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We developed a novel culture-based test, the Rapid CAZ/AVI NP test, for rapid identification of ceftazidime/avibactam susceptibility/resistance in Enterobacterales. This test is based on glucose metabolization upon bacterial growth in the presence of a defined concentration of ceftazidime/avibactam (128/53 µg/mL). Bacterial growth is visually detectable by a red to yellow color change of red phenol, a pH indicator. A total of 101 well characterized enterobacterial isolates were used to evaluate the test performance. This test showed positive percent agreement of 100% and negative percent agreement of 98.5% with overall percent agreement of 99%, by comparison with the MIC gradient strip test (Etest) taken as the reference standard method. The Rapid CAZ/AVI NP test had only 1.5% major errors and 0% extremely major errors. This test is rapid (result within 2 hours 45 minutes), reliable, affordable, easily interpretable, and easy to implement in clinical microbiology laboratories without requiring any specific equipment.
Assuntos
Compostos Azabicíclicos , Ceftazidima , Gammaproteobacteria , Ceftazidima/farmacologia , Enterobacteriaceae , LaboratóriosRESUMO
The increasing use of ceftazidime-avibactam has led to the emergence of a wide range of ceftazidime-avibactam-resistant blaKPC-2 variants. Particularly, the conventional carbapenemase phenotypic assay exhibited a high false-negative rate for KPC-2 variants. In this study, three colloidal gold immunoassays, including the Gold Mountainriver CGI test, Dynamiker CGI test and NG-Test CARBA5, and GeneXpert Carba-R, were used to detect the presence of KPC-2 carbapenemase and its various variants in 42 Klebsiella pneumoniae strains. These strains covered blaKPC-2 (13/42) and 16 other blaKPC-2 variants including blaKPC-12 (1/42), blaKPC-23 (1/42), blaKPC-25 (1/42), blaKPC-33 (6/42), blaKPC-35 (1/42), blaKPC-44 (1/42), blaKPC-71 (1/42), blaKPC-76 (8/42), blaKPC-78 (1/42), blaKPC-79 (1/42), blaKPC-100 (1/42), blaKPC-127 (1/42), blaKPC-128 (1/42), blaKPC-144 (1/42), blaKPC-157 (2/42), and blaKPC-180 (1/42). For KPC-2 strains, all four assays showed 100% negative percentage agreement (NPA) and 100% positive percentage agreement (PPA) with sequencing results. For all 16 KPC-2 variants, GeneXpert Carba-R showed 100% NPA and 100% PPA, and the three colloidal gold immunoassays showed 100% NPA, while the PPAs of the Gold Mountainriver CGI test, Dynamiker CGI test, and NG-Test CARBA5 were 87.5%, 87.5%, and 68.8%, respectively. We also found a correlation between the mutation site in the amino acid of the variants and false-negative results by colloidal gold immunoassays. In conclusion, the GeneXpert Carba-R has been proven to be a reliable method in detecting KPC-2 and its variants, and the colloidal gold immunoassay tests offer a practical and cost-effective approach for their detection. For the sample with a negative result by a colloidal gold immunoassay test but not matching the drug-resistant phenotype, it is recommended to retest using another type of kit or the GeneXpert Carba-R assay, which can significantly improve the accuracy of detection.
Assuntos
Coloide de Ouro , Infecções por Klebsiella , Klebsiella pneumoniae , beta-Lactamases , beta-Lactamases/genética , Klebsiella pneumoniae/genética , Imunoensaio/métodos , Humanos , Coloide de Ouro/química , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/diagnóstico , Sensibilidade e Especificidade , Proteínas de Bactérias/genética , Testes de Sensibilidade MicrobianaRESUMO
BACKGROUND: Vibrio vulnificus exists as one of the most serious foodborne pathogens for humans, and rapid and sensitive detection methods are needed to control its infections. As an emerging method, The Loop-Mediated Isothermal Amplification (LAMP) assay has been applied to the early detection of various foodborne pathogens due to its high efficiency, but sample preprocessing still prolongs the complete detection. To optimize the detection process, our study established a novel sample preprocessing method that was more efficient compared to common methods. RESULT: Using V. vulnificus as the detecting pathogen, the water-lysis-based detecting LAMP method shortened the preprocessing time to ≤ 1 min with 100% LAMP specificity; the detection limits of the LAMP assay were decreased to 1.20 × 102 CFU/mL and 1.47 × 103 CFU/g in pure culture and in oyster, respectively. Furthermore, the 100% LAMP specificity and high sensitivity of the water-lysis method were also obtained on detecting V. parahaemolyticus, V. alginolyticus, and P. mirabilis, revealing its excellent LAMP adaption with improvement in sensitivity and efficiency. CONCLUSION: Our study provided a novel LAMP preprocessing method that was more efficient compared to common methods and possessed the practical potential for LAMP application in the future.
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Técnicas de Diagnóstico Molecular , Vibrio vulnificus , Humanos , Vibrio vulnificus/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Água , Manejo de Espécimes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND AND OBJECTIVES: Malaria continues to be a significant public health concern in India, with several regions experiencing endemicity and sporadic outbreaks. The prevalence of malaria in blood donors, in India, varies between 0.02% and 0.07%. Common techniques to screen for malaria, in blood donors and patients, include microscopic smear examination and rapid diagnostic tests (RDTs) based on antigen detection. The aim of this study was to evaluate a new fully automated analyser, XN-31, for malaria detection, as compared with current practice of using RDT. MATERIALS AND METHODS: Cross-sectional analytical study was conducted to evaluate clinical sensitivity and specificity of new automated analyser XN-31 among blood donors' samples and clinical samples (patients with suspicion of malaria) from outpatient clinic collected over between July 2021 and October 2022. No additional sample was drawn from blood donor or patient. All blood donors and patients' samples were processed by malaria rapid diagnostic test, thick-smear microscopy (MIC) and the haematology analyser XN-31. Any donor blood unit incriminated for malaria was discarded. Laboratory diagnosis using MIC was considered the 'gold standard' in the present study. Clinical sensitivity and specificity of XN-31 were compared with the gold standard. RESULTS: Fife thousand and five donor samples and 82 diagnostic samples were evaluated. While the clinical sensitivity and specificity for donor samples were 100%, they were 72.7% and 100% for diagnostic samples. CONCLUSION: Automated haematology analysers represent a promising solution, as they can deliver speedy and sensitive donor malaria screening assessments. This method also has the potential to be used for pre-transfusion malaria screening along with haemoglobin estimation.
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Doadores de Sangue , Malária , Humanos , Índia , Malária/diagnóstico , Malária/sangue , Estudos Transversais , Feminino , Masculino , Sensibilidade e Especificidade , Adulto , Testes Hematológicos/métodos , Testes Hematológicos/instrumentaçãoRESUMO
BACKGROUND: Currently, culture methods are commonly used in clinical tests to detect pathogenic fungi including Candida spp. Nonetheless, these methods are cumbersome and time-consuming, thereby leading to considerable difficulties in diagnosis of pathogenic fungal infections, especially in situations that respiratory samples such as alveolar lavage fluid and pleural fluid contain extremely small amounts of microorganisms. The aim of this study was to elucidate the utility and practicality of microfluidic chip technology in quick detection of respiratory pathogenic fungi. METHODS: DNAs of clinical samples (mainly derived from sputa, alveolar lavage fluid, and pleural fluid) from 64 coastal patients were quickly detected using microfluidic chip technology with 20 species of fungal spectrum and then validated by Real-time qPCR, and their clinical baseline data were analyzed. RESULTS: Microfluidic chip results showed that 36 cases infected with Candida spp. and 27 cases tested negative for fungi, which was consistent with Real-time qPCR validation. In contrast, only 16 cases of fungal infections were detected by the culture method; however, one of the culture-positive samples tested negative by microfluidic chip and qPCR validation. Moreover, we found that the patients with Candida infections had significantly higher rates of platelet count reduction than fungi-negative controls. When compared with the patients infected with C. albicans alone, the proportion of males in the patients co-infected with multiple Candidas significantly increased, while their platelet counts significantly decreased. CONCLUSIONS: These findings suggest that constant temperature amplification-based microfluidic chip technology combined with routine blood tests can increase the detection speed and accuracy (including sensitivity and specificity) of identifying respiratory pathogenic fungi.
Assuntos
Micoses , Infecções Respiratórias , Masculino , Humanos , Microfluídica , Fungos/genética , Micoses/diagnóstico , Candida/genética , Candida albicans , Sensibilidade e Especificidade , Infecções Respiratórias/diagnósticoRESUMO
Babesia duncani, responsible for human babesiosis, is one of the most important tick-borne intraerythrocytic pathogens. Traditionally, babesiosis is definitively diagnosed by detecting parasite DNA in blood samples and examining Babesia parasites in Giemsa-stained peripheral blood smears. Although these techniques are valuable for determining Babesia duncani, they are often time-consuming and laborious. Therefore, developing rapid and reliable B. duncani identification assays is essential for subsequent epidemiological investigations and prevention and control. In this study, a cross-priming amplification (CPA) assay was developed, combined with a vertical flow visualization strip, to rapidly and accurately detect B. duncani infection. The detection limit of this method was as low as 0.98 pg/µl of genomic DNA from B. duncani merozoites per reaction at 59 °C for 60 min. There were no cross-reactions between B. duncani and other piroplasms infective to humans and mammals. A total of 592 blood samples from patients bitten by ticks and experimental infected hamsters were accurately assessed using CPA assay. The average cost of the CPA assay is as low as approximately $ 0.2 per person. These findings indicate that the CPA assay may therefore be a rapid screening tool for detection B. duncani infection, based on its accuracy, speed, and cost-effectiveness, particularly in resource-limited regions with a high prevalence of human babesiosis.
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Bombyx mori nucleopolyhedrovirus (BmNPV) is highly contagious and poses a serious threat to sericulture production. Because there are currently no effective treatments for BmNPV, a rapid and simple detection method is urgently needed. This paper describes an electrochemical immunosensor for the detection of BmNPV. The immunosensor was fabricated by covalently immobilizing anti-BmNPV, a biorecognition element, onto the surface of the working gold electrode via 11-mercaptoundecanoic acid (MUA)/ß-mercaptoethanol (ME) hybrid self-assembled monolayers. Electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM) were used to characterize the electrochemical performance and morphology of the immunosensor, respectively. Under optimum conditions, the developed immunosensor exhibited a linear response to BmNPV polyhedrin in the range of 1 × 102-1 × 108 fg/mL, with a low detection limit of 14.54 fg/mL. The immunosensor also exhibited remarkable repeatability, reproducibility, specificity, accuracy, and regeneration. Normal silkworm blood was mixed with BmNPV polyhedrin and analyzed quantitatively using this sensor, and the recovery was 92.31 %-100.61 %. Additionally, the sensor was used to analyze silkworm blood samples at different time points after BmNPV infection, and an obvious antigen signal was detected at 12 h post infection. Although this result agreed with that provided by the conventional polymerase chain reaction (PCR) method, the electroanalysis method established in this study was simpler, shorter in detection period, and lower in material cost. Furthermore, this innovative electrochemical immunosensor, developed for the ultra-sensitive and rapid detection of BmNPV, can be used for the early detection of virus-infected silkworms.
Assuntos
Técnicas Biossensoriais , Bombyx , Nucleopoliedrovírus , Nucleopoliedrovírus/isolamento & purificação , Técnicas Biossensoriais/métodos , Animais , Bombyx/virologia , Técnicas Eletroquímicas/métodos , Imunoensaio/métodosRESUMO
Bloodstream infections (BSIs) caused by multidrug-resistant bacteria are a critical life-threatening challenge which necessitates the urgency to trigger life-saving treatment in a timely manner. This study aimed to evaluate the time required for rapid detection of carbapenemase-producing Enterobacterales (CPE) directly from blood culture bottles to optimize empirical treatment of BSI, especially in pediatric and infant patients, using a cost-effective method. This study included 419 Gram-negative bacteria, of which Klebsiella pneumoniae and Escherichia coli were the most common CPE causing BSI in pediatric and neonatal patients. Phenotypic and genotypic resistance of the selected isolates (45 K. pneumoniae and 9 E. coli) were determined by VITEK-2 Compact system and PCR, respectively. BACT/ALERT bottles were spiked with isolates. Finally, colorimetric RESIST-BC assay and Vitek-2 compact system were evaluated for the rapid detection of carbapenem-resistant bacteria directly from positive blood culture bottles. All selected isolates were phenotypically resistant to carbapenems. PCR showed that blaNDM and blaOXA-48 were present in all isolates, blaVIM was present in 44.4%, while blaKPC and blaIMP were entirely absent. The RESIST-BC kit showed good agreement with PCR for blaNDM and blaOXA-48, demonstrating high sensitivity and specificity, but not with blaVIM. These findings point out that RESIST-BC assay demonstrated an exceptionally short detection time for CPE, completing all cases within the first hour after the blood culture bottles flagged positive. It is also superior in providing a clue for clinicians on antibiotic combinations that can be administered, depending on the type of ß-lactamases detected, promptly and efficiently, with low expenses.
Assuntos
Antibacterianos , Proteínas de Bactérias , Hemocultura , beta-Lactamases , Humanos , beta-Lactamases/genética , Proteínas de Bactérias/genética , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/enzimologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/genética , Lactente , Criança , Bacteriemia/microbiologia , Bacteriemia/diagnóstico , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/sangue , Reação em Cadeia da Polimerase/métodos , Recém-NascidoRESUMO
Seafood products are globally consumed, and there is an increasing demand for the quality and safety of these products among consumers. Some seafoods are easily contaminated by marine biotoxins in natural environments or cultured farming processes. When humans ingest different toxins accumulated in seafood, they may exhibit different poisoning symptoms. According to the investigations, marine toxins produced by harmful algal blooms and various other marine organisms mainly accumulate in the body organs such as liver and digestive tract of seafood animals. Several regions around the world have reported incidents of seafood poisoning by biotoxins, posing a threat to human health. Thus, most countries have legislated to specify the permissible levels of these biotoxins in seafood. Therefore, it is necessary for seafood producers and suppliers to conduct necessary testing of toxins in seafood before and after harvesting to prohibit excessive toxins containing seafood from entering the market, which therefore can reduce the occurrence of seafood poisoning incidents. In recent years, some technologies which can quickly, conveniently, and sensitively detect biological toxins in seafood, have been developed and validated, these technologies have the potential to help seafood producers, suppliers and regulatory authorities. This article reviews the seafood toxins sources and types, mechanism of action and bioaccumulation of marine toxins, as well as legislation and rapid detection technologies for biotoxins in seafood for official and fishermen supervision.
Assuntos
Doenças Transmitidas por Alimentos , Toxinas Marinhas , Animais , Humanos , Toxinas Marinhas/toxicidade , Alimentos Marinhos/análise , Bioacumulação , Doenças Transmitidas por Alimentos/epidemiologia , Proliferação Nociva de AlgasRESUMO
We developed a facile strategy for the fabrication of red fluorescent carbon nanodots (R-CDs) and demonstrated their applications for Al3+ sensing. Red-emission carbon dots (CDs) were synthesized using a simple hydrothermal treatment with citric acid and urea as precursors, manifesting intriguing red-emission behaviour at 610 nm. With increasing Al3+ concentration, the fluorescence band at 610 nm decreased gradually. Monitoring the intrinsic fluorescence variation (I610nm ), as-prepared CDs were developed as an effective platform for fluorescent Al3+ sensing, with a linear range of 0.5-60.0 µM and a detection limit of 3.0 nM. More importantly, R-CDs have been applied successfully to the analysis of Al3+ in actual samples with satisfactory recoveries in the range 97.12-102.05%, which indicated that obtained CDs could be implemented as an effective tool for the identification and detection of Al3+ in actual samples.
Assuntos
Pontos Quânticos , Corantes Fluorescentes , Carbono , Solubilidade , Espectrometria de Fluorescência , ÁguaRESUMO
Fluorene nucleus derivatives show great potential for building outstanding fluorescence probes. In this paper, a novel fluorescent probe was developed by reacting with fluorene core with azacyclobutane, which exhibits typical solvation chromogenic effect in solvent. The fluorescence of the probe quenched in highly polar solvent. Based on this phenomenon, a novel fluorescence system for trace water was constructed. The response of this probe was fast (30 s) and sensitive for the detection of trace water in organic solvents, and the detection limit of water content in DMSO reached 0.13%. In addition, the probe can also be made as a test strip combined with homemade portable device and a smartphone for rapid detection of trace water. The luminescence mechanism of the probe is theoretically calculated based on time-contained density functional theory (TDDFT). To showcase its practicality, it has been applied for the detection of trace water in honey and alcohol by dipstick. This method provides a new idea for designing efficient fluorescent probes based on dipstick and mobile phone rapid detection.
Assuntos
Fluorenos , Corantes Fluorescentes , Espectrometria de Fluorescência , Água , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Fluorenos/química , Água/química , Estrutura Molecular , Limite de Detecção , Teoria da Densidade Funcional , Fluorescência , Poluentes Químicos da Água/análiseRESUMO
Rice blast, caused by Pyricularia oryzae, is one of the most destructive rice diseases worldwide. Using resistant rice varieties is the most cost-effective way to control rice blast. Consequently, it is critical to monitor the distribution frequency of avirulence (Avr) genes in rice planting fields to facilitate the breeding of resistant rice varieties. In this study, we established a rapid recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) detection system for the identification of AvrPik, Avr-Piz-t, and Avr-Pi9. The optimized reaction temperature and duration were 37°C and 20 min, indicating that the reaction system could be initiated by body temperature without relying on any precision instruments. Specificity analysis showed that the primer and probe combinations targeting the three Avr genes exhibited a remarkable specificity at genus-level detection. Under the optimized condition, the lower detected thresholds of AvrPik, Avr-Piz-t, and Avr-Pi9 were 10 fg/µl, 100 fg/µl, and 10 pg/µl, respectively. Notably, the detection sensitivity of the three Avr genes was much higher than that of PCR. In addition, we also successfully detected the presence of AvrPik, Avr-Piz-t, and Avr-Pi9 in the leaf and panicle blast lesions with the RPA-LFD detection system. In particular, the genomic DNA was extracted using the simpler PEG-NaOH rapid extraction method. In summary, we developed an RPA detection system for AvrPik, Avr-Pi9, and Avr-Piz-t, combined with the PEG-NaOH rapid DNA extraction method. The innovative approach achieved rapid, real-time, and accurate detection of the three Avr genes in the field, which is helpful to understand the distribution frequency of the three Avr genes in the field and provide theoretical reference for the scientific layout of resistant rice varieties.
Assuntos
Ascomicetos , Oryza , Doenças das Plantas , Doenças das Plantas/microbiologia , Oryza/microbiologia , Ascomicetos/genética , Ascomicetos/patogenicidade , Técnicas de Amplificação de Ácido Nucleico/métodos , Virulência/genética , Genes Fúngicos/genéticaRESUMO
A novel strategy based on gradient porous hollow fiber membrane (GPF) is proposed for the modular assembly of enzyme-nanozyme cascade systems. The porous structure of GPF provided sufficient specific surface area, while the gradient structure effectively minimized the leaching of enzymes and nanozymes. To enhance stability, we prepared and immobilized metal-organic framework (MOF) nanozymes, resulting in the fabrication of GPF-MOF with excellent stability and reusability for colorimetric H2O2 detection. To improve specificity and expand the detection range, micro-crosslinked natural enzymes were modularly assembled, using glucose oxidase as the model enzyme. The assembled system, GPF-mGOx@MOF, achieved a low detection limit of 0.009 mM and a linear range of 0.2 to 11 mM. The sensor retained 87.2% and 80.7% of initial activity after being stored for 49 days and 9 recycles, respectively. Additionally, the reliability of the biosensor was validated through glucose determination of human blood and urine samples, yielding comparable results to a commercial glucose meter.
Assuntos
Estruturas Metalorgânicas , Humanos , Estruturas Metalorgânicas/química , Glucose/química , Peróxido de Hidrogênio/química , Reprodutibilidade dos Testes , Glucose Oxidase/químicaRESUMO
The detection of botulinum neurotoxin A (BoNT/A) endopeptidase activity by pregnancy test paper based on human chorionic gonadotropin (hCG)-functionalized peptide-modified magnetic nanoparticles (MNs) is described for the first time. HCG-functionalized SNAP-25 peptide substrate with hydrolysis recognition sites was optimally designed. HCG can be recognized by pregnancy test strips. BoNT/A light chain (BoNT-LcA) is the central part of the endopeptidase function in holotoxin, which can specifically hydrolyze SNAP-25 peptide to release the hCG-peptide probe, and the hCG-peptide probe released can be quantitatively detected by pregnancy test strips, achieving indirect determination of BoNT/A. By quantifying the T-line color intensity of test strips, the visual detection limit for BoNT-LcA is 12.5 pg/mL, and the linear range of detection for BoNT-LcA and BoNT/A holotoxin was 100 pg/mL to 1 ng/mL and 25 to 250 ng/mL. The ability of the method to quantify BoNT/A was validated in human serum samples. This method shows the potential for sensitive detecting BoNT/A and has prospects for the diagnosis and prognosis of clinical botulism.
Assuntos
Toxinas Botulínicas Tipo A , Glicosídeos , Nanopartículas de Magnetita , Testes de Gravidez , Triterpenos , Humanos , Feminino , Gravidez , Endopeptidases , Gonadotropina CoriônicaRESUMO
Ofloxacin (OFL) is widely used in animal husbandry and aquaculture due to its low price and broad spectrum of bacterial inhibition, etc. However, it is difficult to degrade and is retained in animal-derived food products, which are hazardous to human health. In this study, a simple and efficient method was developed for the detection of OFL residues in meat products. OFL coupled with amino magnetic beads by an amination reaction was used as a stationary phase. Aptamer AWO-06, which showed high affinity and specificity for OFL, was screened using the exponential enrichment (SELEX) technique. A fluorescent biosensor was developed by using AWO-06 as a probe and graphene oxide (GO) as a quencher. The OFL detection results could be obtained within 6 min. The linear range was observed in the range of 10-300 nM of the OFL concentration, and the limit of the detection of the sensor was 0.61 nM. Furthermore, the biosensor was stored at room temperature for more than 2 months, and its performance did not change. The developed biosensor in this study is easy to operate and rapid in response, and it is suitable for on-site detection. This study provided a novel method for the detection of OFL residues in meat products.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Produtos da Carne , Animais , Humanos , Ofloxacino/química , Alérgenos , Aptâmeros de Nucleotídeos/química , Separação Imunomagnética , Técnicas Biossensoriais/métodos , Técnica de Seleção de Aptâmeros/métodosRESUMO
Conventional spherical nucleic acid enzymes (SNAzymes), made with gold nanoparticle (AuNPs) cores and DNA shells, are widely applied in bioanalysis owing to their excellent physicochemical properties. Albeit important, the crowded catalytic units (such as G-quadruplex, G4) on the limited AuNPs surface inevitably influence their catalytic activities. Herin, a hybridization chain reaction (HCR) is employed as a means to expand the quantity and spaces of G4 enzymes for their catalytic ability enhancement. Through systematic investigations, we found that when an incomplete G4 sequence was linked at the sticky ends of the hairpins with split modes (3:1 and 2:2), this would significantly decrease the HCR hybridization capability due to increased steric hindrance. In contrast, the HCR hybridization capability was remarkably enhanced after the complete G4 sequence was directly modified at the non-sticky end of the hairpins, ascribed to the steric hindrance avoided. Accordingly, the improved SNAzymes using HCR were applied for the determination of AFB1 in food samples as a proof-of-concept, which exhibited outstanding performance (detection limit, 0.08 ng/mL). Importantly, our strategy provided a new insight for the catalytic activity improvement in SNAzymes using G4 as a signaling molecule.
Assuntos
Nanopartículas Metálicas , Ácidos Nucleicos , Aflatoxina B1 , Ouro , Hibridização de Ácido NucleicoRESUMO
Phalaenopsis orchids are one of the most popular ornamental plants. More than thirty orchid viruses have been reported, and virus-infected Phalaenopsis orchids significantly lose their commercial value. Therefore, the development of improved viral disease detection methods could be useful for quality control in orchid cultivation. In this study, we first utilized the MinION, a portable sequencing device based on Oxford Nanopore Technologies (ONT) to rapidly detect plant viruses in Phalaenopsis orchids. Nanopore sequencing revealed the presence of three plant viruses in Phalaenopsis orchids: odontoglossum ringspot virus, cymbidium mosaic virus, and nerine latent virus (NeLV). Furthermore, for the first time, we detected NeLV infection in Phalaenopsis orchids using nanopore sequencing and developed the reverse transcription-recombinase polymerase amplification (RT-RPA)-CRISPR/Cas12a method for rapid, instrument-flexible, and accurate diagnosis. The developed RT-RPA-CRISPR/Cas12a technique can confirm NeLV infection in less than 20 min and exhibits no cross-reactivity with other viruses. To determine the sensitivity of RT-RPA-CRISPR/Cas12a for NeLV, we compared it with RT-PCR using serially diluted transcripts and found a detection limit of 10 zg/µL, which is approximately 1000-fold more sensitive. Taken together, the ONT platform offers an efficient strategy for monitoring plant viral pathogens, and the RT-RPA-CRISPR/Cas12a method has great potential as a useful tool for the rapid and sensitive diagnosis of NeLV.
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Amaryllidaceae , Infecção Latente , Sequenciamento por Nanoporos , Orchidaceae , Sistemas CRISPR-Cas , Reações Cruzadas , RecombinasesRESUMO
The detection of pathogens in medical wastewater is crucial due to the high content of pathogenic microorganisms that pose significant risks to public health and the environment. Medical wastewater, which includes waste from infectious disease and tuberculosis facilities, as well as comprehensive medical institutions, contains a variety of pathogens such as bacteria, viruses, fungi, and parasites. Traditional detection methods like nucleic acid detection and immunological assays, while effective, are often time-consuming, expensive, and not suitable for rapid detection in underdeveloped areas. Electrochemical biosensors offer a promising alternative with advantages including simplicity, rapid response, portability, and low cost. This paper reviews the sources of pathogens in medical wastewater, highlighting specific bacteria (e.g., E. coli, Salmonella, Staphylococcus aureus), viruses (e.g., enterovirus, respiratory viruses, hepatitis virus), parasites, and fungi. It also discusses various electrochemical biosensing techniques such as voltammetry, conductometry, impedance, photoelectrochemical, and electrochemiluminescent biosensors. These technologies facilitate the rapid, sensitive, and specific detection of pathogens, thereby supporting public health and environmental safety. Future research may should pay more attention on enhancing sensor sensitivity and specificity, developing portable and cost-effective devices, and innovating detection methods for diverse pathogens to improve public health protection and environmental monitoring.
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Técnicas Biossensoriais , Técnicas Eletroquímicas , Águas Residuárias , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Águas Residuárias/virologia , Águas Residuárias/microbiologia , Águas Residuárias/análise , Vírus/isolamento & purificação , Bactérias/isolamento & purificação , Humanos , Monitoramento Ambiental/métodos , Fungos/isolamento & purificaçãoRESUMO
The increasing number of patients with chronic wounds requires the development of quick and accurate diagnostics methods. One of the key and challenging aspects of treating ulcers is to control wound infection. Early detection of infection is essential for the application of suitable treatment methods, such as systemic antibiotics or other antimicrobial agents. Clinically, the most frequently used method for detecting microorganisms in wounds is through a swab and culture on appropriate media. This test has major limitations, such as the long bacterial growth time and the selectivity of bacterial growth. This article presents an overview of molecular methods for detecting bacteria in wounds, including real-time polymerase chain reaction (rtPCR), quantitative polymerase chain reaction (qPCR), genotyping, next-generation sequencing (NGS), and loop-mediated isothermal amplification (LAMP). We focus on the LAMP method, which has not yet been widely used to detect bacteria in wounds, but it is an interesting alternative to conventional detection methods. LAMP does not require additional complicated equipment and provides the fastest detection time for microorganisms (approx. 30 min reaction). It also allows the use of many pairs of primers in one reaction and determination of up to 15 organisms in one sample. Isothermal amplification of DNA is currently the easiest and most economical method for microbial detection in wound infection. Direct visualization of the reaction with dyes, along with omitting DNA isolation, has increased the potential use of this method.
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DNA , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Infecção dos Ferimentos , Humanos , Primers do DNA , Infecção dos Ferimentos/diagnóstico , Bactérias/genéticaRESUMO
Pesticide residues are currently a prominent concern for food safety, and the development of a rapid, convenient, and accurate method for detecting pesticide residues is crucial to ensure the quality of agricultural products. In this study, a small molecule fluorescent probe based on biphenyl disulfonic acid (BDSA) was designed and prepared, and a sensitive, specific, and rapid detection method for diquat (DQ) and paraquat (PQ) was developed. The fluorescent molecule (BDSA-NDA) was synthesized through amide reaction between BDSA and 1,8-naphthalic anhydride, which exhibited cyan fluorescence (480 nm) when excited at 305 nm in aqueous solution with a large Stokes shift (>150 nm). Diquat and paraquat were found to quench the fluorescence of the probe through internal filtration effect (IFE) and photoelectron transfer (PET). Moreover, diquat possessed a large conjugated structure that emitted fluorescence at 340 nm which was assembled into a pair of ratio fluorescence with BDSA-NDA. Under optimized experimental conditions, the developed method achieved detection limits of 0.003 mg/L for diquat and 0.202 mg/L for paraquat. Furthermore, it could identify paraquat doped in diquat formulations. Additionally, when applied to environmental water samples as well as rice and urine, this detection method demonstrated good recovery rates (water: 96.2-100.6 %, rice: 93.5-101.9 %, urine: 96-103.7 %), meeting actual sample detection requirements effectively. This work presents a novel approach for rapidly detecting diquat and paraquat residues which holds practical application value in areas such as pesticide residue analysis in foods, environmental or clinical samples.