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

Lingxiang Xin, Haojie Wang, Yunhao Hu, Yan Liu, Wensheng Yao, Xiuli Wang, Jian Li, Yuanjie Liu, Rendong Tong, Qi Wang, Youlong Lu, Liangquan Zhu
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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.

Abstract Image

用于检测猪链球菌、猪链球菌血清 2 型和寄生璃色菌的一步法多重实时聚合酶链反应测定的建立与应用
本文旨在建立一种多重实时聚合酶链式反应(PCR)检测方法,用于同时检测猪链球菌(SS)、猪链球菌血清型 2(SS2)和寄生璃色菌(GPS)。本研究根据 SS(gdh)、SS2(cps2j)和 GPS(infB)的特定序列设计了三对引物和三个探针。结果表明,该检测方法与其他猪病原体(大肠杆菌、多杀性巴氏杆菌、金黄色葡萄球菌、无乳链球菌、肺炎链球菌、胸膜肺炎放线杆菌、肺炎支原体、粪肠球菌;化脓性链球菌)无交叉反应。结果显示,SS、SS2 和 GPS 的 R2 值分别为 0.999、0.992 和 0.990。gdh 的多重实时 PCR 检测效率为 93.816%,cps2j 为 105.260%,infB 为 93.175%。灵敏度结果表明,在 10 个拷贝/μL 的条件下即可检测到 SS、SS2 和 GPS。重复性结果表明,SS、SS2 和 GPS 的测定内变异系数和测定间变异系数均为 2%。根据 ROC 曲线确定的 SS、SS2 和 GPS 的最佳临界值分别为 35.085、35.620 和 34.940。曲线下面积分别为 0.943、0.968 和 0.958。总共分析了 88 份临床样本。结果显示,SS 的阳性率为 11.364%(10/88),SS2 为 20.455%(18/88),GPS 为 18.182%(16/88)。总之,所开发的一步法多重实时 PCR 分析法可能是早期检测 SS、SS2 和 GPS 的一种有价值的工具,具有很高的特异性和灵敏度。
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