Additional file 1 of Characterization of porcine sapelovirus prevalent in western Jiangxi, China

Additional file 1: Supplementary Fig. 1. Development of a SYBR Green I-based real-time PCR method for PSV detection. (a) Standard curves generated from the mean cycle threshold (CT) values obtained against the diluted plasmid standards (log 10 copy number). The correlation coefficient (R2) and the equation of the regression curve (Y) were calculated. Equation: y=-5.1538x+50.716; correlation coefficient: R2=0.9996. (b) Melting curve analysis of real-time PCR based on SYBR Green I. The Tm of PSV real-time PCR was 84.82 °C. (c) Specificity of the PSV SYBR Green I real-time PCR. Only the PSV-HuN1 strain showed a high-intensity fluorescent signal. CSFV, PRRSV, PRV, PEDV, PTV, and JEV did not show specific amplification. (d) Sensitivity of the SYBR Green I real-time PCR. The 10-fold serial dilutions of pMD19-T-PSV plasmids ranging from 5.22×108-5.22×101 copies/μL marked as 1-8, respectively. (e) The agarose gel electrophoresis results of conventional PCR. DNA marker of 5000 bp was used. The 10-fold serial dilutions of pMD19-T-PSV plasmids ranging from 5.22×108-5.22 copies/μL in lanes 1-9, respectively; lane 10 contains the negative control.

附加文件1：补充图1。基于SYBR Green I染料的PSV实时聚合酶链式反应（real-time PCR）检测方法的建立。（a）标准曲线：以梯度稀释的质粒标准品的平均循环阈值（CT, Cycle Threshold）值绘制，横坐标为log₁₀拷贝数。计算得到回归曲线方程与相关系数（R²），方程为y=-5.1538x+50.716，相关系数R²=0.9996。（b）熔解曲线分析：针对基于SYBR Green I染料的实时PCR开展熔解曲线分析，PSV实时PCR的解链温度（Tm, Melting Temperature）为84.82℃。（c）特异性验证：PSV的SYBR Green I实时PCR特异性检测结果显示，仅PSV-HuN1毒株呈现高强度荧光信号；猪瘟病毒（CSFV, Classical Swine Fever Virus）、猪繁殖与呼吸综合征病毒（PRRSV, Porcine Reproductive and Respiratory Syndrome Virus）、伪狂犬病病毒（PRV, Pseudorabies Virus）、猪流行性腹泻病毒（PEDV, Porcine Epidemic Diarrhea Virus）、猪捷申病毒（PTV, Porcine Teschovirus）及乙型脑炎病毒（JEV, Japanese Encephalitis Virus）均未出现特异性扩增。（d）灵敏度验证：将pMD19-T-PSV质粒进行10倍梯度稀释，浓度范围为5.22×10⁸~5.22×10¹ copies/μL，分别标记为1~8号样本。（e）常规PCR琼脂糖凝胶电泳结果：采用5000 bp DNA分子量标准。1~9号泳道为浓度从5.22×10⁸~5.22 copies/μL的10倍梯度稀释pMD19-T-PSV质粒，10号泳道为阴性对照。