Tool For Detection Of Pathogens Research Articles

Viral pathogen surveillance in wastewater using metagenomic approaches: obstacles and prospects

Abstract Background Wastewater monitoring offers an important tool for pathogen detection and tracking within large populations at a minimized cost and labor. Untargeted metagenomic approaches for the detection and sequencing of viruses still face limitations. In this study we tested the application of the sequence-independent single-primer amplification (SISPA) protocol in conjunction with nanopore sequencing to the study of viromes in wastewater samples. Methods Influent wastewater was collected from four Wastewater Treatment Plants located in the Lisbon region and Alentejo, Portugal in 2019 and 2023. Samples were processed with different methods including filtration, flocculation, Nanotraps or absorvent tampons. We applied the SISPA protocol, that includes a random amplification step, followed by nanopore sequencing on flow cells R9.4.1. We used the CZ ID (https://czid.org) pipeline for metagenomic analysis and taxonomic reporting. Results A large heterogeneity was found between samples in sequencing output (ranging from 17 thousand to 12 million reads) and the proportion of sequence reads classified as viral (ranging from 0.06% to 4% per sample). Viral taxa with higher number of reads were Picornavirales, dsDNA and ssDNA phages, Mimiviridae and Circoviridae. Known viral pathogens included Rotavirus A, Orthopoxvirus, Human betaherpesvirus and Avian orthoavulavirus. Conclusions Metagenomic studies of environmental samples generally exhibit a low proportion of viral reads, even after viral enrichment. A substantial sequencing effort is thus required to achieve a comprehensive virome characterization. As alternative, a more targeted approach, e.g. to target families of viruses of interest, may be considered. Funding: Fundação para a Ciência e Tecnologia (FCT): GHTM - UID/04413/2020; LA-REAL - LA/P/0117/2020; https://doi.org/10.54499/CEECINST/00102/2018/CP1567/CT0040 to SGS. Research council of the Vrije Universiteit Brussel (OZR-VUB): OZR3863BOF to PL. Key messages • Enrichment for viruses in metagenomic approaches remain challenging in very complex samples as are influent wastewaters. • Virome characterization still necessitates high sequencing efforts but may be used as a complementary environmental survey tool integrated with more targeted approaches.

A dataset for developing proteomic tools for pathogen detection via differential cell lysis of whole blood samples

This data descriptor presents a curated dataset for pathogen detection and identification (Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans) directly from whole-blood samples. The dataset was created using differential cell lysis combined with rapid extraction, digestion, and mass spectrometry-based proteomics. Our method offers a rapid diagnostic alternative to traditional culture, enabling timely disease management, such as sepsis. Highlighting our dataset’s uniqueness, it features a three-tier structure: Spectral Libraries of Pathogens for identifying peptide peaks for putative biomarkers; Spiked pathogen in blood MS data for biomarker panel optimization through varied concentration samples; and Parallel Reaction Monitoring (PRM) data from sepsis patients for validating our biomarker panel, achieving 83.3% sensitivity within seven hours without microbial enrichment culture. This dataset serves as a comprehensive reference for bioinformatic tool development and biomarker panel proposals, advancing microbial detection, antimicrobial resistance, and epidemiological studies.

A label-free strategy for direct detection of Staphylococcus aureus in complex matrixes based on RCA and aptamer

Given the significant threat to human health posed by Staphylococcus aureus, rapid and accurate quantification of S. aureus is essential to prevent and diagnose foodborne diseases and to ensure the safety of food consumption. Here, we developed a novel, simple，and label-free biosensor for quantitatively detecting S. aureus by integrating an allosteric aptamer probe and a rolling circle amplification technique. Pathogen S. aureus-specific binding could induce the structural change of the allosteric aptamer beacon to expose the primer for the padlock probe. Then, the primer could trigger the RCA reaction to produce a long-chain singles-tranded DNA harboring repeating Thioflavin T (ThT) aptamer, leading to powerful fluorescence signals. The assay demonstrates sensitivity, specificity, and accuracy, showing its potential as an alternative tool for pathogen detection in different fields, such as food safety monitoring.

Development of a Colorimetric and SERS Dual-Signal Platform via dCas9-Mediated Chain Assembly of Bifunctional Au@Pt Nanozymes for Ultrasensitive and Robust Salmonella Assay.

Timely screening for harmful pathogens is a great challenge in emergencies where traditional culture methods suffer from long assay time and alternative methods are limited by poor accuracy and low robustness. Herein, we present a dCas9-mediated colorimetric and surface-enhanced Raman scattering (SERS) dual-signal platform (dCas9-CSD) to address this challenge. Strategically, the platform used dCas9 to accurately recognize the repetitive sequences in amplicons produced by loop-mediated isothermal amplification (LAMP), forming nucleic acid frameworks that assemble numerous bifunctional gold-platinum (Au@Pt) nanozymes into chains on the surface of streptavidin-magnetic beads (SA-MB). The collected Au@Pt converted colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue oxidized TMB (oxTMB) via its Pt shell and then enhanced the Raman signal of oxTMB by its Au core. Therefore, the presence of Salmonella could be dexterously converted into cross-validated colorimetric and SERS signals, providing more reliable conclusions. Notably, dCas9-mediated secondary recognition of amplicons reduced background signal caused by nontarget amplification, and two-round signal amplification consisting of LAMP reaction and Au@Pt catalysis greatly improved the sensitivity. With this design, Salmonella as low as 1 CFU/mL could be detected within 50 min by colorimetric and SERS modes. The robustness of dCas9-CSD was further confirmed by various real samples such as lake water, cabbage, milk, orange juice, beer, and eggs. This work provides a promising point-of-need tool for pathogen detection.

The effects of sequencing strategies on Metagenomic pathogen detection using bronchoalveolar lavage fluid samples

ObjectivesMetagenomic next-generation sequencing (mNGS) is a powerful tool for pathogen detection. The accuracy depends on both wet lab and dry lab procedures. The objective of our study was to assess the influence of read length and dataset size on pathogen detection. MethodsIn this study, 43 clinical BALF samples, which tested positive via clinical mNGS and were consistent with the diagnosis, were subjected to re-sequencing on the Illumina NovaSeq 6000 platform. The raw re-sequencing data, consisting of 100 million (M) paired-end 150 bp (PE150) reads, were divided into simulated datasets with eight different data sizes (5 M, 10 M, 15 M, 20 M, 30 M, 50 M, 75 M, 100 M) and five different read lengths (single-end 50 bp (SE50), SE75, SE100, PE100, and PE150). Both Kraken2 and IDseq bioinformatics pipelines were employed to analyze the previously diagnosed pathogens in the simulated data. Detection of pathogens was based on read counts ranging from 1 to 10 and RPM values ranging from 0.2 to 2. ResultsOur results revealed that increasing dataset sizes and read lengths can enhance the performance of mNGS in pathogen detection. However, a larger data sizes for mNGS require higher economic costs and longer turnaround time for data analysis. Our findings indicate 20 M reads being sufficient for SE75 mode to achieve high recall rates. Additionally, high nucleic acid loads in samples can lead to increased stability in pathogen detection efficiency, reducing the impact of sequencing strategies. The choice of bioinformatics pipelines had a significant impact on recall rates achieved in pathogen detection. ConclusionsIncreasing dataset sizes and read lengths can enhance the performance of mNGS in pathogen detection but increase the economic and time costs of sequencing and data analysis. Currently, the 20 M reads in SE75 mode may be the best sequencing option.

Understanding the role of Francisella halioticida in mussel mortalities in France: an integrative approach.

Since 2014, mass mortalities of mussels Mytilus spp. have occurred in production areas on the Atlantic coast of France. The aetiology of these outbreaks remained unknown until the bacterium Francisella halioticida was detected in some mussel mortality cases. This retrospective study was conducted to assess the association between F. halioticida and these mussel mortalities. Mussel batches (n = 45) from the Atlantic coast and English Channel were selected from archived individual samples (n = 863) collected either during or outside of mortality events between 2014 and 2017. All mussels were analysed by real-time PCR assays targeting F. halioticida; in addition, 185 were analysed using histological analysis and 178 by 16S rRNA metabarcoding. F. halioticida DNA was detected by real-time PCR and 16S rRNA metabarcoding in 282 and 34 mussels, respectively. Among these individuals, 82% (real-time PCR analysis) and 76% (16S rRNA metabarcoding analysis) were sampled during a mortality event. Histological analyses showed that moribund individuals had lesions mainly characterized by necrosis, haemocyte infiltration and granulomas. Risk factor analysis showed that mussel batches with more than 20% of PCR-positive individuals were more likely to have been sampled during a mortality event, and positive 16S rRNA metabarcoding batches increased the strength of the association with mortality by 11.6 times. The role of F. halioticida in mussel mortalities was determined by reviewing the available evidence. To this end, a causation criteria grid, tailored to marine diseases and molecular pathogen detection tools, allowed more evidence to be gathered on the causal role of this bacterium in mussel mortalities.

Integrating PEGylated peptide-oriented bacteria-imprinted matrix and PdPt bimetallic-doped imidazolium zeolite framework-8 for sensitive detection of Escherichia coli with smartphone readout system

Escherichia coli (E. coli) is one of the major foodborne pathogens, and its efficient isolation and detection from complex samples are crucial. Herein, we fabricated a novel hydrophilic-PEGylated peptide-oriented bacteria-imprinted PDMS matrix (HPBIM) for highly efficient capture of E. coli from complex samples. The antibody-free HPBIM possessed a hierarchical structure of imprinted cavities, which were inlaid with peptide-functionalized SiO2 (SiO2@peptide). Meanwhile, PEGylation was performed on the non-imprinted region of HPBIM to limit the non-specific absorption. So HPBIM could specifically capture target E. coli due to the synergistic effect of peptide affinity, bacteria-imprinting and PEGylation of non-imprinted region. Additionally, a new polyethylene glycol-covered / boronic acid-modified PdPt bimetallic-doped ZIF-8 (PBPPZ) was prepared for electively labeling E. coli captured by HPBIM. PBPPZ exhibited excellent peroxidase-like activity and could generate sensitive output signals, which could be further quantitatively determined by smartphone readout device. To prove the practicability, we proposed an innovative HPBIM-PBPPZ strategy, in which the HPBIM first selectively captured E. coli, then the PBPPZ specific labeled E. coli and oxidized colorless substrate into colored product, and finally the change in color was quantitatively determined by a smartphone readout device equipped with a Color Grab APP. The biomacromolecule-free HPBIM-PBPPZ strategy exhibited excellent sensitivity with limit of detection of 69 CFU mL−1 for grapefruit juice and 257 CFU mL−1 for milk, and was successfully applied in the determination of E. coli in spiked sample with recovery and relative standard deviation in the range of 88.1–106.7% and 4.0–7.8%, respectively. Therefore, we believe that the HPBIM-PBPPZ strategy has great potential to become a powerful tool for the ultrasensitive detection of pathogens in complex biological samples.

Electrochemical biosensors for pathogenic microorganisms detection based on recognition elements.

The worldwide spread of pathogenic microorganisms poses a significant risk to human health. Electrochemical biosensors have emerged as dependable analytical tools for the point-of-care detection of pathogens and can effectively compensate for the limitations of conventional techniques. Real-time analysis, high throughput, portability, and rapidity make them pioneering tools for on-site detection of pathogens. Herein, this work comprehensively reviews the recent advances in electrochemical biosensors for pathogen detection, focusing on those based on the classification of recognition elements, and summarizes their principles, current challenges, and prospects. This review was conducted by a systematic search of PubMed and Web of Science databases to obtain relevant literature and construct a basic framework. A total of 171 publications were included after online screening and data extraction to obtain information of the research advances in electrochemical biosensors for pathogen detection. According to the findings, the research of electrochemical biosensors in pathogen detection has been increasing yearly in the past 3years, which has a broad development prospect, but most of the biosensors have performance or economic limitations and are still in the primary stage. Therefore, significant research and funding are required to fuel the rapid development of electrochemical biosensors. The overview comprehensively evaluates the recent advances in different types of electrochemical biosensors utilized in pathogen detection, with a view to providing insights into future research directions in biosensors.

Enhancing Pathogen Detection Methods through a Novel Molecular Diagnostic Approach with CRISPR/Cas Technology

Molecular diagnostics is widely recognized as one of the most efficient approaches for detecting and characterizing microorganisms. This method relies on the identification of specific nucleic acid sequences and enables the quick and precise determination of pathogen presence in various samples. Conventional methods for pathogen detection rely on the culture and identification of the pathogen in a laboratory setting, which can be time-consuming and expensive. Molecular diagnostic methods, such as PCR and DNA sequencing, have emerged as powerful alternatives to culture-based methods, offering greater sensitivity and specificity. However, these methods are still limited by their reliance on costly equipment and specialized expertise. In recent years, the CRISPR/Cas technology has emerged as a powerful tool for genome editing and manipulation, as well as for molecular diagnostics. This review presents a novel approach for improving pathogen detection methods through the utilization of CRISPR/Cas technology. The proposed method offers several distinct advantages over existing molecular diagnostic techniques. Notably, it provides enhanced specificity and accuracy, thereby minimizing the occurrence of false-positive results. Additionally, this method can rapidly and effectively detect pathogens, making it particularly attractive for use in clinical and laboratory settings. Therefore, molecular diagnostics using CRISPR diagnostics based on Cas12a is a powerful tool for pathogen detection and improving the accuracy and speed of diagnosis. Its prospects for future use are wide-ranging and may encompass many areas of life sciences.

Simultaneous diagnosis of tuberculous pleurisy and malignant pleural effusion using metagenomic next-generation sequencing (mNGS)

BackgroundMetagenomic next-generation sequencing (mNGS) has become a powerful tool for pathogen detection, but the value of human sequencing reads generated from it is underestimated.MethodsA total of 138 patients with pleural effusion (PE) were diagnosed with tuberculous pleurisy (TBP, N = 82), malignant pleural effusion (MPE, N = 35), or non-TB infection (N = 21), whose PE samples all underwent mNGS analysis. Clinical TB tests including culture, Acid-Fast Bacillus (AFB) test, Xpert, and T-SPOT, were performed. To utilize mNGS for MPE identification, 25 non-MPE samples (20 TBP and 5 non-TB infection) were randomly selected to set human chromosome copy number baseline and generalized linear modeling was performed using copy number variant (CNV) features of the rest 113 samples (35 MPE and 78 non-MPE).ResultsThe performance of TB detection was compared among five methods. T-SPOT demonstrated the highest sensitivity (61% vs. culture 32%, AFB 12%, Xpert 35%, and mNGS 49%) but with the highest false-positive rate (10%) as well. In contrast, mNGS was able to detect TB-genome in nearly half (40/82) of the PE samples from TBP subgroup, with 100% specificity. To evaluate the performance of using CNV features of the human genome for MPE prediction, we performed the leave-one-out cross-validation (LOOCV) in the subcohort excluding the 25 non-MPE samples for setting copy number standards, which demonstrated 54.1% sensitivity, 80.8% specificity, 71.7% accuracy, and an AUC of 0.851.ConclusionIn summary, we exploited the value of human and non-human sequencing reads generated from mNGS, which showed promising ability in simultaneously detecting TBP and MPE.

Using dCas9 as an intermediate bridge of loop-mediated isothermal amplification-based lateral flow colorimetric biosensor for point-of-care Salmonella detection

Foodborne pathogens pose a serious threat to human health worldwide. Combining isothermal amplification techniques (e.g., loop-mediated isothermal amplification, LAMP) with lateral flow biosensor (LFB) is ideal for point-of-care testing of foodborne pathogens, but challenges remain in distinguishing target amplicons and by-products. Here, we developed a portable lateral flow colorimetric biosensor, termed CALL (dCas9-assisted LAMP-based LFB), using dCas9 as an intermediate bridge to address these challenges. As a proof-of-concept, we chose Salmonella as the model. Strategically, LAMP amplicons of the target gene contain many contiguous repeats that can be recognized and unlocked by dCas9/single-guide RNA, which then serve as scaffolds to assemble many gold nanoparticles (GNPs) into chainlike structures on the test line. This strategy eliminates false positives caused by by-products and enables all repeats of LAMP amplicon to participate in capturing GNPs, greatly improving specificity and sensitivity. The biosensor integrates rapid extraction, LAMP amplification, dCas9-based assembly and LFB analysis without requiring complex equipment, enabling on-site screening of Salmonella as low as 41 CFU/mL in 40 min. The practicability of the biosensor was further demonstrated by testing real foods. The success of CALL provides a promising direction for developing foodborne pathogen detection tools.

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Amplification-Free COVID-19 Detection by Digital Droplet REVEALR.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, exposed a pressing need for new public health tools for pathogen detection, disease diagnosis, and viral genotyping. REVEALR (RNA-encoded viral nucleic acid analyte reporter) is an isothermal DNAzyme-based point-of-care diagnostic that functions with a detection limit of ∼10 copies/μL when coupled with a preamplification step and can be utilized for viral genotyping of SARS-CoV-2 variants of concern through base pair mismatch recognition in a competitive binding format. Here, we describe an advanced REVEALR platform, termed digital droplet REVEALR (ddREVEALR), that can achieve direct viral detection and absolute sample quantitation utilizing a signal amplification strategy that relies on chemical modifications, DNAzyme multiplexing, and volume compression. Using an AI-assisted image-based readout, ddREVEALR was found to achieve 95% positive predictive agreement from a set of 20 nasal pharyngeal swabs collected at UCI Medical Center in Orange, California. We propose that the combination of amplification-free and protein-free analysis makes ddREVEALR a promising application for direct viral RNA detection of clinical samples.

CRISPR-Cas- and Aptamer-based Systems for Diagnosing Pathogens: A Review

Pathogenic infections cause severe clinical illnesses in humans and animals. Increased encounters between humans and animals and constant environmental changes exacerbate the transmission of zoonotic infectious diseases. Recently, the World Health Organization has declared some zoonotic epidemics as public health emergencies of international concern. Hence, rapid and accurate detection of the causative pathogen is particularly essential in combating emerging and re-emerging infectious diseases. Traditional pathogen detection tools are time-consuming, costly, and require skilled personnel, which greatly hinder the development of rapid diagnostic tests, particularly in resource-constrained regions. Clustered regularly interspaced short palindromic repeats (CRISPR-)-Cas- and aptamer-based platforms have replaced traditional pathogen detection methods. Herein we review two novel next-generation core pathogen detection platforms that are utilized for clinical and foodborne pathogenic microorganisms: CRISPR-Cas-based systems, including dCas9, Cas12a/b, Cas13, and Cas14; and aptamer-based biosensor detection tools. We highlight CRISPR-Cas- and aptamer-based techniques and compare the strengths and weaknesses. CRISPR-Cas-based tools require cumbersome procedures, such as nucleic acid amplification and extraction, while aptamer-based tools require improved sensitivity. We review the combination of CRISPR-Cas- and aptamer-based techniques as a promising approach to overcome these deficiencies. Finally, we discuss Cas14-based tools as functionally stronger platforms for the detection of non-nucleic acid targets.

Evaluation of the ability of commercial disinfectants to degrade free nucleic acid commonly targeted using molecular diagnostics

Polymerase chain reaction (PCR) is an essential tool for rapid detection of pathogens, but is susceptible to cross-contamination by residual nucleic acid, leading to false-positive results. Adequate surface decontamination would help prevent this, but most protocols target infectious microbes rather than free nucleic acid. The aim of this study was to evaluate the ability of commercial surface disinfectants to degrade different representative classes of nucleic acid. Commercial surface disinfectants with various active ingredients, as well as 10% chlorine bleach, were tested. Nucleic acid was dried on to stainless steel coupons and treated with disinfectant for 0-4min prior to neutralization and quantification by quantitative reverse transcription PCR. The effective disinfectants were also evaluated in the presence of organic load. Only dilute chlorine bleach and the hypochlorite-based commercial disinfectant significantly degraded any type of free nucleic acid. Hydrogen-peroxide- and quaternary-ammonium-based disinfectants gave <1 log reduction after 4min for all targets. Results were time-dependent for each target, which underscores the importance of adequate contact time. Organic load appeared to have little impact on the efficacy of hypochlorite-based disinfectants for nucleic acid degradation. This study demonstrates the importance of proper selection and application of disinfectant to remove residual nucleic acid when processing samples for molecular diagnostic testing.

Developing tools for rapid detection of pathogens

Developing tools for rapid detection of pathogens

327. Understanding Current Clinical Scenarios for Plasma Microbial Cell-free DNA Diagnostic Testing toward Informing Diagnostic Stewardship

Abstract Background Plasma microbial cell-free (mcfDNA) testing has emerged as a promising tool for unbiased detection of pathogens in patients with suspected infections. A systematic evaluation of the patient populations in which this test is used may provide insight into applications for clinical use. We describe demographic and clinical characteristics of patients tested with plasma mcfDNA linked to the Premier Healthcare Database (PHD). Methods A retrospective cross-sectional analysis was conducted using the PHD: a US hospital-based, service-level, all-payer database containing information primarily from geographically diverse communities, teaching hospitals, and health systems. Patients with plasma mcfDNA testing 4/1/2018-12/31/2020 were deterministically linked to the PHD. Patient characteristics were collected via chargemaster data, ICD-10 diagnosis, and procedure codes. Immunocompromised (IC) status was identified using 2021 Agency for Healthcare Research (AHRQ) ICD-based definitions. The AHRQ Elixhauser Comorbidity Index score was derived using ICD-10 codes to indicate comorbidity burden. Results A total of 778 patients in the PHD underwent plasma mcfDNA testing during the study period, 88% of whom were tested in the inpatient setting (Table 1). Thirteen percent had a discharge diagnosis of sepsis with an unspecified organism. Median inpatient length of stay was 10 days with a median of 4 days from time of admission to plasma mcfDNA testing. Forty-seven percent of patients were IC with a majority (62%) having an Elixhauser score ≥ 7, suggestive of a high comorbidity burden (Table 2). Two hundred forty-nine (32%) inpatients required intensive care unit (ICU) level care for a median 15 days. Conclusion Plasma mcfDNA testing is primarily being used in the inpatient setting in a wide variety of clinical scenarios, particularly among seriously ill and immunocompromised patients, who tend to have broad infectious disease differentials and high morbidity and mortality risks. Understanding the current populations, indications, and timing of mcfDNA testing may contribute to developing diagnostic stewardship research and guidelines to optimize impact on clinical outcomes. Disclosures Constance Lau, MPH, Karius, Inc.: Current employee|Karius, Inc.: Stocks/Bonds T. Matthew Hill, PharmD, PhD, Karius, Inc: Paid employee|Karius, Inc: Stocks/Bonds Frederick S. Nolte, PhD, D(ABMM), F(AAM), Karius: Employee.

2223. A comparative analysis of the detection of UTI pathogens via culture method and the Open Array-nanofluidic real time PCR method

Abstract Background Urinary tract infections (UTI) are the second most prevalent microbial infection, impacting 150 million people globally every year. Traditionally, urine culture is considered the gold standard for UTI pathogen detection. However, molecular techniques, such as real time multiplex PCR tests targeting multiple pathogens, are becoming the alternative diagnostic tools for rapid detection of pathogens with the potential to provide quick diagnosis and enable targeted treatment. Due to the serious economic and healthcare utilization burden UTIs pose, early pathogen detection with a rapid turn-around-time to results has the potential to be instrumental for improving patient care and outcomes. Methods A total of 300 deidentified patient samples that were previously tested via urine culture (ARUP laboratories, Utah) were subjected to real time PCR molecular testing employing the nanofluidic Open Array ® platform (HealthTrackRX, Texas). Statistical analyses were performed using R version 3.6.0. Results Among 300 urine specimens studied, culture detected pathogens in 183 patient samples (61%), and 117 samples were deemed negative. Culture and PCR results demonstrated an overall agreement of 75.3% (n=226) with 59% positive (n=177) and 16.3% negative (n=49). Results for 24.7% samples (n=74) were discordant. A total of 2% (n=6) were culture positive and PCR negative, and 22.7% (n=68) were culture negative and PCR positive. Agreement between PCR and culture positive results was 0.97, 95%CI (0.94, 0.99) (177/183). Among the PCR positive samples (n=245), 32.7% were poly-microbial (n=80) and 67.3% were mono-microbial (n=165). In comparison, among culture positive samples (n=183), 33.3% (n=61) were polymicrobial and 65.6% (n=120) monomicrobial. Conclusion Our study demonstrates that multiplex real time PCR-based detection of UTI bacterial pathogens has positive agreement with the traditional urine culture method. PCR based testing has the potential to deliver quick pathogen identification to enable targeted treatment options and medication adjustments. Further studies will correlate semi-quantitative cfu/mL culture values with semi-quantitative copies/mL PCR values to further refine result reporting and test performance. Disclosures Pallavi Upadhyay, PhD, HealthTrackRx: Stocks/Bonds Fahida Surar, B.S., HealthTrackRx: Salaried Employee Geun Kim, M.S., HealthTrackRx: Salaried Employee Jay Reddy, PhD, HealthTrackRx: Stocks/Bonds Barbara D. Alexander, MD, Astellas: Advisor/Consultant|HealthtrackRx: Advisor/Consultant|HealthtrackRx: Grant/Research Support|Scynexis: Grant/Research Support|UpToDate: Advisor/Consultant Kimberly Hanson, MD, MHS, FIDSA, HealthTrackRx: Advisor/Consultant|HealthTrackRx: Clinical Advisory Board Member Vijay Singh, PhD, HealthTrackRx: Stocks/Bonds.

Development of a CRISPR/Cas9-integrated lateral flow strip for rapid and accurate detection of Salmonella

Rapid and accurate detection of foodborne pathogens is crucial for safeguarding human health. Herein, we constructed a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-integrated lateral flow strip (Cas9-LFS) for detecting Salmonella . A primer set targeting the fimA gene of Salmonella spp. was used to amplify all Salmonella serovars. After extraction, the genomic DNA of Salmonella was amplified with unlabelled forward primer and FITC-labelled reverse primer, thus producing numerous FITC-labelled amplicons that can be precisely recognized by Cas9 complexed with a specific single-guide RNA (sgRNA). When loaded on the LFS, the complexes of amplicons and Cas9/sgRNA can act as bonds to anchor gold nanoparticles to the test line, resulting in a visible color change. This design makes Cas9-LFS more powerful than conventional LFS in eliminating false-positives caused by primer dimers. By using Salmonella Typhimurium as a reference strain, Cas9-LFS achieved a limit of detection as low as 10 2 CFU/mL and showed good accuracy in distinguishing Salmonella -contaminated milk. We expect this unique strategy to open a new window for building versatile pathogen detection tools. • A CRISPR/Cas9-integrated LFS (Cas9-LFS) was constructed for accurate and visual detection of Salmonella. • Primer-dimer interference that is inevitable in conventional amplifications can be totally overcome with the method. • As low as 10 2 CFU/mL of Salmonella can be well measured with the constructed method. • Salmonella -contaminated milk samples have been measured with considerable accuracy.

LAMP-Based Point-of-Care Biosensors for Rapid Pathogen Detection

Seeking optimized infectious pathogen detection tools is of primary importance to lessen the spread of infections, allowing prompt medical attention for the infected. Among nucleic-acid-based sensing techniques, loop-mediated isothermal amplification is a promising method, as it provides rapid, sensitive, and specific detection of microbial and viral pathogens and has enormous potential to transform current point-of-care molecular diagnostics. In this review, the advances in LAMP-based point-of-care diagnostics assays developed during the past few years for rapid and sensitive detection of infectious pathogens are outlined. The numerous detection methods of LAMP-based biosensors are discussed in an end-point and real-time manner with ideal examples. We also summarize the trends in LAMP-on-a-chip modalities, such as classical microfluidic, paper-based, and digital LAMP, with their merits and limitations. Finally, we provide our opinion on the future improvement of on-chip LAMP methods. This review serves as an overview of recent breakthroughs in the LAMP approach and their potential for use in the diagnosis of existing and emerging diseases.