qPCR-based Method Research Articles

Development and optimization of the qPCR-based rapid detection method for Chinese species of Sanghuangporus

Sanghuangporus, well known as ‘Sanghuang’, is a macrofungal genus accommodating 11 edible and medicinal species in China. With the prosperous development of ‘Sanghuang’ business in China, the fraudulent practice naming the products of other morphologically and phylogenetically related species as ‘Sanghuang’ happens frequently, which disturbs the market order of ‘Sanghuang’ business. To promote the healthy industrial development of Sanghuangporus and also prevent the loss of this macrofungal resource, we develop a qPCR-based rapid detection method in this study. Internal transcribed spacer sequences of Fuscoporia, Inonotus, Phellinus, Sanghuangporus and Tropicoporus were aligned to design Sanghuangporus-specific amplification primers and probe, which can specifically distinguish all 11 Chinese species of Sanghuangporus from species of other closely related genera. The concentrations of Sanghuangporus-specific primers and probe were optimized as 0.7 μmol/L and 0.5 μmol/L, respectively, where the qPCR has the minimum detectable DNA template concentration of 10-3 ng/μL. In summary, the current qPCR-based detection method was specific, sensitive and rapid for Chinese species of Sanghuangporus.

Comparison of different diagnostic protocols for the detection of Toxocara spp. in faecal samples of cats and dogs

BackgroundToxocara canis and Toxocara cati are parasitic nematodes that occur worldwide. As embryonated Toxocara spp. eggs in the environment pose a zoonotic risk, especially for children, optimal diagnostic approaches are necessary for effective disease response and management, including surveillance. However, little is known about the performance of different diagnostic protocols for detecting Toxocara spp. in the faeces of cats and dogs, hampering movement towards an optimal diagnostic process. This study aimed to compare detection methods, including a newly developed sequential sieving protocol (SF-SSV) and a high-throughput multiplex qPCR-based method to facilitate epidemiological studies.MethodsSpecies-specific Toxocara spp. egg suspensions and canine and feline faecal samples from the field were used to estimate analytical and diagnostic sensitivity of the protocols. The performance of two automated DNA extraction protocols using enzymatic and mechanical lysis were compared by multiplex qPCR, targeting both T. canis and T. cati-specific genomic sequences. All samples were examined by microscopy-based techniques, the sedimentation flotation technique (SF) and a newly developed SF-SSV for the detection, enrichment and purification of parasite eggs. The costs and processing times necessary for all protocols were estimated and compared for both single samples and sets of 100 samples.ResultsTo detect Toxocara spp. eggs, SF-SSV showed the highest analytical sensitivity and a significantly higher diagnostic sensitivity than the DNA detection methods. Mechanical lysis performed better than enzymatic lysis for automated DNA extraction. In automated DNA extraction, 96-well plates performed better than 24-well plates. DNA detection and microscopy-based parasitological methods showed substantial agreement between the results generated by each method. Microscopy-based techniques required the lowest costs and least hands-on time for a single sample. However, when costs and labour were estimated for a set of 100 samples, the DNA detection protocol using 96-well plates for extraction revealed costs similar to SF-SSV and the fastest processing times.ConclusionsSF-SSV was superior in terms of analytical and diagnostic sensitivity for the detection of Toxocara spp. eggs. For larger sets of samples, multiplex qPCR-based DNA detection represents an alternative to microscopy-based methods, based on the possibility of faster sample processing at similar costs to SF-SSV, and the ability to provide species-specific diagnoses.Graphical

Development of an RPA-CRISPR/Cas12a Assay for Rapid and Sensitive Diagnosis of Plant Quarantine Fungus Setophoma terrestris.

Setophoma terrestris is an important phytopathogenic fungus listed by China as a harmful fungus subject to phytosanitary import control. This pathogen is a threat to a wide range of plants, particularly as the causal agent of onion pink root rot, one of the most severe diseases of onions. In order to provide rapid identification and early warning of S. terrestris and prevent its spread, we have developed a rapid, accurate, and visually intuitive diagnostic assay for this pathogen, by utilizing recombinase polymerase amplification (RPA), coupled with CRISPR/Cas12a cleavage and fluorescence-based detection systems or paper-based lateral flow strips. The developed RPA-CRISPR/Cas12a assay exhibited remarkable specificity for the detection of S. terrestris. Moreover, this protocol can detect the pathogen at a sensitivity level of 0.01 pg/μL, which significantly outperforms the 1 pg/μL sensitivity achieved by the existing qPCR-based detection method. The entire diagnostic procedure, including DNA extraction, the RPA reaction, the Cas12a cleavage, and the result interpretation, can be accomplished in 40 min. Furthermore, the successful application of the assay in infected plant samples highlighted its potential for rapid and accurate pathogen detection in agricultural settings. In summary, this RPA-CRISPR/Cas12a diagnostic method offers a potentially valuable technological solution for quarantine and disease management.

Sensitive quantification of fibroblast activation protein and high-throughput screening for inhibition by FDA-approved compounds

Fibroblast activation protein (FAP) has been extensively studied as a cancer biomarker for decades. Recently, small-molecule FAP inhibitors have been widely adopted as a targeting moiety of experimental theranostic radiotracers. Here we present a fast qPCR-based analytical method allowing FAP inhibition screening in a high-throughput regime. To identify clinically relevant compounds that might interfere with FAP-targeted approaches, we focused on a library of FDA-approved drugs. Using the DNA-linked Inhibitor Antibody Assay (DIANA), we tested a library of 2,667 compounds within just a few hours and identified numerous FDA-approved drugs as novel FAP inhibitors. Among these, prodrugs of cephalosporin antibiotics and reverse transcriptase inhibitors, along with one elastase inhibitor, were the most potent FAP inhibitors in our dataset. In addition, by employing FAP DIANA in the quantification mode, we were able to determine FAP concentrations in human plasma samples. Together, our work expands the repertoire of FAP inhibitors, analyzes the potential interference of co-administered drugs with FAP-targeting strategies, and presents a sensitive and low-consumption ELISA alternative for FAP quantification with a detection limit of 50 pg/ml.

Development and application of a qPCR-based method coupled with spore trapping to monitor airborne pathogens of wheat causing stripe rust, powdery mildew, and Fusarium head blight

Development and application of a qPCR-based method coupled with spore trapping to monitor airborne pathogens of wheat causing stripe rust, powdery mildew, and Fusarium head blight

291 Supra-maximal Extent of Resection is Associated With Improved Progression-Free Survival in Patients With IDH Wild-Type Glioblastoma Once Negative TERT Promoter Molecular Margins are Achieved

INTRODUCTION: The standard of care for isocitrate dehydrogenase (IDH) wild-type GBM involves safe maximal resection with subsequent chemoradiation. TERT promoter (TERTp) mutations are clonal in >85% of IDH wild-type GBMs, detectable in all tumor cells. Advances in surgical management have focused on optimizing maximal resection given the positive correlation between extent of resection (EOR) and post-operative outcomes. METHODS: TERTp mutation detection in margin specimens was performed via a novel 30min qPCR-based method. A machine learning algorithm incorporating preoperative anatomic and intraoperative characteristics was developed using 148 patients to predict EOR. This model was prospectively applied to 44 patients to assess the correlation between a TERTp mutation negative margin and survival in cases when GTR was predicted. RESULTS: Retrospective analysis of 148 patients who underwent resection of GBM revealed significant improvement in 36-month survival with residual volume (RV) <4.9 cc). The primary predictive characteristics for achieving this RV were tumor volume, ventricular contact, multifocality, and Sawaya grade. Prospective analysis of 44 patients showed that, in cases where GTR was predicted and achieved, 26% obtained a TERTp negative margin. Although small, this group's PFS was significantly prolonged (0% vs 75% event-free at 30 months, p = 0.02), and a numerically superior prolonged OS was observed, while survival in GTR with positive molecular margin was indistinguishable from STR. CONCLUSIONS: These findings provide novel evidence supporting the utility of supramaximal resection targeting a “molecular margin” to maximize postoperative survival for a subset of patients with “localized” GBM. Further study is warranted to assess intraoperative, quantitative testing of TERTp mutation in GBM specimens to guide marginal resection in these patients.

Assessment of crAssphage as a human fecal source tracking marker in the lower Great Lakes

CrAssphage or crAss-like phage ranks as the most abundant phage in the human gut and is present in human feces-contaminated environments. Due to its high human specificity and sensitivity, crAssphage is a potentially robust source tracking indicator that can distinguish human fecal contamination from agricultural or wildlife sources. Its suitability in the Great Lakes area, one of the world's most important water systems, has not been well tested. In this study, we tested a qPCR-based quantification method using two crAssphage marker genes (ORF18-mod and CPQ_064) at Toronto recreational beaches along with their adjacent river mouths. Our results showed a 71.4 % (CPQ_064) and 100 % (ORF18-mod) human sensitivity for CPQ_064 and ORF18-mod, and a 100 % human specificity for both marker genes. CrAssphage was present in 57.7 % or 71.2 % of environmental water samples, with concentrations ranging from 1.45 to 5.14 log10 gene copies per 100 mL water. Though concentrations of the two marker genes were strongly correlated, ORF18-mod features a higher human sensitivity and higher positive detection rates in environmental samples. Quantifiable crAssphage was mostly present in samples collected in June and July 2021 associated with higher rainfall. In addition, rivers had more frequent crAssphage presence and higher concentrations than their associated beaches, indicating more frequent and greater human fecal contamination in the rivers. However, crAssphage was more correlated with E. coli and Enterococcus at the beaches than in the rivers, suggesting human fecal sources may be more predominant in driving the increases in E. coli and Enterococcus at the beaches when impacted by river plumes.

Multilab Validation Report for the Verification and Subtyping of Listeria monocytogenes Using qPCR

Listeria monocytogenes (Lm) is a Gram-positive bacterium that causes invasive listeriosis, an illness with high mortality and hospitalization rates. Due to the severity of illness associated with Lm, rapid identification and characterization of isolates from foods and the food-processing environment are critical to properly identify and track the pathogen and quickly remove adulterated foods from the market. Prior methods can rely on time-consuming biochemical or sera-agglutination assays to perform these tasks. Development of a high-throughput method that would rapidly perform these tasks is critical to improve response to contamination events. Previously, a single laboratory validation of a qPCR-based method was presented that could rapidly verify Lm isolates and characterize them into six molecular serogroups. In the current study, a multi-laboratory validation (MLV) was performed to evaluate the reliability of the qPCR method for identification and serogrouping of Lm isolates. Sixteen collaborating laboratories independently analyzed a panel of 43 blinded isolates plus three control strains using the qPCR method. This panel was comprised of representatives for non-Listeria (n = 7), Listeria sp. (n = 8), and Lm (n = 28) strains. The Lm isolates contained representatives of the six serogroups: 2A, 2B, 2C, 4B, NT, and 4bV/IVb-v1, with five strains for each serogroup except 4bV/IVb-v1 (n = 3). The results generated by 16 laboratories showed high sensitivity, specificity, and accuracy, generally ≥97%, for both the genus-species and serogrouping qPCRs. Results from one laboratory lowered the sensitivity of the non-Listeria group to 93%. These results indicated the method was highly reliable. However, only the previously evaluated serogroups were tested within the MLV panel, though there is the potential for other serogroup results. Sequence Read Archive (SRA) files for Lm isolates were evaluated to determine the frequency of other potential serogroup profiles. This effort identified a low percentage of isolates with atypical qPCR serogroups (0.30%) that are consistent with Lm and were generally associated with lineage II and the natural environment. In summary, the results indicate that the proposed qPCR method is reliable and has a high degree of sensitivity, accuracy, and specificity, while also decreasing hands-on analysis time and increasing throughput of the analysis.

Shark and ray genome size estimation: methodological optimization for inclusive and controllable biodiversity genomics

Estimate of nuclear DNA content serves as an independent tool for validating the completeness of whole genome sequences and investigating the among-species variation of genome sizes, but for some species, the requirement of fresh cells makes this tool highly inaccessible. Here we focused on elasmobranch species (sharks and rays), and using flow cytometry or quantitative PCR (qPCR), estimated the nuclear DNA contents of brownbanded bamboo shark, white spotted bamboo shark, zebra shark, small-spotted catshark, sandbar shark, slendertail lanternshark, basking shark, megamouth shark, red stingray, and ocellate spot skate. Our results revealed their genome sizes spanning from 3.18 pg (for ocellate spot skate) to 13.34 pg (for slendertail lanternshark), reflecting the huge variation of genome sizes already documented for elasmobranchs. Our qPCR-based method ‘sQuantGenome’ enabled accurate genome size estimation without using live cells, which has been a severe limitation with elasmobranchs. These findings and our methodology are expected to contribute to better understanding of the diversity of genome sizes in elasmobranchs even including species with limited availability of fresh tissue materials. It will also help validate the completeness of already obtained or anticipated whole genome sequences.

Potential Association of Cytochrome P450 Copy Number Alteration in Tumour with Chemotherapy Resistance in Lung Adenocarcinoma Patients.

Resistance to anticancer agents is a major obstacle to efficacious tumour therapy and responsible for high cancer-related mortality rates. Some resistance mechanisms are associated with pharmacokinetic variability in anticancer drug exposure due to genetic polymorphisms of drug-metabolizing cytochrome P450 (CYP) enzymes, whereas variations in tumoural metabolism as a consequence of CYP copy number alterations are assumed to contribute to the selection of resistant cells. A high-throughput quantitative polymerase chain reaction (qPCR)-based method was developed for detection of CYP copy number alterations in tumours, and a scoring system improved the identification of inappropriate reference genes that underwent deletion/multiplication in tumours. The copy numbers of both the target (CYP2C8, CYP3A4) and the reference genes (ALB, B2M, BCKDHA, F5, CD36, MPO, TBP, RPPH1) established in primary lung adenocarcinoma by the qPCR-based method were congruent with those determined by next-generation sequencing (for 10 genes, slope = 0.9498, r2 = 0.72). In treatment naïve adenocarcinoma samples, the copy number multiplication of paclitaxel-metabolizing CYP2C8 and/or CYP3A4 was more prevalent in non-responder patients with progressive disease/exit than in responders with complete remission. The high-throughput qPCR-based method can become an alternative approach to next-generation sequencing in routine clinical practice, and identification of altered CYP copy numbers may provide a promising biomarker for therapy-resistant tumours.

Rapid detection and quantification of Aphanomyces cochlioides in sugar beet

Sugar beet (Beta vulgaris L.) is an economically important crop in temperate climates providing nearly 30% of sugar production worldwide. The oomycete Aphanomyces cochlioides is the causative agent of seedling damping-off and root rot disease in sugar beet. The pathogen is responsible for plant degeneration and drastic yield losses in all major sugar beet producing areas. The identification of resistant germplasm is essential to reduce the use of chemical treatments as well as the costs of protective measures and to effectively limit the damage caused by the pathogen. In this study we aimed to establish a qPCR-based method to quantify the pathogen DNA in infected plants and to predict the resistance levels of different sugar beet genotypes in response to A. cochlioides. The difference in the response to A. cochlioides isolates with different geographical origins was investigated. In addition, confocal microscopy was performed in order to observe the spatial and temporal colonization pattern in infected seedlings of susceptible and partially resistant breeding lines. The research presented in this article provides a tool to understand the progress of the infection in infected tissues and to identify the genetic background of resistance to A. cochlioides that can be used to support breeding programs.

Molecular Real-Time PCR Monitoring of Onion Fusarium Basal Rot Chemical Control.

Fusarium basal rot disease (FBR) is a destructive threat to onion crops around the globe. It causes seedlings' death, development disruption, and pre- and post-harvest bulb infection and rotting, with a concern for toxin infestation. It is an emerging disease in Israel, with new reports from farms nationwide. Recently, we reported on a full-season pot experiment to protect two leading commercial cultivars against FBR chemically. Here, we present new real-time qPCR molecular tracking of the pathogens inside the host plant and compare the infection levels to a deep analysis of the impacts of this experiment's treatments on plant growth and health indexes. The new findings reveal variations within each treatment's effectiveness regarding sprout development and bulb ripening stages. For instance, in the yellow Orlando cv., high protection was obtained with Azoxystrobin + Tebuconazole (Az-Te) in sprouts against F. oxysporum f. sp. cepae and with Fludioxonil + Sedaxen in mature plants against Fusarium acutatum. Thus, combining these fungicides may protect plants throughout their lifecycle. Also, Prochloraz at low dose was highly efficient in the Orlando cv. Still, to shield red Noam cv. plants from both pathogens, increasing this fungicide concentration towards the season-ending should be preferred. The qPCR tracking showed that all chemical treatments tested could reduce infection from pathogens by 80-90%, even with compounds such as Az-Te that were less effective. This implies that the pesticide was effective but probably phytotoxic to the plants, and thus, lower dosages must be considered. The molecular-based analysis discloses the high infection ability of F. oxysporum f. sp. cepae compared to F. acutatum in both cultivars. It also indicates an antagonism between those species in the Orlando cv. and synergism in the Noam cv. The current work reveals weak and strong points in chemical FBR protection and offers new ways to improve its application. The qPCR-based method enables us to closely monitor the pathogenesis and efficacy of chemical-preventing treatments and optimize crop-protection protocols.

Comparative Methods for Quantification of Sulfate-Reducing Bacteria in Environmental and Engineered Sludge Samples.

This study aimed to compare microscopic counting, culture, and quantitative or real-time PCR (qPCR) to quantify sulfate-reducing bacteria in environmental and engineered sludge samples. Four sets of primers that amplified the dsrA and apsA gene encoding the two key enzymes of the sulfate-reduction pathway were initially tested. qPCR standard curves were constructed using genomic DNA from an SRB suspension and dilutions of an enriched sulfate-reducing sludge. According to specificity and reproducibility, the DSR1F/RH3-dsr-R primer set ensured a good quantification based on dsrA gene amplification; however, it exhibited inconsistencies at low and high levels of SRB concentrations in environmental and sulfate-reducing sludge samples. Ultimately, we conducted a qPCR method normalized to dsrA gene copies, using a synthetic double-stranded DNA fragment as a calibrator. This method fulfilled all validation criteria and proved to be specific, accurate, and precise. The enumeration of metabolically active SRB populations through culture methods differed from dsrA gene copies but showed a plausible positive correlation. Conversely, microscopic counting had limitations due to distinguishing densely clustered organisms, impacting precision. Hence, this study proves that a qPCR-based method optimized with dsrA gene copies as a calibrator is a sensitive molecular tool for the absolute enumeration of SRB populations in engineered and environmental sludge samples.

Associations of Telomere Length and Change With Cognitive Decline Were Modified by Sex and Race: The REGARDS Study.

We examined the associations of baseline telomere length (TL) and TL change with cognitive function over time in older US adults, as well as differences by sex and race. A total of 1820 cognitively healthy individuals (median baseline age: 63years) were included. Telomere length was measured using qPCR-based method at baseline and among 614 participants in the follow-up examination 10years later. Cognitive function was assessed by a four-test battery every 2years. In multivariable-adjusted linear mixed models, longer baseline TL and smaller attrition/lengthening of TL over time were associated with better Animal Fluency Test score. Longer baseline TL was also linearly associated with better Letter Fluency Test score. The observed associations were consistently more pronounced in women than men and in Black compared to White participants. Telomere length may be a biomarker that predicts long-term verbal fluency and executive function, particularly in women and Black Americans.

A qPCR Method to Assay Endonuclease Activity of Cas9-sgRNA Ribonucleoprotein Complexes.

The CRISPR-Cas system has emerged as the most efficient genome editing technique for a wide range of cells. Delivery of the Cas9-sgRNA ribonucleoprotein complex (Cas9 RNP) has gained popularity. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR)-based assay to quantify the double-strand break reaction mediated by Cas9 RNP. To accomplish this, the dextransucrase gene (dsr) from Leuconostoc citreum was selected as the target DNA. The Cas9 protein was produced using recombinant Escherichia coli BL21, and two sgRNAs were synthesized through in vitro transcription to facilitate binding with the dsr gene. Under optimized in vitro conditions, the 2.6 kb dsr DNA was specifically cleaved into 1.1 and 1.5 kb fragments by both Cas9-sgRNA365 and Cas9-sgRNA433. By monitoring changes in dsr concentration using qPCR, the endonuclease activities of the two Cas9 RNPs were measured, and their efficiencies were compared. Specifically, the specific activities of dsr365RNP and dsr433RNP were 28.74 and 34.48 (unit/μg RNP), respectively. The versatility of this method was also verified using different target genes, uracil phosphoribosyl transferase (upp) gene, of Bifidobacterium bifidum and specific sgRNAs. The assay method was also utilized to determine the impact of high electrical field on Cas9 RNP activity during an efficient electroporation process. Overall, the results demonstrated that the qPCR-based method is an effective tool for measuring the endonuclease activity of Cas9 RNP.

A new duplex qPCR-based method to quantify Mycoplasma mycoides in complex cell culture systems and host tissues

Bacterial pathogen-host interactions are a complex process starting with adherence and colonization followed by a variety of interactions such as invasion or cytotoxicity on one hand and pathogen recognition, secretion of proinflammatory/antibacterial substances and enhancing the barrier function of epithelial layers on the other hand. Therefore, a variety of in vitro, ex vivo and in vivo models have been established to investigate these interactions. Some in vitro models are composed of different cell types and extracellular matrices such as tissue explants or precision cut lung slices. These complex in vitro models mimic the in vivo situation more realistically, however, they often require new and more sophisticated methods for quantification of experimental results. Here we describe a multiplex qPCR-based method to quantify the number of bacteria of Mycoplasma (M.) mycoides interacting with their hosts in an absolute manner as well as normalized to the number of host cells. We choose the adenylate kinase (adk) gene from the pathogen and the Carcinoembryonic antigen-related cell adhesion molecule 18 (CEACAM18) gene from the host to determine cell numbers by a TaqMan-based assay system. Absolute copy numbers of the genes are calculated according to a standard containing a defined number of plasmids containing the sequence which is amplified by the qPCR. The new multiplex qPCR therefore allows the quantification of M. mycoides interacting with host cells in suspension, monolayer, 3D cell culture systems as well as in host tissues.

A qPCR-based method to detect the eel parasitic nematode Anguillicola crassus in intermediate and final hosts.

Being able to systematically detect parasitic infection, even when no visual signs of infection are present, is crucial to the establishment of accurate conservation policies. The nematode Anguillicola crassus infects the swimbladder of anguillid species and is a potential threat for eel populations. In North America, naïve hosts such as the American eel Anguilla rostrata are affected by this infection. The accidental introduction of A. crassus following restocking programs may contribute to the actual decline of the American eel in Canada. We present a quantitative real time PCR-based method to detect A. crassus infection in final and intermediate hosts. We tested two protocols on samples from different geographical origins in Canada: 1) a general detection of A. crassus DNA in pools of young final hosts (glass eels) or crustacean intermediate hosts 2) a detection at the individual scale by analyzing swim bladders from elvers, or from adult yellow and silver eels. The DNA of A. crassus was detected in one pool of zooplankton (intermediate host) from the Richelieu River (Montérégie-Québec), as well as in individual swim bladders of 13 elvers from Grande and Petite Trinité rivers (Côte-Nord-Québec). We suggest that our qPCR approach could be used in a quantitative way to estimate the parasitic burden in individual swim bladders of elvers. Our method, which goes beyond most of previous developed protocols that restricted the diagnosis of A. crassus to the moment when it was fully established in its final host, should help to detect early A. crassus infection in nature.

Same-day Enterococcus qPCR results of recreational water quality at two Toronto beaches provide added public health protection and reduced beach days lost

ObjectivesWe evaluated the potential impacts from using a rapid same-day quantitative polymerase chain reaction (qPCR) monitoring method for beach posting outcomes at two Toronto beaches.MethodsIn total, 228 water samples were collected at Marie Curtis Park East and Sunnyside Beaches over the 2021 summer season. Water samples were processed using the USEPA 1609.1 Enterococcus qPCR-based method. Escherichia coli (E. coli) culture data and daily beach posting decisions were obtained from Toronto Public Health.ResultsNo significant correlation was observed between previous-day and same-day (retrospective) E. coli enumeration results at any Sunnyside Beach transect, and only relatively low (R = 0.41–0.56) or no significant correlation was observed at sampling transects for Marie Curtis Park East Beach. Comparing our same-day Enterococcus qPCR data to Toronto’s 2-day E. coli geometric mean beach posting decisions, we noted the need for additional postings for 1 (2%) and 3 (8%) missed health-risk days at Sunnyside and Marie Curtis Park East Beaches, respectively. The qPCR data also pointed to incorrect postings for 12 (31%) and 6 (16%) lost beach days at Sunnyside and Marie Curtis Park East Beaches, respectively.ConclusionApplication of a rapid Enterococcus qPCR method at two Toronto beaches revealed 5% of beach posting decisions were false negatives that missed health-risk days, while 23% of decisions were false positives resulting in lost beach days. Deployment of the rapid same-day qPCR method offers the potential to reduce both health risks and unnecessary beach postings.

Abstract 215: A novel enzymatic library preparation workflow that dramatically reduces artifacts associated with damaged FFPE samples

Abstract Clinical oncology heavily relies on formalin-fixed, paraffin-embedded (FFPE) tissue samples for histology and molecular characterization. The chemical and physical modifications of nucleic acids introduced during fixation, storage and purification negatively impact molecular profiling and vary from sample-to-sample. Conventional sonication and ligation-based library preparation is considered the gold-standard approach for FFPE samples, but it is time-consuming and expensive. Importantly, these processes introduce artifacts that impact downstream analysis and interpretation (Haile et al., 2019). We have developed a novel fragmentation chemistry that virtually eliminates hairpin artifacts, achieving levels on par with non-FFPE control samples. Our fragmentation method is highly scalable, exhibits minimal sequence bias, and reduces cost and workflow inefficiencies associated with mechanical shearing. In this study, we developed a unified method for library preparation from FFPE samples which produces similar insert sizes across variable input mass and quality of FFPE samples. We carefully evaluated the performance of this method relative to a sonication-based approach employing the KAPA HyperPrep kit. Libraries were constructed from 50 to 200 ng of FFPE DNA, inputs typically used for NGS, ranging from low-to high-quality as assessed using a qPCR-based method and DNA integrity (DIN). Targeted sequencing was performed using a 37 kb custom oncology hybridization capture panel to investigate molecular complexity. Our workflow virtually eliminated hairpin artifacts that were present in up to 4.5% of reads in sonication-based libraries. Soft-clipping was also 3- to 7-fold lower in libraries prepared with the Watchmaker kit relative to sonicated DNA libraries, improving overall sequencing economy. Furthermore, the mean target coverage achieved with the Watchmaker kit was comparable to or higher than sonication libraries using the same input mass. Because input masses were normalized post-shearing, which typically results in 20-40% sample loss, coverage with our approach is significantly higher relative to sonication, if normalizing to pre-sheared DNA input. Watchmaker DNA Library Preparation with Fragmentation enables high-quality DNA library preparation from damaged FFPE samples, producing high target coverage, uniform insert size, and minimizing sequencing artifacts to improve sensitivity and specificity. This approach is highly scalable and automatable, enabling various oncology applications. Citation Format: Giulia Corbet, Philip Benson, Kailee Reed, Skyler Mishkin, Thomas Harrison, Kristin Scott, Zane Jaafar, Kristina Giorda, Josh Haimes, Martin Ranik, Brian Kudlow. A novel enzymatic library preparation workflow that dramatically reduces artifacts associated with damaged FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 215.

380 A Rapid CSF Assay Accelerates Diagnosis and Treatment Initiation for CNS Neoplasms

INTRODUCTION: The low yields of CSF studies and serologies in current diagnostic workflows for suspected CNS neoplasms often lead to neurosurgical biopsies of high-risk lesions. To address this need, we developed an assay to rapidly identify hallmark mutations of CNS neoplasms in CSF. METHODS: Targeted Rapid Sequencing (TetRS) was developed as a qPCR-based method to detect IDH1/2, TERT promoter, BRAF, H3F3A, and MYD88 mutations in CSF-derived DNA within 80 minutes of collection. CSF was prospectively collected over 18 months from 98 patients with suspected new CNS neoplasms. Results were utilized in clinical decision-making. RESULTS: 40 of 98 patients were ultimately diagnosed with a neoplasm. TetRS detected a mutation in 15 out of 40 patients with a neoplasm (37.5%), including cases of CNS lymphoma, glioblastoma, IDH-mutant brainstem glioma and H3K27M-mutant diffuse midline glioma. Detection of a mutation accelerated diagnosis in comparison to cases where a mutation was not detected (median 0.5 vs 10.5 days, p < 0.00001). In multiple cases, a positive TetRS result obviated the need for biopsy and enabled initiation of disease-directed therapy. In one such case, detection of the MYD88 L265P variant was diagnostic of CNS lymphoma, enabling methotrexate and rituximab initiation within 3 days of admission. Results of repeated CSF samplings correlated with treatment response and recurrence. In another case, detection of the IDH1 R132G variant was diagnostic of IDH-mutant brainstem glioma, enabling IDH inhibitor and radiation therapy, with clinical and radiographic response. CONCLUSIONS: The targeted cell-free DNA panel for CSF presented here is a novel tool to rapidly, cost-effectively and broadly diagnose CNS neoplasia, initiate therapies and follow disease progression in the absence of neurosurgical tissue sampling.