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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]

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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]