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The soilborne pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is currently devastating banana production worldwide. Once introduced, it is not possible to eradicate the pathogen from soils where it can survive for decades. The only management option available then is to replace Foc TR4-susceptible with -resistant varieties. Timely detection of the pathogen, however, is an important strategy to prevent the introduction of Foc TR4 into new areas and prevent its spread from infested sites. In this study, a single-tube detection technique was developed by combining recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a technology (RPA-Cas12a) for detection of Foc TR4. The RPA-Cas12a assay was conducted isothermally, had a sensitivity of up to 10 fg target DNA and did not cross react with any of the 76 non-target isolates included in the specificity testing. The RPA-Cas12a assay detected Foc TR4 from naturally infected banana samples collected in the field and visualization was possible with the naked eye under LED blue light transillumination. The method can be integrated with inexpensive fluorescent or electronic detection devices to accelerate Foc TR4 in-field detection and, thereby, fast-track disease containment strategies. [ABSTRACT FROM AUTHOR]

Objective: To establish a rapid detection method for canine brucellosis using recombinase-aided amplification (RAA) technology. Methods: The outer membrane protein 25 gene fragment (Omp25) of Brucella canis was targeted. Primers and fluorescent probes were designed and synthesized, and recombinant plasmids were constructed as standards. The RAA assay was optimized by screening primers and establishing a fluorescent reaction system. Sensitivity was analyzed using plasmid standards with varying copy numbers. Specificity was tested using genomes from Brucella canis, Brucella suis, Brucella melitensis, Brucella abortus, Staphylococcus aureus, pathogenic Escherichia coli, Salmonella enteritidis, Shigella spp. , Proteus mirabilis, and Listeria monocytogenes. Reproducibility was evaluated using plasmid standards from the same and different batches. Results: The optimized RAA system used primers bOmp25-F2/bOmp25-R2 and probe bOmp25-P, with a constant reaction temperature of 39°C for 15 minutes. The detection sensitivity was 1 copy/μL. No cross-reaction was observed with other Brucella species or pathogenic bacteria, indicating high specificity. Intra-batch variability was below 1.00%, and inter-batch variability was below 2.00%. The positive detection coincidence rate of RAA was significantly higher than that of commercial real-time fluorescence quantitative PCR (100% VS 86.96%, P<0.05). Conclusion: The RAA-based rapid detection method for Brucella canis is suitable for clinical rapid testing. It offers advantages such as quick detection, high sensitivity, strong specificity, and good reproducibility. This method provides new insights for the rapid detection of canine brucellosis and the precise diagnosis of other pet diseases, making it suitable for promotion and application. [ABSTRACT FROM AUTHOR]

BACKGROUND: Accurate identification of meat species is critical to prevent economic fraud and safeguard public health. The use of inappropriate meat sources, such as murine, poses significant health risks because of potential contamination with pathogens and allergens, leading to foodborne illnesses. The present study aimed to develop a novel real‐time enzymatic recombinase amplification (ERA) method for the rapid and specific detection of murine DNA in meat products. RESULTS: A novel ERA primer and probe set was designed, targeting a murine‐specific single‐copy nuclear gene identified through bioinformatics analysis. The assay demonstrates high specificity, showing no amplification in commonly consumed meats, other animals or major crops. Additionally, it exhibits remarkable sensitivity, detecting as few as five copies of murine genomic DNA. For practical application, the ERA method could effectively identify mouse DNA in laboratory‐prepared samples at concentrations as low as 0.5% and also quantify samples with mouse DNA content as low as 5%. It also accurately detects the presence of murine‐derived ingredients in commercially available meat products. The detection process is straightforward, utilizing a simple isothermal device for incubation, blue light excitation and a smartphone camera for result interpretation. This rapid analysis can be completed within 20 min. CONCLUSION: The newly developed real‐time ERA method provides a valuable tool for standardizing meat trade practices, promoting food safety and enhancing consumer confidence in the authenticity of meat products. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Based on recombinase polymerase amplification (RPA) detection technology (combined with (lateral flow dipstick, LFD)), it is aimed to establish a dual recombinase polymerase amplification method for the rapid identification of Pasteurella multocida (Pm) and Actinobacillus pleuropneumoniae (APP). The conserved fragments of Pm kmt1 gene and APP ApxIV gene were selected for the amplification of target fragments. Eight pairs of primers for Pm and APP, and one probe for KMT1Pn and APP323Pn were designed. A single RPA-Basic primer screening test was performed. The reaction time and temperature of double RPA were optimized. The optimal primers and probe matching systems of dual RPA-LFD were explored. Dual RPA sensitivity and specificity tests were performed. The method was used to detect 60 clinical samples. The results of the primer screening test showed that the primers had the strongest specificity and the highest amplification efficiency for ApxIV2698F/ApxIV3020R and KMT1F/KMT1R. The method had the best amplification efficiency at a reaction temperature of 37ºC and a reaction time of 35 min. The optimal primer ratio of KMT1F/KMT1R and ApxIV2698F/ ApxIV3020R was 2 μL : 1.5 μL, and the optimal probe ratio of KMT1Pn and APP323Pn was 0.6 μL : 0.4 μL. The minimum detection limit of dual RPA-Basic and RPA-LFD sensitivity test was 10-6 ng/μL. The specific test results showed no cross-reaction with enteropathogenic Escherichia coli, Salmonella, Glaesserella parasuis, Staphylococcus aureus, Streptococcus suis, Aeromonas hydrophila. Using 60 clinical samples of suspected Pm and/or APP infection to evaluate the detection system, the detection rate of dual RPA-Basic and RPA-LFD is higher than that of PCR, indicating that they have strong practicability. This study successfully established a dual RPA-Basic and RPA-LFD detection method for Pm and APP, which can be used for the rapid differential diagnosis of Pm and APP mixed infection in clinical. [ABSTRACT FROM AUTHOR]

Mycoplasma hyopneumoniae (M. hyopneumoniae) is one of the primary agents involved in porcine respiratory disease complex, and circulates in the swine industry worldwide. The prevention and control of M. hyopneumoniae is complicated. Thus, a recombinase-aided amplification (RAA) assay coupled with the clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas12a system was established for the detection of M. hyopneumoniae. The most suitable primer pairs and CRISPR RNA (crRNA) were screened and selected for the RAA-CRISPR/Cas12a detection system. We have achieved a detection limit of 1 copy/µL and 5 copies/µL per reaction for the RAA-CRISPR/Cas12a-fluorescence assay and RAA-CRISPR/Cas12a-lateral flow assay (LFA), respectively. Furthermore, the RAA-CRISPR/Cas12a system displayed no cross-reactivity with other respiratory pathogens. The performance of the RAA-CRISPR/Cas12a system was compared with PCR as recommended by the Chinese national standard (GB/T 35909-2018) and qPCR as recommended by the Chinese entry–exit inspection and quarantine industry standard (SN/T4104-2015) for clinical samples, and good consistency with these methods was observed. Above all, the methods shed a light on the convenient, portable, visual, highly sensitive and specific detection of M. hyopneumoniae , demonstrating a great application potential for on-site monitoring of M. hyopneumoniae in the field. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background: Endophthalmitis is an ophthalmologic emergency requiring accurate and rapid diagnosis for treatment. Currently, the diagnosis commonly relies on culture and molecular biology, which falls short of clinical rapid diagnosis. The purpose of this study was to evaluate the feasibility of a self-build Microfluidic Real-time Polymerase Chain Reaction (RT-PCR) System for rapidly identifying potential pathogens of endophthalmitis. Methods: This study included 22 patients who presented to Shenzhen Eye Hospital and the Ophthalmology Department of the Affiliated Hospital of Guizhou Medical University in China between January 2023 and March 2024. The samples were cultured using conventional methods and underwent Microfluidic RT-PCR and metagenomic next-generation sequencing (mNGS). Results: The Microfluidic RT-PCR System identified pathogens in 11 of 22 cases (50.00%), compared with 40.91% for microbiology culture. 14 cases (63.64%) had concordant results, and 5 cases were positive for the microfluidic system only. The agreements between culture and microfluidic system, as well as culture and mNGS were 100.00% (6/6) and 50.00% (3/6), respectively. The average waiting time for the microfluidic system was about 30 min if excepting DNA extraction time, which was much shorter than 2.88 days for culture and 1.57 days for mNGS. Conclusion: The microfluidic-based RT-PCR system was preliminarily proved to be a sensitive, easy-to-operate, and rapid in-hospital technology. It is expected to become a rapid diagnostic platform for endophthalmitis. [ABSTRACT FROM AUTHOR]

Simple Summary: Feline calicivirus (FCV) is widely prevalent in domestic cats around the world. As an RNA virus, FCV is highly mutable and prevalent. The spread of the virus mainly causes upper respiratory tract infections by low pathogenic FCV, or even, leads to death by virulent systemic FCV, which poses a serious threat to felines. Currently, there is no specific drug for this virus. Efficacious FCV vaccines protect against severe disease but not against infection. What is more, due to the variability of the ORF2 of the virus, the mutation of FCV happens at high frequencies during transportation, which result in the low cross-protection of the vaccine. And the vaccine is ineffective which can only alleviate some of the clinical symptoms, especially in some virulent systemic FCV infection cases. Consequently, there is an urgent need to develop a method that allows rapid detection of the virus for control and prevention of FCV. In this study, we designed the CRISPR/Cas13a-LFD reaction system and evaluated its specificity, sensitivity, reproducibility, and clinical reliability after establishing and optimizing, which provides an efficient tool for the early diagnosis of FCV. Feline calicivirus (FCV) is one of the most common viral pathogens in domestic cats worldwide, which mainly causes upper respiratory tract infections in felines and seriously threatens the health of felines. Consequently, it is crucial to establish a rapid detection method to efficiently take control and prevent the spread of FCV. To construct the Cas13a-RAA-LFD reaction system, this study specifically designed recombinase-aided amplification (RAA) primers added with a T7 promoter and CRISPR RNA (crRNA), which were both based on the FCV relatively conserved sequence. The Cas13a protein cleaved the reporting probes only when crRNA recognized the target sequence. The results could be directly observed by lateral flow dipsticks (LFDs). To evaluate this system, factors such as RAA amplification time, Cas13a protein concentration, crRNA concentration, and CRISPR reaction time were optimized. Then, a comparison of the coincidence rate for clinical samples between this method and the polymerase chain reaction (PCR) agarose electrophoresis method was performed to evaluate the reliability of the method. Eventually, the results indicated that the target gene could be effectively amplified by the Cas13a-RAA-LFD method, and the results could be visually observed by LFD. The method could detect FCV specifically, whilst having no cross-reaction with other common viruses which infect felines, such as feline parvovirus (FPV), feline coronavirus (FCoV) and feline herpesvirus (FHV). This method is extremely sensitive and has been validated to detect viral nucleic acids down to 100 copies/μL. The good reproducibility and stability of the method were also verified by this study. Testing of clinical samples proved that the coincidence rate of clinical detection reached 96.39%. In summary, this study established a simplistic, efficient, accurate, and visualized FCV detection method, which can be utilized for early prevention and control of FCV. [ABSTRACT FROM AUTHOR]

An immunochromatographic test system based on the indirect competitive format has been developed for the rapid control of cyanotoxin microcystin-LR (MC-LR) in drinking water. To increase the assay sensitivity, a hydroquinone-based reduction of silver ions on the surface of gold nanoparticles as part of the immune complexes formed on the immunochromatographic membrane has been carried out. This stage allows for the enhancement of the colouration in test strip zones. The developed test system is characterised by an instrumental limit of detection of 0.05 ng/mL, a cut-off of 0.5 ng/mL, and a working range of the detectable concentrations of 0.08–0.42 ng/mL. The assay duration was 17 min. The applicability of the test system for MC-LR detection in drinking water is demonstrated. The assay recoveries vary from 90% to 120%. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The livestock industry uses ofloxacin, an antibiotic, to prevent several animal diseases; however, the overdose of ofloxacin used in animal farming treatments may appear in food products and cause some adverse human health effects. Hence, there is an immediate need to develop a method suitable for on site large-scale detection of ofloxacin residues in animal-derived foods. This study aimed to prepare a monoclonal antibody with high sensitivity and affinity for ofloxacin by re-synthesizing the ofloxacin hapten and synthesizing the corresponding complete antigen. The IC50 of the enzyme-linked immunosorbent assay (ic-ELISA) was 0.13 ng/mL, and the detection limit was 0.033 ng/mL. The visual detection limit of the established colloidal gold immunochromatographic test strip, for the visual detection of actual samples, was 1 ng/g. In summary, this work establishes a rapid detection method of ofloxacin residues on the basis of colloidal gold immunochromatography that is suitable for actual detection. [ABSTRACT FROM AUTHOR]

Two immunochromatographic assays based on hematite (α-Fe2O3) nanoparticles were developed for rapid detection of aflatoxin M1 (AFM1) and Escherichia coli O157:H7 in the milk. Hematite nanoparticles were prepared and utilized as colored signal labels. The competitive format was applied for AFM1 detection, while the sandwich format for E. coli O157:H7 determination. Under optimal conditions, the assay exhibited a visual detection limit of 0.10 ng/mL and quantitative determination limit of 0.017 ng/mL for AFM1. For E. coli O157:H7, a visual detection limit of 2.9 × 105 CFU/mL was achieved. The entire determination process could be accomplished within 15 min. Compared with traditional nanolabels, hematite nanoparticles showed advantages of facile preparation, high stability and easy bioconjugation. The developed assays provided effective and rapid screening strategies for AFM1 and E. coli O157:H7 in milk samples with high sensitivity and specificity. [ABSTRACT FROM AUTHOR]