct visualisation of positive reactions in permanently closed reaction tubes. The optimised method was validated using complex foods (boiled sausage, instant soup, and chocolate) with known amounts of soybean. Further, its applicability was shown in 12 processed retail foods. Results were verified by orthogonal qPCR. The enhanced LAMP method allowed detection at or below 10 mg soybean per kg processed food. The method provides rapid and easy-to-use screening without the need for detection equipment. Hence, it may serve to verify the presence of soybean ingredients and support a risk-based precautionary labelling of non-ingredient soybean in compound foods. Also, as determination of clinical reaction thresholds before and after allergen immunotherapy (AIT) is both inclusion and exclusion criterion for clinical trials and success parameter of AIT, the method may allow verification of calculable soybean content in provocation meals and thus a standardised administration for threshold determination before and after AIT.

Artificially Intelligent Nanogap for Rapid DNA Sequencing: A Machine Learning Aided Quantum Tunneling Approach

Artificially Intelligent Nanogap for Rapid DNA Sequencing: A Machine Learning Aided Quantum Tunneling Approach

Comparative analysis of two Rapid DNA technologies for the processing of blood and saliva-based samples

Rapid DNA technologies recently gained significant momentum as a means to generate DNA profiles faster than with standard laboratory workflows. Initially developed for the analysis of buccal reference samples, applications are being considered for other types of forensic samples. In this study, an identical set of 150 blood and saliva-based samples was processed using two different Rapid DNA technologies, the Applied BioSystems™ RapidHIT™ ID System using the RapidINTEL™ sample cartridge and the ANDE™ 6C Rapid DNA Analysis™ System using the I-Chip. A subset of samples were subjected to alternative collection methods or sample pre-treatments to determine the optimal strategy for each instrument. An equivalent sample set was also processed using a conventional DNA analysis workflow. The sensitivity range of the two Rapid DNA technologies was comparable based on blood and saliva dilution series, with both technologies able to generate full profiles from samples typically yielding 5–10 ng of DNA when processed using conventional DNA analysis. The brand of cotton swabs used for Rapid DNA analysis had an impact on the results for both systems. Differences were observed in success rate between the two systems when processing blood (on fabrics, FTA paper or hard surfaces) and saliva-based samples (drink containers, FTA paper, chewing gum, cigarette butt filter paper) and depended on the sample type. Importantly, deviating from the manufacturer’s instructions for sample collection and pre-treatment was more detrimental to the ANDE 6C results. The quality of DNA profiles, as assessed using heterozygote peak height ratios, interloci balance and artifact presence, confirmed the results to be reliable and acceptable for single source samples. Profiling results were obtained when samples were reprocessed using the same Rapid DNA technology or conventional DNA analysis. Secondary analysis using a substitute software (GeneMapper ID-X v1.5) to recover additional genetic information was shown to be feasible. Finally, a comparison between the Applied Biosystems™ RapidHIT™ ID System Software v1.3.1 and v1.3.2 was also performed. Findings of this study could assist those interested in using Rapid DNA technology for blood or saliva-based samples, in various settings and for different applications.

Abstract P3063: Selective Release Of Mitochondrial Dna After Brief Myocardial Ischemia

Objective: Mitochondrial DNA (mtDNA) is one of several pro-inflammatory damage-associated molecular patterns released from necrotic myocytes during myocardial infarction (MI). Based on our recent observation of a ~50-fold rise in circulating mtDNA after brief whole-body ischemia in a porcine model of cardiac arrest without ongoing MI, we hypothesized that ischemia-induced mtDNA release can occur in the absence of necrosis. Accordingly, we measured circulating mtDNA levels before and after localized myocardial ischemia of insufficient duration to produce myocyte necrosis in swine. Methods: Swine (n=6) were subjected to a 10-minute occlusion of the left anterior descending (LAD) coronary artery based on prior evidence that this duration of ischemia elicits myocardial injury without necrosis. Peripheral venous and coronary sinus (CS) blood samples collected at baseline (BL) as well as 1-hour and 24-hours after reperfusion were analyzed via qPCR to quantify mtDNA and nuclear DNA (nucDNA) release. Results were compared to a separate group of swine (n=6) subjected to a 75-minute LAD occlusion to produce MI. Results: Prolonged ischemia led to a significant rise in CS levels of mtDNA (6.2 ± 3.6-fold vs. BL; p<0.05) and nucDNA (5.4 ± 2.0-fold vs. BL; p<0.05) 1 hour after reperfusion, consistent with rapid non-selective DNA release after myocyte necrosis. In contrast, brief ischemia was associated with detectable elevations in CS mtDNA (4.6 ± 1.4-fold vs. BL; p<0.05) but not nucDNA (2.1 ± 0.5-fold vs. BL; p=0.35) at this timepoint. However, the magnitude of DNA release after brief or prolonged ischemia was not sufficient to increase circulating mtDNA or nucDNA, as peripheral venous levels of mtDNA and nucDNA did not differ from BL at 1-hour or 24-hours post-reperfusion in either group. Conclusion: Brief myocardial ischemia is associated with selective release of mtDNA into circulation, providing a potential mechanism by which immune activation may occur after myocardial injury even when myocyte necrosis is absent. The magnitude of mtDNA release after brief myocardial ischemia is ~10-fold lower than that detected in peripheral blood after resuscitation from cardiac arrest, suggesting that mtDNA is released from other organs after brief whole-body ischemia.

CRISPR/Cas12a combined with RPA for detection of T. gondii in mouse whole blood

BackgroundToxoplasma gondii is an opportunistic protozoan that is ubiquitous in humans and animals. It can invade any human organ and cause severe diseases, including toxoplasma ophthalmopathy, meningoencephalitis, and liver necrosis. Porcine toxoplasmosis is prevalent in China. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas (CRISPR-Associated Protein) systems are widely used for gene editing and pathogen detection. CRISPR-based diagnostics are molecular assays that have been developed to detect parasites with high sensitivity and specificity.MethodsThis study aimed to establish a combined CRISPR/Cas12a and RPA rapid detection method for T. gondii by targeting the B1 gene and 529 bp repeat element (529 RE). The detection results could be visualized by the fluorescence or lateral flow strips (LFS). The sensitivity and specificity of the method were evaluated, and T. gondii-infected mouse blood was used for detection.ResultsThe results indicated that the established method for T. gondii detection was satisfactory, with a detection limit of 1.5 cp/μl for the two loci. Moreover, the B1 gene could detect 1 tachyzoite per reaction, and the 529 RE could detect 0.1 tachyzoite per reaction, consistently with the highly sensitive nested polymerase chain reaction (PCR) results. The method was suitable for strains, including RH, and did not cross-react with other protozoa DNA with similar habits. The T. gondii-infected mouse blood samples were all positive for T. gondii at 1, 3, and 5 days post infection (dpi).ConclusionsThis study established a rapid, sensitive, and time-saving DNA detection method for T. gondii that has the potential to be an alternative tool for T. gondii detection in the field.Graphical abstract

Rapid and Sensitive Human-Specific DNA Quantitation Using a Microfluidic Amplification Module at the Point-of-Care.

A rapid microfluidic human-specific DNA quantitation assay module was developed for chip-based amplification of the human TH01 and Alu loci in the presence of PicoGreen. The method makes use of the thermal cycler and 488 nm Solid State laser-based optical train that are components of the fully-integrated, sample-in to results out, ANDE Rapid Nucleic Acid Analysis system. The assay was effective in quantitating human DNA from a variety of sample types, including blood, buccal, and forensic touch samples mixed with varying amounts of non-human DNA. The 28-cycle TH01 and 10-cycle Alu reactions were completed in 18 minutes and 7 minutes, respectively. The observed limit of detection (LOD) of the assay is approximately 0.3 ng, and the flexibility of assay design allows an LOD of as little as 0.005 femtograms.Clinical Relevance-We have developed a fully-integrated, sample-in to results-out, Rapid Nucleic Acid Analysis system that characterizes nucleic acid fragments (whether generated by PCR, rt-PCR, sequencing, or SNP reactions) by electrophoresis in plastic microfluidic channels. Here we describe the development, characterization, and validation of the microfluidic quantitation module. The quantitation module is the first that can be incorporated into integrated microfluidic workflows for the analysis of highly-multiplexed clinical diagnostic assays interrogating hundreds of genomic targets in a single sample. In particular, the use of a microfluidic quantitation module allows reaction volumes, thermal cycling conditions, and electrophoretic injection protocols to be determined based on nucleic acid content during and throughout fully-automated processing-dramatically enhancing the power of the fully-automated diagnostic system.

A Miniaturized System for Rapid, Isothermal Detection of SARS-CoV-2 in Human and Environmental Samples.

We report a small-footprint cost-effective isothermal rapid DNA amplification system, with integrated microfluidics for automated sample analysis and detection of SARS-CoV-2 in human and environmental samples. Our system measures low-level fluorescent signals in real-time during amplification, while maintaining the desired assay temperature on a low power, portable system footprint. A unique soft microfluidic chip design was implemented to mitigate thermocapillary effects and facilitate optical alignment for automated image capture and signal analysis. The system-on-board prototype, coupled with the LAMP primers designed by BioCoS, was sensitive enough to detect large variations in viral loads of SARS-CoV-2 corresponding to a threshold cycle range of 16 to 39. Furthermore, tested samples consisted of a broad range of viral strains and lineages identified in Canada during 2021-2022. Clinical specimens were collected and tested at the Kingston Health Science Centre using a clinically validated PCR assay, and variants were determined using whole genome sequencing.

Design and Analytical Evaluation of a Rapid Plasma Screening Assay for Circulating Human Papillomavirus DNA via Thermostable Enzyme Chemistries.

Infection with oncogenic strains of human papillomavirus (HPV), such as HPV-16 and HPV-18, can lead to malignant progression and tumorigenesis. As an adjunct to traditional invasive tissue sampling methods, the use of modern thermostable enzyme chemistries can aid in the development of innovative assay workflows to extract and detect circulating HPV DNA (cHPV-DNA) in liquid biopsies. In this work, we first successfully generated a model system to replicate fragmented cHPV-DNA in human plasma. Using this model system, we designed a novel thermostable enzyme chemistry-based cHPV-DNA assay for rapid clinical HPV screening and robustly evaluated its analytical assay performance. Our findings demonstrated that the use of thermostable enzymes provided faster cHPV-DNA extraction and amplification, leading to an overall three-fold improvement in overall assay time as compared to the current standard assay workflow and achieving clinically relevant levels of analytical specificity, sensitivity, and precision for accurate cHPV-DNA detection with excellent 100% sensitivity and specificity in contrived human plasma specimens. In summary, we have devised a rapid laboratory workflow to facilitate the emerging use of liquid biopsies for minimally invasive, rapid, and scalable HPV DNA testing. With facile assay modifications, our thermostable enzyme-based cHPV-DNA assay can be utilized for the detection of other clinically high-risk HPV genotypes.

Synthetic gRNA/Cas9 ribonucleoprotein targeting HBV DNA inhibits viral replication.

The presence of hepatitis B virus (HBV) covalently closed circular (ccc) DNA (cccDNA), which serves as a template for viral replication and integration of HBV DNA into the host cell genome, sustains liver pathogenesis and constitutes an intractable barrier to the eradication of chronic HBV infection. The current antiviral therapy for HBV infection, using nucleos(t)ide analogues (NAs), can suppress HBV replication but cannot eliminate integrated HBV DNA and episomal cccDNA. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 is a powerful genetic tool that can edit integrated HBV DNA and minichromosomal cccDNA for gene therapy, but its expression and delivery require a viral vector, which poses safety concerns for therapeutic applications in humans. In the present study, we used synthetic guide RNA (gRNA)/Cas9-ribonucleoprotein (RNP) as a nonviral formulation to develop a novel CRISPR/Cas9-mediated gene therapy for eradicating HBV infection. We designed a series of gRNAs targeting multiple specific HBV genes and tested their antiviral efficacy and cytotoxicity in different HBV cellular models. Transfection of stably HBV-infected human hepatoma cell line HepG2.2.15 with HBV-specific gRNA/Cas9 RNPs resulted in a substantial reduction in HBV transcripts. Specifically, gRNA5 and/or gRNA9 RNPs significantly reduced HBV cccDNA, total HBV DNA, pregenomic RNA, and HBV antigen (HBsAg, HBeAg) levels. T7 endonuclease 1 (T7E1) cleavage assay and DNA sequencing confirmed specific HBV gene cleavage and mutations at or around the gRNA target sites. Notably, this gene-editing system did not alter cellular viability or proliferation in the treated cells. Because of their rapid DNA cleavage capability, low off-target effects, low risk of insertional mutagenesis, and readiness for use in clinical application, these results suggest that synthetic gRNA/Cas9 RNP-based gene-editing can be utilized as a promising therapeutic drug for eradicating chronic HBV infection.

A new multiplex qPCR assay to detect and differentiate big cat species in the illegal wildlife trade

All species of big cats, including tigers, cheetahs, leopards, lions, snow leopards, and jaguars, are protected under the Convention on the International Trade in Endangered Species (CITES). This is due in large part to population declines resulting from anthropogenic factors, especially poaching and the unregulated and illegal trade in pelts, bones, teeth and other products that are derived from these iconic species. To enhance and scale up monitoring for big cat products in this trade, we created a rapid multiplex qPCR test that can identify and differentiate DNA from tiger (Panthera tigris), cheetah (Acinonyx jubatus), leopard (Panthera pardus), lion (Panthera leo), snow leopard (Panthera uncia), and jaguar (Panthera onca) in wildlife products using melt curve analysis to identify each species by its unique melt peak temperature. Our results showed high PCR efficiency (> 90%), sensitivity (detection limit of 5 copies of DNA per PCR reaction) and specificity (no cross amplification between each of the 6 big cat species). When paired with a rapid (< 1 h) DNA extraction protocol that amplifies DNA from bone, teeth, and preserved skin, total test time is less than three hours. This test can be used as a screening method to improve our understanding of the scale and scope of the illegal trade in big cats and aid in the enforcement of international regulations that govern the trade in wildlife and wildlife products, both ultimately benefiting the conservation of these species worldwide.

Introducing the New Editorial Team

Introducing the New Editorial Team

ИТОГИ ПИЛОТНОГО ПРОЕКТА ПО НЕОНАТАЛЬНОМУ СКРИНИНГУ СПИНАЛЬНОЙ МЫШЕЧНОЙ АТРОФИИ В САНКТ-ПЕТЕРБУРГЕ: ОРГАНИЗАЦИОННЫЕ, ДИАГНОСТИЧЕСКИЕ И КЛИНИЧЕСКИЕ АСПЕКТЫ

Spinal muscular atrophy (SMA) is one of the most frequent and severe hereditary diseases that leads to the destruction of motor neurons in the spinal cord and muscle weakness and is resulted from mutations in the SMN1 gene. The purpose of this research was to identify homo- and heterozygous mutations in the SMN1 gene in newborns. Materials and methods used: a multicenter prospective open continuous screening study of newborns at the age of 4 DoL (for full-term)/7 DoL (for preterm). The screening was carried out in Dec. 30, 2021-Nov. 17, 2022 on the basis of 21 obstetrics facilities located within the City of Saint Petersburg (Russia). Results: 36,140 participants (17,687 (49%) boys/18,453 (51%) girls) of the neonatal screening; 77 (0.21%) parents/caretakers have refused to participate. A technique for detecting homo- and heterozygous deletions in the SMN1 gene was developed based on rapid DNA extraction from dried blood spots followed by the real-time PCR. The project had identified 4 patients with SMA, two of whom had two and the other two had three copies of the SMN2 gene. The period of time needed to set the diagnosis ranged from 7th to 14th DoL. All of these patients have received the gene replacement therapy for a period of 3 to 5 months. There was a positive trend as a result of the therapy in all of the cases. 772 SMA carriers were also identified during the screening. 51% of their parents/caretakers have been followingly consulted on the possible risks of the disease. The frequency of SMA in the City of Saint Petersburg was 1 in 9035 newborns with a carrier frequency of 1 in 47. The sensitivity and specificity of the test were determined, which were 99.74% and 100%, respectively. Conclusion: SMA screening has been tested and put into practice. The conducted project of neonatal screening for SMA in the City of Saint Petersburg has demonstrated the readiness of its residents and the municipal healthcare system, the practitioners and organizers to this type of medical solution. The described developed methodology has demonstrated its effectiveness in terms of early detection of patients and carriers of this disease.

Pristine Multi-walled carbon nanotubes for a rapid and efficient plasmid DNA clarification

Therapeutic approaches based on nucleic acids to modulate cell activity have recently gained attention. These molecules arise from complex biotechnological processes, requiring effective manufacturing strategies, high purity, and precise quality control to be used as biopharmaceuticals. One of the most critical and time-consuming steps for nucleic acids-based biotherapeutics manufacturing is their purification, mainly due to the complexity of the extracts. In this study, a simple, efficient, and reliable method to isolate and clarify plasmid DNA (pDNA) from complex samples is described. The method is based on the selective capture of RNA and other impurities, using pristine carbon nanotubes (CNTs). Multi-walled CNTs (MWCNTs) with different diameters were studied to determine their adsorption capacity and to address their ability to interact and distinguish between nucleic acids. The results revealed that MWCNTs preferentially interact with RNA and that smaller MWCNTs present a higher adsorption capacity, as expected by the higher specific surface area. Overall, this study showed that MWCNTs significantly reduce the levels of impurities, namely RNA, gDNA, and proteins, by approximately 83.6 % compared to their initial level, enabling the recovery of clarified pDNA in solution while maintaining its stability throughout the recovery process. This method facilitates the pre-purification of pDNA for therapeutic applications.

A CRISPR-Cas12a-based platform for ultrasensitive rapid highly specific detection of Mycobacterium tuberculosis in clinical application.

Tuberculosis, caused by Mycobacterium tuberculosis (MTB), is the second leading cause of death after COVID-19 pandemic. Here, we coupled multiple cross displacement amplification (MCDA) technique with CRISPR-Cas12a-based biosensing system to design a novel detection platform for tuberculosis diagnosis, termed MTB-MCDA-CRISPR. MTB-MCDA-CRISPR pre-amplified the specific sdaA gene of MTB by MCDA, and the MCDA results were then decoded by CRISPR-Cas12a-based detection, resulting in simple visual fluorescent signal readouts. A set of standard MCDA primers, an engineered CP1 primer, a quenched fluorescent ssDNA reporter, and a gRNA were designed targeting the sdaA gene of MTB. The optimal temperature for MCDA pre-amplification is 67°C. The whole experiment process can be completed within one hour, including sputum rapid genomic DNA extraction (15 minutes), MCDA reaction (40 minutes), and CRISPR-Cas12a-gRNA biosensing process (5 minutes). The limit of detection (LoD) of the MTB-MCDA-CRISPR assay is 40 fg per reaction. The MTB-MCDA-CRISPR assay does not cross reaction with non-tuberculosis mycobacterium (NTM) strains and other species, validating its specificity. The clinical performance of MTB-MCDA-CRISPR assay was higher than that of the sputum smear microscopy test and comparable to that of Xpert method. In summary, the MTB-MCDA-CRISPR assay is a promising and effective tool for tuberculosis infection diagnosis, surveillance and prevention, especially for point-of-care (POC) test and field deployment in source-limited regions.

Simple and Rapid DNA Isolation from Shrimps by Using Single tube extraction buffer with Single step

Simple and Rapid DNA Isolation from Shrimps by Using Single tube extraction buffer with Single step

Polγ coordinates DNA synthesis and proofreading to ensure mitochondrial genome integrity.

Accurate replication of mitochondrial DNA (mtDNA) by DNA polymerase γ (Polγ) is essential for maintaining cellular energy supplies, metabolism, and cell cycle control. To illustrate the structural mechanism for Polγ coordinating polymerase (pol) and exonuclease (exo) activities to ensure rapid and accurate DNA synthesis, we determined four cryo-EM structures of Polγ captured after accurate or erroneous incorporation to a resolution of 2.4-3.0 Å. The structures show that Polγ employs a dual-checkpoint mechanism to sense nucleotide misincorporation and initiate proofreading. The transition from replication to error editing is accompanied by increased dynamics in both DNA and enzyme, in which the polymerase relaxes its processivity and the primer-template DNA unwinds, rotates, and backtracks to shuttle the mismatch-containing primer terminus 32 Å to the exo site for editing. Our structural and functional studies also provide a foundation for analyses of Polγ mutation-induced human diseases and aging.

A self-powered biosensing system based on triboelectric nanogenerator for rapid bacterial DNA detection

A self-powered biosensing system based on triboelectric nanogenerator (TENG) for bacterial DNA detection is invented and a vertical contact-separation TENG was designed to provide a steady power supply to a detection bioreactor. A carboxyl-functionalized capture probe was modified on etched indium tin oxide (ITO) glass, with which target bacterial DNA could be recognized. Meanwhile, multi-walled carbon-nanotubes (CNT)-modified signal probe was designed to amplify the detection signal due to its excellent electrical conductivity. The self-powered biosensing system could measure the variation in output voltage and be highly sensible with a low detection limit at 0.084 pM of target DNA. In addition, a mathematical model based on the Freundlich isotherm was built to simulate the DNA detection process, and it was feasible to simulate the calibration equation and detection range. A portable self-powered biosensing device was made by integrating miniaturized TENG and bioreactor in a 3D-printed mold, with which the presence of bacterial DNA could be instantly indicated by LED (light-emitting diode). This study provided a useful platform for quick bacterial DNA detection and paved the way for further development of pocket diagnosis with TENG-based biosensing systems.

Rapid and scalable DNA extraction and real-time PCR assay from boxwood tissue for the detection of Calonectria pseudonaviculata, causal agent of boxwood blight.

Boxwood blight can be challenging to detect in the field, especially when symptoms are mild, thus requiring large numbers of plants to be screened. Therefore, a rapid diagnostic assay that can detect the pathogen from large amounts of plant tissue would be useful. Here, we present a crude DNA extraction protocol that is rapid and scalable. The DNA extraction protocol can process large volumes of tough boxwood tissue rapidly without using cetyltrimethylammonium bromide (CTAB) or phenol-chloroform to remove inhibitors. Additionally, to detect the boxwood blight pathogen, Calonectria pseudonaviculata, we developed a TaqMan probe to use with previously described PCR primers for a real-time PCR assay. The assay's limit of detection was determined by diluting symptomatic boxwood leaves in non-diseased leaves and by adding spores to non-diseased leaves to simulate diagnostic scenarios. The assay was able to detect the pathogen in symptomatic leaves diluted up to 1×104 to 1×105 fold in non-diseased leaves, and from as low as 1,000 to 10,000 spores added to 1.2 g of non-diseased leaves. The ability to extract DNA from large volumes of plant tissue facilitates screening more plant tissue using the real-time PCR assay without increasing the number of samples to process in the lab.