Development of a Droplet Digital PCR for the Detection of Haemophilus parasuis
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Abstract:
Specific primers and one TaqMan probes were designed according to the conserved sequence of the OMP P2 gene of Haemophilus parasuis (HPS) in this study. An accurate quantitative droplet digital PCR (ddPCR) method was developed through the optimization of reaction conditions, specificity, sensitivity and reproducibility tests, analysis of clinical samples, and comparison with the real-time quantitative PCR (qPCR). Under the optimized reaction conditions, the results showed that ddPCR exhibited no cross-reactions with Actinobacillus pleuropneumoniae, Streptococcus suis, Bacillus subtilis, Escherichia coli, Salmonella, Staphylococcus aureu, Porcine circovirus 2, and swine fever virus. The developed method had a higher sensitivity than qPCR with a correlation coefficient (R2) of 0.996, a good linearity, detection limit of positive plasmid as 2.647 copies/μL, and coefficient of variation as 3.29%. The quantitative analysis results of clinical samples showed that ddPCR possessed advantages of high sensitivity and good specificity with a detection rate slightly higher than that for qPCR. The ddPCR developed in this study can accurately and quantitatively detect HPS, which will provide a useful reference for HPS related research.
