Molecular Approaches For Detection Research Articles

Reliability of the chemical, metabolic, and molecular methods in discriminating aflatoxigenic from non-aflatoxigenic Aspergillus isolates

Aflatoxins, potent carcinogens produced by Aspergillus species (A. flavus and A. parasiticus), are primarily consumed through dietary sources. Aflatoxin management using non-aflatoxigenic Aspergillus to outcompete their aflatoxigenic congeneric to achieve low residual toxin levels is a promising biological control (BC) field intervention. A prerequisite for the BC program is the accurate delineation of aflatoxigenic fungi from their non-aflatoxigenic relatives. In this study, we use three methods: Chemical (Dichlorvos-ammonia), Metabolic (ELISA coupled live cultures), and Molecular (10 aflatoxin biosynthetic genes) to determine the aflatoxigenicity of Aspergillus spp. previously collected from Eastern Kenya, an aflatoxin hotspot. Thirty-six different Aspergillus isolates were identified and studied microscopically from a total of 314 fungal cultures. The chemical method reported 21 aflatoxigenic and 15 non-aflatoxigenic isolates, while the metabolic technique distinguished 20 aflatoxigenic and 16 non-aflatoxigenic isolates. A large proportion (97.22 %) of the isolates harboured the aflR, aflO, and aflM genes, while 91.67 % of the isolates possessed the aflS gene. All isolates (100 %) harboured the aflD, aflQ, aflP, and aflK genes. ELISA-coupled live cultures positively correlated with Dichlorvos-ammonia (p > 0.01), only differing in one sample, indicating a strong concurrence between the two methods at segregating toxigenic A. flavus isolates. Cluster analysis based on Multiple Correspondence revealed that the non-aflatoxigenic group exhibited similar characteristics correlating with the absence of aflR, aflS, aflI, and aflO genes. These findings indicate the reliability of the Chemical and Metabolic methods and can function as confirmatory assays to complement the molecular detection approach in determining aflatoxigenicity among Aspergillus spp.

Rapid and sensitive diagnosis of live Mycobacterium tuberculosis using clustered regularly interspaced short palindromic repeat‐Cas13a point‐of‐care RNA testing

AbstractMycobacterium tuberculosis (MTB) is the causal pathogen of tuberculosis (TB). Rapid and accurate detection of live MTB is important for transmission control and patient treatment. Here, we described a clustered regularly interspaced short palindromic repeat (CRISPR)‐Cas13a‐based molecular diagnosis approach for rapid and specific detection of live MTB. This detection method, which we termed CRISPR‐Live‐MTB, contained two consecutive reactions including nuclear acid sequence‐based amplification (NASBA) and CRISPR‐Cas13a collateral cleavage reaction. CRISPR‐Live‐MTB could efficiently detect MTB single‐stranded RNA (ssRNA) in 2 hours with high specificity over double‐stranded DNA (dsDNA). Importantly, CRISPR‐Live‐MTB exhibited a limit of detection of 2.4 copies for MTB ssRNA, which was 1000 times lower than that of the clinically used NASBA method. Moreover, lateral flow was integrated into the CRISPR‐Live‐MTB method to enable point‐of‐care testing application with a sensitivity of 95% and a specificity of 100%. Overall, our study demonstrated the feasibility of CRISPR‐Live‐MTB as a rapid, sensitive, and specific approach for live MTB detection.

Universal digital high-resolution melting for the detection of pulmonary mold infections.

Improvements in diagnostics for invasive mold infections are urgently needed. This work presents a new molecular detection approach that addresses technical and workflow challenges to provide fast pathogen detection, identification, and quantification that could inform treatment to improve patient outcomes.

Establishment and application of a rapid diagnostic method for BVDV and IBRV using recombinase polymerase amplification-lateral flow device.

Bovine Viral Diarrhea Virus (BVDV) and Infectious Bovine Rhinotracheitis Virus (IBRV) are the two most prevalent infectious diseases in cattle. They both can cause persistent infection and immunosuppression, resulting in significant economic losses in the livestock industry. Therefore, rapid detection of early BVDV and IBRV infections is crucial. In this study, a method for the rapid detection of BVDV and IBRV was established by using recombinase polymerase amplification (RPA) combined with lateral flow device (LFD). By optimizing the temperature and time conditions of the RPA reaction, the sensitivity, specificity, and clinical performance were evaluated. The results indicated that the RPA reaction could be completed at 40°C within 25 min. The LOD for BVDV and IBRV by RPA-LFD were 5.1 × 101 copies/μL and 6.65 × 101 copies/μL, respectively, with no cross-reactivity observed with other viruses such as CSFV, BRSV, BPIV3, BRV, and BCoV. Testing of 32 clinical samples showed consistent results between RPA-LFD and qPCR. The RPA-LFD method established in this study can be used for the rapid clinical detection of BVDV and IBRV, which providing a rapid and convenient molecular biology approach for on-site rapid detection and epidemiological investigations. Simultaneously, it offers technical support for the prevention and control of these viruses.

Quantitative Assessment of Intracellular Effectors and Cellular Response in RAGE Activation.

The review delves into the methods for the quantitative assessment of intracellular effectors and cellular response of Receptor for Advanced Glycation End products (RAGE), a vital transmembrane receptor involved in a range of physiological and pathological processes. RAGE bind to Advanced Glycation End products (AGEs) and other ligands, which in turn activate diverse downstream signaling pathways that impact cellular responses such as inflammation, oxidative stress, and immune reactions. The review article discusses the intracellular signaling pathways activated by RAGE followed by differential activation of RAGE signaling across various diseases. This will ultimately guide researchers in developing targeted and effective interventions for diseases associated with RAGE activation. Further, we have discussed how PCR, western blotting, and microscopic examination of various molecules involved in downstream signaling can be leveraged to monitor, diagnose, and explore diseases involving proteins with unique post-translational modifications. This review article underscores the pressing need for advancements in molecular approaches for disease detection and management involving RAGE.

A novel sensitive hexaplex high-resolution melt assay for identification of five human Plasmodium species plus internal control

BackgroundThe diagnosis of malaria infection in humans remains challenging, further complicated by mixed Plasmodium species infections, potentially altering disease severity and morbidity. To facilitate appropriate control measures and treatment, rapid, sensitive, and specific detection assays, including those for the second minor species, would be required. This study aimed to develop a multiplex high-resolution melting (hexaplex PCR-HRM) assay with seven distinct peaks corresponding to five Plasmodium species of the Plasmodium genus, and an internal control to limit false negatives providing quality assurance testing results. MethodsFive species-specific primers for human malaria species were designed targeting on the Plasmodium 18 small subunit ribosomal RNA (18S rRNA) and mitochondrial genes. The hexaplex PCR-HRM was developed for the simultaneous and rapid detection and differentiation of five human Plasmodium spp. The limit of detection (LoD), sensitivity, and specificity of the assay were evaluated. Artificial mixing was used to assess the ability to determine the second minor species. Furthermore, a hexaplex PCR-HRM assay was used to identify 120 Plasmodium-infected clinical isolates from Kanchanaburi, Western Thailand, where malaria is endemic. ResultsThe hexaplex PCR-HRM assay detected the targeted genome of five Plasmodium species at levels as low as 2.354–3.316 copies/uL with 91.76 % sensitivity and 98.04 % specificity. In artificial mixing, the assay could detect minority parasite species at 0.001 % of the predominant parasite population. Plasmodium vivax infections (99 %) accounted for the majority of malaria cases in Kanchanaburi, Thailand. ConclusionsThe developed hexaplex PCR-HRM assay we present in this study is a novel approach for multiplexing the Plasmodium genus and detecting five Plasmodium species with the advantage of detecting second minority parasite species. The developed one-step assay without any nesting protocols would reduce the risks of cross-contamination. Moreover, it also provides a simple, sensitive, specific, and low-cost approach for optional molecular detection of malaria.

CRISPR-Cas Detection Coupled with Isothermal Amplification of Bursaphelenchus xylophilus.

The pine wood nematode (PWN), Bursaphelenchus xylophilus, causes significant damage to pine trees and, thus, poses a serious threat to pine forests worldwide, particularly in China, Korea, and Japan. A fast, affordable, and ultrasensitive detection of B. xylophilus is urgently needed for disease diagnosis. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have reshaped molecular diagnosis, with high speed, precision, specificity, strength, efficiency, and versatility. Herein, we established two isothermal diagnostics methods based on CRISPR-based platforms (CRISPR/Cas12a and CRISPR/Cas13a) for B. xylophilus-specific detection via fluorescence or lateral-flow strip readout. The guide RNA and CRISPR RNA were designed to target the 5S ribosomal DNA intergenic spacer sequences region of B. xylophilus. Recombinase-aided amplification was used for preamplification whose reaction condition was 37°C for 15 min. The sensitivity of CRISPR/Cas12a could reach 94 copies/µl of plasmid DNA, or 2.37 copies/µl of purified genomic DNA (gDNA) within 45 min at 37°C, while the sensitivity of CRISPR/Cas13a was 1,000 times higher than that of CRISPR/Cas12a of plasmid DNA in 15 min or 100 times higher of purified gDNA at the minimum reaction time of 4 min via fluorescence measurement. The CRISPR/Cas12a assay enabled the detection of 0.01 PWNs per 100 mg of pine wood, 10 times higher than that of the CRISPR/Cas13a assay. This work enriches molecular detection approaches for B. xylophilus and provides huge potential for ultrasensitive and rapid methods to detect B. xylophilus in pine wood, facilitating point-of-sample diagnostic processing for pine wilt disease management.

Proteomics analyses of herbicide-tolerant genetically modified, conventionally, and organically farmed soybean seeds

The present study compared genetically modified (GM) crops with crops from different farming practices using high-resolution tandem mass spectrometry (HR-MS) and proteomics bioinformatics tools. In a previously published study, a number of significant differences regarding nutritional and elemental composition between a selection of GM, non-GM conventionally farmed, and organic soybeans have been found. In the present study, the proteome-level equivalence of the same samples was assessed using HR-MS. Direct comparison of tandem mass spectra and bottom-up proteomics bioinformatics indicated that proteomes of all samples investigated were very similar overall, with only a few distinct protein expression clusters obtained for GM and organic samples. Standard bottom-up proteome analyses identified 1025 soy proteins; of these 39 were found to be differentially expressed (p < 0.01) between GM, non-GM conventionally farmed, and organically farmed soybeans. Subsequent bioinformatics analyses of these proteins highlighted several potentially affected biochemical pathways that could contribute to the compositional differences reported earlier. In addition, protein markers separating conventionally, and organically farmed soybean seeds were found and peptide markers for the detection of GM soy in food and feed samples are described. Taken together, the data presented here shows that HR-MS based proteomics approaches can be used for the detection of transgenic events in food and feed grade soy, the differentiation of organically and conventionally farmed plants, and provide mechanistic explanations of effects observed on the phenotypic level of GM plants. HR-MS and proteomic bioinformatics thus should be considered key tools when developing molecular panel approaches for detection and safety assessments of novel crop varieties destined for use in feed and food.

Electrophoretic Assembly of Antibody–Antigen Complexes Facilitates 1000 Times Improvement in the Limit of Detection of Serological Paper-Based Assay

Serological assays detect the presence of specific antibodies in blood. There are urgent and important applications for serological point-of-care (POC) assays. However, available detection methods are either insufficiently sensitive or too complex for POC settings. Here, we demonstrate that lateral flow immunoassay (LFIA), which is arguably the simplest universal molecular detection approach, can serve as a methodological platform for highly sensitive serological POC assays if combined with a simple, fast, and inexpensive electrophoretic step. In this work, we compared such electrophoretically driven LFIA (eLFIA) with conventional LFIA for the detection of immunoglobulins G against hepatitis B and C in serum. The limit of detection of eLFIA was proven to be 1000 times lower than that of conventional LFIA and sufficiently low to support clinical serological tests. eLFIA takes less than 10 min, requires only a minor accessory powered by a small 9 V battery, and can be performed by an untrained person in the POC environment using a 3 μL specimen of finger-prick capillary blood.

Molecular Approaches for Detection of Trichoderma Green Mold Disease in Edible Mushroom Production.

Due to the evident aggressive nature of green mold and the consequently huge economic damage it causes for producers of edible mushrooms, there is an urgent need for prevention and infection control measures, which should be based on the early detection of various Trichoderma spp. as green mold causative agents. The most promising current diagnostic tools are based on molecular methods, although additional optimization for real-time, in-field detection is still required. In the first part of this review, we briefly discuss cultivation-based methods and continue with the secondary metabolite-based methods. Furthermore, we present an overview of the commonly used molecular methods for Trichoderma species/strain detection. Additionally, we also comment on the potential of genomic approaches for green mold detection. In the last part, we discuss fast screening molecular methods for the early detection of Trichoderma infestation with the potential for in-field, point-of-need (PON) application, focusing on isothermal amplification methods. Finally, current challenges and future perspectives in Trichoderma diagnostics are summarized in the conclusions.

Global distribution, traditional and modern detection, diagnostic, and management approaches of Rhizoctonia solani associated with legume crops.

Sustainable development relies heavily on a food system that is both safe and secure. Several approaches may lead to sustainability and food safety. An increase in the cultivation of legume crops is one of the approaches for enhancing agricultural viability and ensuring adequate food supply. Legumes may increase daily intake of fiber, folate, and protein as substitutes for meat and dairy. They are also crucial in various intercropping systems worldwide. However, legume production has been hampered by Rhizoctonia solani due to its destructive lifestyle. R. solani causes blights, damping off, and rotting diseases in legume crops. Our knowledge of the global distribution of R. solani associated with legume crops (alfalfa, soybean, chickpea, pea, lentil, common bean, and peanut), detection, diagnosis, and management of legume crops diseases caused by R. solani is limited. Traditional approaches rely on the incubation of R. solani, visual examination of symptoms on host legume crops, and microscopy identification. However, these approaches are time-consuming, require technical expertise, fail to detect a minimal amount of inoculum, and are unreliable. Biochemical and molecular-based approaches have been used with great success recently because of their excellent sensitivity and specificity. Along with conventional PCR, nested PCR, multiplex PCR, real-time PCR, magnetic-capture hybridization PCR, and loop-mediated isothermal amplification have been widely used to detect and diagnose R. solani. In the future, Next-generation sequencing will likely be used to a greater extent to detect R. solani. This review outlines global distribution, survival, infection and disease cycle, traditional, biochemical, molecular, and next-generation sequencing detection and diagnostic approaches, and an overview of the resistant resources and other management strategies to cope with R. solani.

Single-molecule force manipulation facilitated multiple rereads of a drug-bound DNA at specified sites by surface-enhanced Raman spectroscopy.

Single-molecule surface-enhanced Raman spectroscopy (SERS) directly probes the vibrational characteristics of individual molecules to uncover the information inaccessible from ensemble measurements. As a surface-sensitive technique, the amplification of SERS signal occurs upon the excitation of localized surface plasmon resonance in metallic nanostructures, which can overcome optical diffraction limit for spatial detections at a nanometer scale. Complementary to this passive detection technique, optical tweezers-based single-molecule force manipulation method enables the active control of a target molecule with the restricted orientation in three dimensions. By applying a stretching force to the target molecule, it provides high-frequency feedback in sub-milliseconds with sub-piconewton force resolution and sub-nanometer distance sensitivity. Combining the active molecular control and the passive molecular vibrational measurement, we have integrated the dual-traps optical tweezers technique with the Raman spectroscopic detection platform. Under precise single-molecule force manipulations, the real-time SERS readout of the specified sites of a drug-bound DNA are recorded and re-recorded multiple times in a programmable manner. Our results showcase the promise of this correlative molecular manipulation and detection approach for real-time, label-free, and multi-dimensional characterizations of a single target biomacromolecule with both high spatial resolution and high site-specified recognition accuracy.

Effect of the focused gap-plasmon mode on tip-enhanced Raman excitation and scattering.

As a powerful molecular detection approach, tip-enhanced Raman scattering (TERS) spectroscopy has the advantages of nanoscale spatial resolution, label-free detection and high enhancement factor, therefore has been widely used in fields of chemistry, materials and life sciences. A TERS system enhanced by the focused gap-plasmon mode composed of Surface Plasmon Polariton (SPP) focus and the metal probe has been reported, however, its underlying enhancement mechanism for Raman excitation and scattering remains to be deeply explored. Here, we focus on the different performances of optical focus and SPP focus in the TERS system, and verify that the cooperation of these two focuses can produce maximum enhancement in a local electromagnetic field. Further, the Purcell effect on sample scattering in such a system is studied for the enhancement of Raman scattering collection in the far field. Finally, the local field enhancement and the sample far-field scattering enhancement are combined to show a full view of the whole process of TERS enhancement. This research can be applied to optimize the excitation and collection of Raman signals in TERS systems, which is of great value for the research and development of TERS technology.

Dendric nanostructures for SARS-CoV-2 proteins detection based on surface enhanced infrared absorption spectroscopy.

Infrared spectroscopy is a non-destructive and rapid characterization tool that can distinguish different viral proteins by spectral details. However, traditional infrared spectroscopy has insufficient absorption signal intensity contrast when measuring low-concentration samples. In this work, surface enhanced infrared absorption (SEIRA) spectroscopy is proposed by deploying a novel nanostructure array as SEIRA substrates. An array of gold dendric nanostructures are designed and fabricated with a precision resonance control to achieve surface enhancement covering a broadband molecular "finger-print" region. The spectral positions of the multiple resonances accurately correspond to the characteristic absorption peaks of the SARS-CoV-2 proteins. An approach for SARS-CoV-2 protein detection based on SEIRA spectroscopy is then proposed. A low concentration detection of 40 μg/ml diluted SARS-CoV-2 nucleocapsid protein is experimentally demonstrated and the enhancement factor (EF) achieved is in good agreement with simulation results. The SEIRA methodology based on broadband resonance nanostructure design provides a systematic approach for sensitive, non-destructive and rapid protein molecular detection, which could be extended to various kind of molecular characterization and biomedical diagnostics.

Nanotechnology for the detection of plant pathogens

Plant pathogens are the important yield-limiting factors, which significantly reduce crop productivity globally, posing serious problems for food security and continues to be the biggest agricultural concern in the world. Even though chemical treatment is still the primary strategy for reducing the incidence of plant disease, their repeated application can cause the pathogens to become less susceptible. Over spraying can also pollute the environment and significantly affect soil microbiota. Therefore, to ensure agricultural sustainability and food security, efficient diagnostic techniques for the rapid identification of plant pathogens in the early stages of infection become crucial. Many molecular approaches for rapid plant pathogen detection have been developed to achieve this goal. However, they are time-consuming, costly, require skilled operators, and are generally unsuitable for in-situ analysis. Plant protection is feasible when any of the nanotechnology tools like microneedle patches, nanopore sequencing, nano barcoding, nano biosensors, quantum dots, nano diagnostic kit equipment, metal nanoparticles, miRNA based nanodiagnosis, and array based nano sensors is used for plant pathogen diagnosis. As they emerge as a potential tool to improve the sensitivity, accuracy, and rapidness of plant pathogen identification, and facilitate high-throughput analysis. The current review focuses on the use of nanotechnology for more quick , inexpensive, and accurate plant pathogens diagnosis.

1669P Monitoring immune checkpoint inhibition in advanced solid tumors using genome-wide cfDNA fragmentomes

The rapid identification of disease progression in patients receiving immune checkpoint inhibitors (ICIs) is challenging using methods that rely on imaging assessments. Here, we investigated genome-wide cell-free DNA (cfDNA) fragmentation profiles to molecularly detect disease progression in the phase I study of entinostat and nivolumab +/- ipilimumab in advanced solid tumors (ETCTN-9844, NCT02453620). Patients with metastatic or unresectable solid tumors received an entinostat run-in 2 weeks prior to the addition of anti-PD-1 and anti-CTLA-4 ICIs. Blood was collected at baseline (50/50 patients) and at 10 weeks after treatment initiation (27/50 patients). Low-coverage (1-2x) whole genome sequencing was performed on cfDNA from plasma samples. Genome-wide cfDNA fragmentation features were included in a Bayesian model to approximate a tumor fraction as compared to best response by RECIST v1.1. Molecular response was defined as reduction (≥ 30%) of the fragmentation based tumor fraction between the baseline and 10-week time point. 50 patients with breast (n = 28), salivary gland (n = 7), gastrointestinal (n = 6), gynecological (n = 4), sarcoma (n = 3), and lung cancer (n = 2) received a median of 4 prior lines of therapy (0-14). Response assessment using molecular analyses and best overall response by RECIST were reported at a mean of 9.3 (5-10.7) and 14.3 weeks (5-73). Molecular responders and non-responders at week 10 experienced a median PFS that was not reached (NR) and 4.5 months (HR = 5.4; 95% CI 1.7-17; p = 0.004) while patients with RECIST response and non-response at week 10 had similar PFS (median 6.4 vs. 6.7 months; HR = 1.7; 95% CI 0.54-5.3; p = 0.36). Among the subgroup of radiographic non-responders with stable disease, PFS for molecular responders was NR while for molecular non-responders it was 5.2 months (p = 0.021). There is a clinical need for non-invasive tests to identify primary or acquired resistance to ICIs, sparing patients from ineffective treatments associated with immune-related adverse events. Here, we demonstrate a cfDNA fragmentation based approach for molecular detection of disease progression that could guide future trials with ICIs.

Molecular Approaches for Detection of Rifampin-Resistant Mycobacterium tuberculosis and Its Associated Risk Factors among General Population

GeneXpert MTB/RIF is a molecular technique for the diagnosis of tuberculosis. This method not only detects M. tuberculosis but also identifies the absence or presence of different drug resistant mutations in DNA fragments like rifampicin resistance. In this study, about 200 septum samples were collected from tuberculosis suspected people of Isfandyar Bukhari District Head Quarter Hospital Attock for screening and determination of Mycobacterium tuberculosis and multi-drug-resistant tuberculosis. The samples were screened for the presence of M. tuberculosis using Fluorescence Microscopy staining followed by GeneXpert MTB/RIF assay. The results were further confirmed by Line Probe Assay and Mycobacterium Growth Indicator Tube 960 techniques. In a total of 200 samples, 45(22.5%) were found positive, 155 (77.5%) were classified negative on culture while 38(19%) were positive and 162(81%) were negative on FM, 33(16.5%) were positive and 167(83.5%) were negative on ZN staining microscopy and 47(23.5%) were noted positive and 153 (76.5%) were negative on GeneXpert. The overall sensitivity of GeneXpert was 07% higher than that of smear microscopy and 4.2 % higher than culture.Different risk factors reported for TB are; malnutrition (23.40%), health care workers (14.89%), immunosuppression (12.76%), unimmunized with Bacille Calmette-Guérin (BCG) vaccine and contact with infected persons (10.63%), diabetes mellitus (8.51%), poverty and foreign-born children are (4.2%). It is concluded that the most common risk factor for TB was malnutrition (23.40%) and the foreign-born children (4.2%) were least affected. GeneXpert assay for identification of tuberculosis was found more sensitive than fluorescence microscopy and for isolation of MTB and rifampicin resistance, GeneXpert RIF assay can efficiently be usedwhich is simple, robust and efficient with minimum handling technique.

Development of gold nanoparticle-based visual assay for rapid detection of Escherichia coli specific DNA in milk of cows affected with mastitis

For early diagnosis of infections, a rapid, sensitive and precise method to identify microbial DNA coming from veterinary clinical samples, such as milk of cows affected with mastitis, is fascinating. We developed an amplification-free visual assay for rapid and sensitive detection of specific DNA from Escherichia coli, particularly uid A gene encoding for beta-glucuronidase, isolated in milk of cows affected with mastitis, based on multiple gold nanoparticles (AuNPs) captured on a magnetic microbead surface, leading to plasmonic signal enhancement, thereby improving overall sensitivity of the assay. This test can be performed in 1–1.5 h after post template DNA preparation and can be visualized with naked eyes without the use of any expensive equipment. The visual assay detects a minimum microbial load of 102 CFU/μl and can identify bacterial DNA as low as 1 pg. This test provides a precise detection tool for E coli in clinical samples, such as milk of cows affected with mastitis, as a quick and user-friendly molecular detection approach.

Molecular approaches for detection and quantification of Vibrio scophthalmi based on recA.

Molecular approaches for detection and quantification of Vibrio scophthalmi based on recA.

Novel Molecular Barcoding for Rapid Pathogen Detection in Infectious Keratitis.

ABSTRACT This proof-of-concept study describes the application of a novel molecular barcoding approach for rapid and comprehensive pathogen detection in infectious keratitis.