Isothermal DNA Amplification Research Articles

Evaluation of an electricity-independent method for IS2404 Loop-mediated isothermal amplification (LAMP) diagnosis of Buruli ulcer in resource-limited settings.

Buruli ulcer (BU) caused by Mycobacterium ulcerans (MU) is a devastating necrotic skin disease. PCR, recommended for confirmation of BU by WHO, requires an adequately equipped laboratory, therefore often delaying timely diagnosis and treatment of BU patients in remote settings. Loop-mediated isothermal amplification (LAMP) is a PCR-based protocol for isothermal amplification of DNA that has been suggested for diagnosis of BU in low-resource settings. This is an exploratory diagnostic test evaluation study, with an embedded qualitative sub-study. Its aims are two-fold: First, to evaluate a simple rapid syringe-based DNA extraction method (SM) in comparison with a more elaborate conventional DNA extraction method (CM), followed by a LAMP assay targeting IS2404 for the detection of MU, either using a commercially available pocket warmer (pw) or a heat block (hb) for incubation. Second, to complement this by exploring the diagnostic workflow for BU at a community-based health centre in an endemic area in rural Ghana as an example of a potential target setting, using interviews with researchers and health care workers (HCWs). Diagnostic test evaluation results are discussed in relation to the requirements of a target product profile (TPP) for BU diagnosis and the target setting. A protocol using SM for DNA extraction followed by IS2404 PCR (IS2404 PCRSM) was able to identify MU DNA in 73 out of 83 BU clinical specimens submitted for diagnosis. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of IS2404 PCRSM were 90.12%, 100%, 100% and 65.21% respectively, as compared to the reference standard IS2404 PCR in combination with a standard extraction protocol for mycobacterial DNA. Evaluation of the LAMP assay on 64 SM DNA extracts showed a sensitivity, specificity, PPV and NPV of 83.6%, 100%, 100% and 50%, respectively, using either pocket warmer (pwLAMPSM) or heat block (hbLAMPSM) for incubation of the reaction, as compared to the same reference standard. The limit of detection of pwLAMPSM was found to be 30 copies of the IS2404 target. Interview findings explored barriers to BU diagnosis and treatment, including perceptions of the disease, costs, and availability of transport. Participants confirmed that a diagnosis at the PoC, in addition to screening based on clinical criteria, would be advantageous in order to prevent delays and loss to follow-up. The high diagnostic and analytic accuracy of the pwLAMP, evaluated by us in combination with a syringe-based DNA extraction method, supports its potential use for the rapid detection of MU in suspected BU samples at the community or primary health care level without reliable electricity supply. Further optimization needs include a lysis buffer, evaluation directly at the PoC and/or other sites, assessing staff training requirements and quality control.

Self-Replicating DNA-Based Nanoassemblies.

The properties of DNA that make it an effective genetic material also allow it to be ideal for programmed self-assembly. Such DNA-programmed assembly has been utilized to construct responsive DNA origami and wireframe nanoassemblies, yet replicating these hybrid nanomaterials remains challenging. Here we report a strategy for replicating DNA wireframe nanoassemblies using the isothermal ligase chain reaction lesion-induced DNA amplification (LIDA). We designed a triangle wireframe structure that can be formed in one step by ring-closing of its linear analog. Introducing a small amount of the wireframe triangle to an excess of the linear analog and complementary fragments, one of which contains a destabilizing abasic lesion, leads to rapid, sigmoidal self-replication of the wireframe triangle via cross-catalysis. Using the same cross-catalytic strategy we also demonstrate rapid self-replication of a hybrid wireframe triangle containing synthetic vertices as well as the self-replication of circular DNA. This work reveals the suitability of isothermal ligase chain reactions such as LIDA to self-replicate complex DNA architectures, opening the door to incorporating self-replication, a hallmark of life, into biomimetic DNA nanotechnology.

Achieving unprecedented stability in lyophilized recombinase polymerase amplification with thermostable pyruvate kinase from Thermotoga maritima

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41°C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. Considering the onsite use of RPA reagents, lyophilized RPA reagents with long storage stability are highly desired. In this study, as one of the approaches to solve this problem, we attempted to use a thermostable pyruvate kinase (PK). PK gene was isolated from a thermophilic bacterium Thermotoga maritima (Tma-PK). Tma-PK was expressed in Escherichia coli and purified from the cells. Tma-PK exhibited higher thermostability than human PK. The purified Tma-PK preparation was applied to RPA as an ATP-regenerating enzyme. Liquid RPA reagent with Tma-PK exhibited the same performance as that with human PK. Lyophilized RPA reagent with Tma-PK exhibited higher performance than that with human PK. Combined with our previous results of RPA reagents of thermostable Pol from a thermophilic bacterium, Aeribacillus pallidus, the results in this study suggest that thermostable enzymes are preferable to mesophilic ones as a component in lyophilized RPA reagents.

A Diagnostic Chip for the Colorimetric Detection of Legionella pneumophila in Less than 3 h at the Point of Need.

Legionella pneumophila has been pinpointed by the World Health Organization as the highest health burden of all waterborne pathogens in the European Union and is responsible for many disease outbreaks around the globe. Today, standard analysis methods (based on bacteria culturing onto agar plates) need several days (~12) in specialized analytical laboratories to yield results, not allowing for timely actions to prevent outbreaks. Over the last decades, great efforts have been made to develop more efficient waterborne pathogen diagnostics and faster analysis methods, requiring further advancement of microfluidics and sensors for simple, rapid, accurate, inexpensive, real-time, and on-site methods. Herein, a lab-on-a-chip device integrating sample preparation by accommodating bacteria capture, lysis, and DNA isothermal amplification with fast (less than 3 h) and highly sensitive, colorimetric end-point detection of L. pneumophila in water samples is presented, for use at the point of need. The method is based on the selective capture of viable bacteria on on-chip-immobilized and -lyophilized antibodies, lysis, the loop-mediated amplification (LAMP) of DNA, and end-point detection by a color change, observable by the naked eye and semiquantified by computational image analysis. Competitive advantages are demonstrated, such as low reagent consumption, portability and disposability, color change, storage at RT, and compliance with current legislation.

The Utilization of the SaLux19-Based Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid and Sensitive Identification of Minute Amounts of a Biological Specimen.

Our research has developed a highly sensitive and simple assay to detect small amounts of animal and human biological material in less than 40 min. The handheld SaLux19 device developed at the Max Planck Institute of Experimental Medicine in Göttingen, Germany, was used to validate our concept. The proposed system uses isothermal amplification of DNA in a rapid assay format. Our results show that the assay can detect Sus scrofa nucleic acids with very high sensitivity and specificity. This detection system has potential for forensic scenarios.

Protein-to-DNA Converter with High Signal Gain.

DNA isothermal amplification techniques have been applied extensively for evaluating nucleic acid inputs but cannot be implemented directly on other types of biomolecules. In this work, we designed a proximity activation mechanism that converts protein input into DNA barcodes for the DNA exponential amplification reaction, which we termed PEAR. Several design parameters were identified and experimentally verified, which included the choice of enzymes, sequences of proximity probes and template strand via the NUPACK design tool, and the implementation of a hairpin lock on the proximity probe structure. Our PEAR system was surprisingly more robust against nonspecific DNA amplification, which is a major challenge faced in existing formats of the DNA-based exponential amplification reaction. The as-designed PEAR exhibited good target responsiveness for three protein models with a dynamic range of 4-5 orders of magnitude down to femtomolar input concentration. Overall, our proposed protein-to-DNA converter module led to the development of a stable and robust configuration of the DNA exponential amplification reaction to achieve high signal gain. We foresee this enabling the use of protein inputs for more complex molecular evaluation as well as ultrasensitive protein detection.

Use of the 23S rRNA gene as a target template in the universal loop-mediated isothermal amplification (LAMP) of genomic DNA from phytoplasmas.

Plant-pathogenic bacteria cause numerous diseases in host plants and can result in serious damage. Timely and accurate diagnostic techniques are, therefore, crucial. While advances in molecular techniques have led to diagnostic systems able to distinguish known plant pathogens at the species or strain level, systems covering larger categories are mostly lacking. In this study, a specific and universal LAMP-based diagnostic system was developed for phytoplasmas, a large group of insect-borne plant-pathogenic bacteria that cause significant agricultural losses worldwide. Targeting the 23S rRNA gene of phytoplasma, the newly designed primer set CaPU23S-4 detected 31 'Candidatus Phytoplasma' tested within 30 min. This primer set also showed high specificity, without false-positive results for other bacteria (including close relatives of phytoplasmas) or healthy plants. The detection sensitivity was ~10,000 times higher than that of PCR methods for phytoplasma detection. A simple, rapid method of DNA extraction, by boiling phytoplasma-infected tissues, was developed as well. When used together with the universal LAMP assay, it enabled the prompt and accurate detection of phytoplasmas from plants and insects. The results demonstrate the potential of the 23S rRNA gene as a versatile target for the LAMP-based universal detection of bacteria at the genus level and provide a novel avenue for exploring this gene as molecular marker for phytoplasma presence detection.IMPORTANCEPhytoplasmas are associated with economically important diseases in crops worldwide, including lethal yellowing of coconut palm, "flavescence dorée" and "bois noir" of grapevine, X-disease in stone fruits, and white leaf and grassy shoot in sugarcane. Numerous LAMP-based diagnostic assays, mostly targeting the 16S rRNA gene, have been reported for phytoplasmas. However, these assays can only detect a limited number of 'Candidatus Phytoplasma' species, whereas the genus includes at least 50 of these species. In this study, a universal, specific, and rapid diagnostic system was developed that can detect all provisionally classified phytoplasmas within 1 h by combining the LAMP technique targeting the 23S rRNA gene with a simple method for DNA extraction. This diagnostic system will facilitate the on-site detection of phytoplasmas and may aid in the discovery of new phytoplasma-associated diseases and putative insect vectors, irrespective of the availability of infrastructure and experimental resources.

Abstract 322: Ultraplex genome and exome sequencing of rare single cells

Abstract Single-cell genome analysis enables researchers to gain novel insights into tumor heterogeneity. Conducting single-cell genomic analysis using next-generation sequencing (NGS) methods has traditionally been challenging since the amount of genomic DNA present in a single cell is limited. PCR-based amplification methods normally have high error rates, low coverage uniformity, and extensive allelic drop-outs making interpretation of SNVs and CNVs challenging.Here we introduce a new whole-genome amplification method which utilizes an optimized isothermal DNA amplification reaction with a Hi-fidelity, error correcting polymerase and downstream library preparation protocol to allow pooling of amplified DNA. The key DNA amplification steps use multiple displacement amplification (MDA) technology to amplify gDNA directly from single cells, nuclei or previously isolated genomic DNA. During the MDA reaction, sample-barcodes are incorporated into the newly synthetized DNA, allowing the amplified DNA to be pooled and processed in to one ultraplex library. This method saves library preparation time, reduces DNA library costs and limits plastic consumable usage.Our current workflow allows 24 samples to be multiplexed in one library and 24 libraries to be produced for a total of 576 samples per sequencing flow cell. This method can be used to investigate chromosomal instability with low pass sequencing or SNV and CNV analysis following whole genome, exome-capture workflows. Citation Format: Ioanna Andreou, Samuel Rulli, Katja Heitz, Silke Huebner, Christin Meerschiff, Eric Lader. Ultraplex genome and exome sequencing of rare single cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 322.

Comparison of Warmstart colorimetric LAMP using heat-treated samples with two Plasmodium-detection methods in Ekiti state, southwestern Nigeria

Background: Malaria remains a deadly disease responsible for numerous deaths worldwide, especially in tropi-cal developing countries. Widely used methods for diagnosis include microscopy, which is laborious, lengthy, and error-prone, and rapid diagnostic tests (RDT), which have recently faced limitations on accuracy. Molecular methods like the polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) have emerged as accurate, sensitive, and rapid ways of detecting the disease. While PCR requires expensive equip-ment, LAMP is less expensive to perform. However, contemporary malaria LAMP still requires DNA extraction kits from patient samples. DNA extraction by heating can be used to lower the cost, time, and level of training required from end-users to perform LAMP in low-resource settings. Methods: Blood samples were screened for Plasmodium parasites using microscopy, followed by a commonly used RDT kit. Heat treatment was used for quick DNA extraction from the blood samples, and LAMP primer sets were then used together with Warmstart colorimetric LAMP reagents for isothermal DNA amplification. Agarose gel electrophoresis was used to confirm that the colorimetric observations in LAMP reactions were due to successful DNA amplification. The data obtained were analysed in GraphPad Prism and subjected to chi-square tests with 95% confidence level. Results: Warmstart colorimetric LAMP using heat-treated blood samples achieved successful DNA amplifica-tion and detected Plasmodium parasites in 43.3% of samples, compared to 40% and 30% Plasmodium parasite detection yielded by antigen RDT kit and microscopy respectively. However, the difference between the percent-ages obtained using the different methods was not statistically significant. LAMP approach further enabled the identification/discrimination of different Plasmodium species. Conclusion: The Warmstart LAMP using heat-treated samples can be used as a rapid, cheaper, and easier-to-perform molecular method for the detection and species-level differentiation of malaria parasites.

Real-time MBDi-RPA using methyl-CpG binding protein 2: A real-time detection method for simple and rapid estimation of CpG methylation status

BackgroundAnalysis of CpG methylation is informative for cancer diagnosis. Previously, we developed a novel method to discriminate CpG methylation status in target DNA by blocking recombinase polymerase amplification (RPA), an isothermal DNA amplification technique, using methyl-CpG binding domain (MBD) protein 2 (MBD2). The method was named MBD protein interference-RPA (MBDi-RPA). In this study, MBDi-RPA was performed using methyl-CpG binding protein 2 (MeCP2), another MBD family protein, as the blocking agent. ResultsMBDi-RPA using MeCP2 detected low levels of CpG methylation, showing that it had higher sensitivity than MBDi-RPA using MBD2. We also developed real-time RPA, which enabled rapid analysis of DNA amplification without the need for laborious agarose gel electrophoresis and used it in combination with MBDi-RPA. We termed this method real-time MBDi-RPA. The method using MeCP2 could determine the abundance ratio of CpG-methylated target DNA simply and rapidly, although highly sensitive detection was challenging. Significance and noveltyReal-time MBDi-RPA using MeCP2 could be potentially useful for estimating CpG methylation status in target DNA prior to more detailed analyses.

DNA digestion and formation of DNA-network structures with Holliday junction-resolving enzyme Hjc_15-6 in conjunction with polymerase reactions

The recently identified novel Holliday junction-resolving enzyme, termed Hjc_15-6, activity investigation results imply DNA cleavage by Hjc_15-6 in a manner that potentially enhances the molecular self-assembly that may be exploited for creating DNA-networks and nanostructures. The study also demonstrates Pwo DNA polymerase acting in combination with Hjc_15-6 capability to produce large amounts of DNA that transforms into large DNA-network structures even without DNA template and primers. Furthermore, it is demonstrated that Hjc_15-6 prefers Holliday junction oligonucleotides as compared to Y-shaped oligonucleotides as well as efficiently cleaves typical branched products from isothermal DNA amplification of both linear and circular DNA templates amplified by phi29-like DNA polymerase. The assembly of large DNA network structures was observed in real time, by transmission electron microscopy, on negative stained grids that were freshly prepared, and also on the same grids after incubation for 4 days under constant cooling. Hence, Hjc_15-6 is a promising molecular tool for efficient production of various DNA origamis that may be implemented for a wide range of applications such as within medical biomaterials, catalytic materials, molecular devices and biosensors.

Multi-enzymatic systems synergize new RCA technique amplified super-long dsDNA from DNA circle

BackgroundIn the field of DNA amplification, there are great challenges in the effectively amplify of long-chain amplification, especially amplification up to several hundred kb level. ResultsA novel technique for the unbiased whole genome amplification from a thimbleful of DNA circles, such as low as 10 ng/ 10 μL of the circular cpDNA or low as 5 ng/ 10 μL of the plasmid, is developed, which can amplify an abundance of the whole genome sequences. Specifically, the new technique that combines rolling-amplification and triple-enzyme system presents a tightly controlled process of a series of buffers/reactions and optimized procedures, that applies from the primer-template duplexes to the Elution step. The result of this technique provides a new approach for extending RCA capacity, where it can reach 200 kb from the circular cpDNA amplification and 150 kb from the plasmid DNA amplification, that demonstrates superior breadth and evenness of genome coverage, high reproducibility, small amplification bias with the amplification efficiency. Significance and noveltyThis new technique will develop into one of the powerful tools for isothermal DNA amplification in vitro, genome sequencing/analysis, phylogenetic analysis, physical mapping, and other molecular biology applications.

A multiplexed, allele-specific recombinase polymerase amplification assay with lateral flow readout for sickle cell disease detection.

Isothermal nucleic acid amplification tests have the potential to improve disease diagnosis at the point of care, but it remains challenging to develop multiplexed tests that can detect ≥3 targets or to detect point mutations that may cause disease. These capabilities are critical to enabling informed clinical decision-making for many applications, such as sickle cell disease (SCD). To address this, we describe the development of a multiplexed allele-specific recombinase polymerase amplification (RPA) assay with lateral flow readout. We first characterize the specificity of RPA using primer design strategies employed in PCR to achieve point mutation detection, and demonstrate the utility of these strategies in achieving selective isothermal amplification and detection of genomic DNA encoding for the healthy βA globin allele, or genomic DNA containing point mutations encoding for pathologic βS and βC globin alleles, which are responsible for most sickle cell disorders. We then optimize reaction conditions to achieve multiplexed amplification and identification of the three alleles in a single reaction. Finally, we perform a small pilot study with 20 extracted genomic DNA samples from SCD patients and healthy volunteers - of the 13 samples with valid results, the assay demonstrated 100% sensitivity and 100% specificity for detecting pathologic alleles, and an overall accuracy of 92.3% for genotype prediction. This multiplexed assay is rapid, minimally instrumented, and when combined with point-of-care sample preparation, could enable DNA-based diagnosis of SCD in low-resource settings. The strategies reported here could be applied to other challenges, such as detection of mutations that confer drug resistance.

Lamp Primer Designing Software: The Overview

The loop-mediated isothermal amplification (LAMP) of DNA is considered as the most promising method for DNA diagnostics of infectious diseases in a “point-of-care testing” format. Currently, the online services and downloadable programs have been developed for design of LAMP primers, some of them (freely available) are overviewed here. Among them, the PrimerExplorer software appears to be mostly used, followed by the NEB LAMP Primer Design Tool that is steadily gaining popularity. The other non-commercial programs such as GLAPD, MorphoCatcher, LAMPrimer iQ, and LAVA have been utilized for LAMP primer design much more rarely. The advantages and disadvantages of the currently existing free software for the construction of LAMP primers, alongside with directions for its further development, are discussed.

Optimization of the molecular detection assay of Salmonella (MDS®) for drag swab samples of aviaries.

Detection of Salmonella sp. is important for the broiler chicken production chain because it is one microorganisms involved in food-borne diseases. Thus, this study performed the optimization of a technique of Loop-mediated isothermal DNA amplification (LAMP) through the 3MTM Molecular Detection Assay 2: Salmonella (MDS®), in accordance with Ordinance number 126 of the Ministry of Agriculture, for the detection of Salmonella sp. in drag swab. The methodology followed ISO 16140-2: 2016, with the analysis naturally contaminated drag swab samples collected from broiler aviaries and artificially contaminated with salmonella ATCCs. Of the 300 samples processed in protocol A (pre-enrichment tetrathionate broth (TT)), 45 were positive for Salmonella sp., 242 negative, one false-positive, and 12 false-negative, while of the 300 samples analyzed in protocol B (pre-enrichment brain-heart infusion broth (BHI)), 40 were positive, 256 negative, one false-positive, and three false-negative. The result for protocol A was a sensitivity of 79%, specificity of 99.6%, Positive Predictive Value (PPV) of 98%, and Negative Predictive Value (NPV) of 95%; and for protocol B, 93% sensitivity, 99.6% specificity, 98% PPV, and 99% NPV. Both protocols were associated with the reference method (p>0.05), concluding that the MDS® can be used for the qualitative detection of Salmonella sp.

Loop-Mediated Isothermal Amplification of DNA (LAMP) as an Alternative Method for Determining Bacteria in Wound Infections.

The increasing number of patients with chronic wounds requires the development of quick and accurate diagnostics methods. One of the key and challenging aspects of treating ulcers is to control wound infection. Early detection of infection is essential for the application of suitable treatment methods, such as systemic antibiotics or other antimicrobial agents. Clinically, the most frequently used method for detecting microorganisms in wounds is through a swab and culture on appropriate media. This test has major limitations, such as the long bacterial growth time and the selectivity of bacterial growth. This article presents an overview of molecular methods for detecting bacteria in wounds, including real-time polymerase chain reaction (rtPCR), quantitative polymerase chain reaction (qPCR), genotyping, next-generation sequencing (NGS), and loop-mediated isothermal amplification (LAMP). We focus on the LAMP method, which has not yet been widely used to detect bacteria in wounds, but it is an interesting alternative to conventional detection methods. LAMP does not require additional complicated equipment and provides the fastest detection time for microorganisms (approx. 30 min reaction). It also allows the use of many pairs of primers in one reaction and determination of up to 15 organisms in one sample. Isothermal amplification of DNA is currently the easiest and most economical method for microbial detection in wound infection. Direct visualization of the reaction with dyes, along with omitting DNA isolation, has increased the potential use of this method.

Detection of high-risk HPV 16 genotypes in cervical cancers using isothermal DNA amplification with electrochemical genosensor

Cervical cancer emerges as the third most prevalent types of malignancy among women on a global scale. Cervical cancer is significantly associated with the persistent infection of human papillomavirus (HPV) type 16. The process of diagnosing is crucial in order to prevent the progression of a condition into a malignant state. The early detection of cervical cancer through initial stage screening is of the utmost significance in both the prevention and effective management of this disease. The present detection methodology is dependent on quantitative polymerase chain reaction (qPCR), which necessitates the use of a costly heat cycler instrument. In this study, we report the development of an electrochemical DNA biosensor integrated with an isothermal recombinase polymerase amplification (RPA) reaction for the detection and identification of the high-risk HPV-16 genotype. The electrochemical biosensor exhibited a high degree of specificity and sensitivity, as evidenced by its limit of detection (LOD) of 0.23 copies/μL of HPV-16 DNA. The validity of this electrochemical platform was confirmed through the analysis of 40 cervical tissues samples, and the findings were consistent with those obtained through polymerase chain reaction (PCR) testing. Our straightforward electrochemical detection technology and quick turnaround time at 75 min make the assay suitable for point-of-care testing in low-resource settings.

Analysis of genetically modified soya and soya products using a fluorescence probe based on PCR-initiated isothermal amplification of G-quadruplex DNA

We proposed a novel strategy for the analysis of genetically modified (GM) soya samples by combining PCR-initiated generation and isothermal amplification of G-quadruplex DNA. PCR primers were designed to consist of a target-specific sequence, a G-quadruplex complement sequence, and a nicking endonuclease recognition site. After the PCR step, the nicking enzyme cut the recognition site, and single stranded G-quadruplex sequences were displaced and further amplified by polymerase during the isothermal amplification reaction. Then, the G-quadruplex sequences combined with N-methyl mesoporphyrin IX to generate strong fluorescence. This method has a detection limit of 0.2% and a linear range from 50% to 0.5% GM content. When exponential isothermal amplification was coupled to the preceding amplification step, the detection limit was further improved to 0.02% GM soya. GM contents were successfully detected at 0.1% GM percentage in soy milk and 0.9% GM percentage in tofu and dried tofu samples. The method may be applied to the analysis of various GM species simply by changing the target sequence in the primers.

BIOM-58. ZERO-WASTE MOLECULAR DIAGNOSTICS FROM BIOPSY NEEDLE WASHES

Abstract BACKGROUND Brain tumor classification has increasingly integrated molecular diagnostics into official criteria. However, biopsy tissue is limited due to the small quantity acquired in eloquent areas of the brain and is, therefore, usually earmarked for histology. We have identified that the fluid used to wash biopsy needles contains excess tumor material that can be diagnostically useful. Characterization of this non-zero-sum waste material provides an opportunity for improvement in the diagnosis and treatment for brain tumor patients. METHODS For suspected or established high grade brain tumor biopsies (n = 37 across a multi-institutional cohort at submission), neurosurgeons were instructed to save fluid used to wash biopsy needles. Samples were processed and characterized in a variety of methods to establish diagnostic utility including (1) cell-free DNA isolation and targeted panel sequencing (n = 13), (2) isothermal amplification of cell-free DNA from raw wash water for rapid diagnostics (n = 2), (3) cell-line generation (n = 24) and, (4) cell-free mutant protein capture and quantification (n = 9). RESULTS (1) 13 wash samples were considered for cf-tDNA extraction and Illumina TruSight Oncology 500 panel sequencing (with extracted DNA ranging from 20ng-40ug). In 2 returned cases (11 pending), concordance was 100% with clinical targeted panel sequencing where panels overlapped. (2) One case suspected of harboring H3K27M was subjected to targeted, loop-mediated isothermal amplification and Oxford Nanopore real-time sequencing, confirming an H3.3K27M diagnosis (11.8% allele fraction) within 1hr35min of receipt of sample from the operating room. (3) 5 cases resulted in primary cell culture generation (4) A novel single-molecule mutant protein quantification technique was performed on 9 cases. RESULTS from all analyses and concordance with outstanding sequencing will be presented. CONCLUSION We demonstrate that biopsy byproducts contain enough high-quality tumor-derived material to perform a variety of useful diagnostics with the potential to have immediate translational impact on standard of care.

Application of recombinant human pyruvate kinase in recombinase polymerase amplification

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41°C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. In this study, we attempted to use pyruvate kinase instead of creatine kinase (CK) that has been consistently used as an ATP-regenerating enzyme in RPA. Human pyruvate kinase M1 (PKM) was expressed in Escherichia coli and purified from the cells. RPA with PKM was performed at 41°C with the invitro synthesized urease subunit β (ureB) DNA from Ureaplasma parvum serovar 3 as a standard DNA. The optimal concentrations of PKM and phosphoenolpyruvate were 20ng/μL and 10mM, respectively. The RPA reaction with PKM was more sensitive than that with CK. PKM exhibited higher thermostability than CK, suggesting that the RPA reagents with PKM are preferable to those with CK for onsite use.