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针对猪传染性胃肠炎病毒(TGEV)S基因的保守序列设计引物,以TEGV疫苗毒株为模板,克隆S基因,并构建重组阳性质粒,优化反应体系和扩增条件,建立基于SYBR GreenⅠ染料的实时荧光定量PCR检测方法,并对其特异性、重复性和灵敏度进行分析。结果显示:建立的检测方法灵敏度可达10拷贝/μL,标准曲线线性关系良好(r=0.997),扩增效率高,具有良好的特异性和重复性。使用该方法对124份临床疑似TGEV病料进行检测,阳性样本有17份,检测样本的阳性率为13.7%。该方法可用于兽医临床上TGEV的快速检测和流行病学分析。
Aiming at the conservative sequence of S gene of swine transmissible gastroenteritis virus (TGEV), primers were designed. The S gene was cloned by using TEGV vaccine strain as template, and a recombinant positive plasmid was constructed. The reaction system and amplification conditions were optimized and SYBR Green Ⅰ dye Real-time fluorescence quantitative PCR detection method, and its specificity, repeatability and sensitivity analysis. The results showed that the sensitivity of the established detection method was 10 copies / μL, the calibration curve had good linearity (r = 0.997), high amplification efficiency, good specificity and repeatability. Using this method, 124 samples of clinical suspected TGEV were detected, 17 samples were positive and the positive rate was 13.7%. The method can be used for rapid detection and epidemiological analysis of veterinary clinical TGEV.