| Summary: | <i>Haemophilus parasuis</i> (<i>H. parasuis,</i> HPS) is a prominent pathogenic bacterium in pig production. Its infection leads to widespread fibrinous inflammation in various pig tissues and organs, often in conjunction with various respiratory virus infections, and leads to substantial economic losses in the pig industry. Therefore, the rapid diagnosis of this pathogen is of utmost importance. In this study, we used recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR) technology to establish a convenient detection and analysis system for <i>H. parasuis</i> that is fast to detect, easy to implement, and accurate to analyze, known as RPA-CRISPR/Cas12a analysis. The process from sample to results can be completed within 1 h with high sensitivity (0.163 pg/μL of DNA template, <i>p</i> < 0.05), which is 10<sup>4</sup> -fold higher than the common PCR method. The specificity test results show that the RPA-CRISPR/Cas12a analysis of <i>H. parasuis</i> did not react with other common pig pathogens, including <i>Streptococcus suis</i> type II and IX, <i>Actinobacillus pleuropneumoniae</i>, <i>Escherichia coli</i>, <i>Salmonella</i>, <i>Streptococcus suis</i>, and <i>Staphylococcus aureus</i> (<i>p</i> < 0.0001). The RPA-CRISPR/Cas12a assay was applied to 15 serotypes of <i>H. parasuis</i> clinical samples through crude extraction of nucleic acid by boiling method, and all of the samples were successfully identified. It greatly reduces the time and cost of nucleic acid extraction. Moreover, the method allows results to be visualized with blue light. The accurate and convenient detection method could be incorporated into a portable format as point-of-care (POC) diagnostics detection for <i>H. parasuis</i> at the field level.
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