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Glaesserella parasuis is a specific bacterial pathogen of Glässer's disease which causes significant economic losses to the swine industry. Dependable and rapid detection of G. parasuis is crucial to prevent and control Glässer's disease outbreaks. In this study, a recombinase-aided amplification (RAA) assay based on the infB gene was developed to rapid detect G. parasuis. The novel method performs isothermal detection at 42°C for real-time analysis or visualization and data analysis (RAA-VDA). The developed assay showed high specificity for G. parasuis detection without cross-reactions to other clinically important swine pathogens. The analytical sensitivity of real-time RAA was 67.17 copies per reaction with 95% reliability, which was comparable to the G. parasuis quantitative real-time PCR (qPCR). However, the detection limit of RAA-VDA was 142.43 copies per reaction with 95% reliability. The coefficient of variation analysis of the intrabatch and interbatch experimental replicate results were less than 4.30% and 6.74%, respectively, indicating the real-time RAA assay had high repeatability and reproducibility. A total of 108 clinical tissue samples were used to evaluate the clinical diagnostic performance. The diagnostic accordance rates of qPCR with real-time RAA and RAA-VDA were 100% and 98.15% (106/108), respectively. This system combined instrumental analysis and visualized analysis to accomplish a new try for rapid detection of G. parasuis in clinical practice. [ABSTRACT FROM AUTHOR]

Pseudorabies (PR) is an acute infectious disease of pigs caused by pseudorabies virus (PRV), which has caused great economic losses to the pig industry worldwide. Reliable and timely diagnose is crucial for the surveillance, control and eradication of PR. Here, a real‐time fluorescent recombinase‐aided amplification (real‐time RAA) assay was established to detect PRV. Primers and probes were designed based on the conserved regions of the PRV gE gene. The assay was specific for the detection of wild‐type PRV, showing no cross‐reactivity with other important porcine viruses (including PRV gE‐deleted vaccine strains). Analytical sensitivity of the assay was three 50% tissue culture infectious doses (TCID50) of PRV DNA per reaction with 95% reliability, which is comparable to that of a PRV‐specific real‐time PCR (qPCR) assay. In diagnosis of 206 clinical tissue samples, the diagnose accordance rate between the real‐time RAA assay and qPCR assay was 97.57% (201/206). Interestingly, the amplified products of real‐time RAA could be visualized under a portable blue light instrument, making it possible for the rapid detection of PRV in resource‐limited settings and on‐site screening. Therefore, our developed real‐time RAA assay is a diagnostic method for the rapid detection of PRV in the field. [ABSTRACT FROM AUTHOR]

Classical swine fever (CSF), which is caused by the CSF virus (CSFV), remains one of the most economically important diseases of the global swine industry. Rapid and reliable detection of CSFV is critical for controlling CSF. In this study, a novel fluorescent probe‐based real‐time reverse transcription recombinase‐aided amplification (rRT‐RAA) assay, targeting a highly conserved position within the 5′ non‐translated region (5′NTR) among all CSFV genotypes, was developed for the detection of CSFV. The assay is highly specific to CSFV and does not cross react with other important viruses. Sensitivity analysis revealed that the assay could detect two 50% tissue culture infectious dose (TCID50) of CSFV RNA per reaction at 95% probability, which is comparable to that of a documentary reverse transcription quantitative PCR (RT‐qPCR) assay for CSFV. The rRT‐RAA assay exhibited good reproducibility, with intra‐ and inter‐assay coefficient of variation values of <8.0%. Of the 135 samples (including 102 clinical tissue samples and 33 different cell culture isolates of CSFV), 50 and 52 samples were tested positive for CSFV by rRT‐RAA and RT‐qPCR, respectively. The coincidence rate between the two assays was 98.5% (133/135). Further linear regression analysis showed a significant correlation between the rRT‐RAA and RT‐qPCR assays with an R2 value of 0.8682. Interestingly, the amplification products of the rRT‐RAA assay could be directly observed with naked eyes under a portable blue light imager, making it possible for an on‐site testing. Our results indicate that the rRT‐RAA assay is a robust diagnostic tool for the rapid detection of CSFV. [ABSTRACT FROM AUTHOR]