The establishment and application of a one-step multiplex real-time polymerase chain reaction assay for the detection of Streptococcus suis, Streptococcus suis serotype 2, and Glaesserella parasuis

Abstract

This article aims to establish a multiplex real-time polymerase chain reaction (PCR) assay for the simultaneous detection of Streptococcus suis (SS), Streptococcus suis serotype 2 (SS2), and Glaesserella parasuis (GPS). In this study, three pairs of primers and three probes were designed based on the specific sequences of SS (gdh), SS2 (cps2j), and GPS (infB). The results showed that the assay was not cross-reacted with other swine pathogens (Escherichia coli, Pasteurella multocida, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae, and Enterococcus faecalis; Streptococcus pyogenes). 10⁸ to 10² copies/μL showed the R² values for SS, SS2, and GPS were 0.999, 0.992, and 0.990, respectively. The multiplex real-time PCR efficiency was 93.816% for gdh, 105.260% for cps2j, and 93.175% for infB. The sensitivity result showed that SS, SS2, and GPS could be detected at 10 copies/μL. The repeatability result showed that intra-assay and inter-assay coefficients of variation of SS, SS2, and GPS were <2%. The best cutoff values for SS, SS2, and GPS were determined from ROC curves to be 35.085, 35.620, and 34.940, respectively. Areas under the curve were 0.943, 0.968, and 0.958. In total, 88 clinical samples were analyzed. The results indicated positive rates of 11.364% (10/88) for SS, 20.455% (18/88) for SS2, and 18.182% (16/88) for GPS. In conclusion, the developed one-step multiplex real-time PCR assay may be a valuable tool for the early detection of the SS, SS2 and, GPS with high specificity and sensitivity.