【摘要】 猪传染性胃肠炎病毒（TGEV）是一种高度传染性的病原体，会导致猪（尤其是仔猪）严重腹泻，造成重大经济损失。为建立一种用于TGEV血清抗体的高特异性、高灵敏度的间接ELISA检测方法，为监测猪传染性胃肠炎病毒在猪场的流行情况以及免疫后抗体水平评价提供技术支撑，以灭活的TGEV HN—2012株全病毒作为固相包被抗原，系统性优化反应参数，确定最佳反应条件。结果表明，建立的间接ELISA检测方法阴阳性临界值为0.439 2，最佳反应条件为：用碳酸盐缓冲液将灭活后的抗原(灭活前病毒滴度1.0×10⁷ TCID₅₀/mL)1∶8稀释，50μL/孔，37℃孵育3 h;2.5%BSA 37℃封闭2 h；血清样本稀释比例为1∶64,37℃孵育45 min；酶标抗体1∶6 000稀释，37℃孵育60 min;TMB显色10 min。该检测方法特异性好，与其他常见猪病毒阳性血清均无交叉反应；敏感性高，高免阳性血清1∶1 024倍稀释时检测结果仍为阳性；批内、批间重复性好，变异系数均小于10%；利用所建立的ELISA方法对100份疑似感染TGEV的猪血清样品进行检测，结果与血清中和试验符合率为98%。对河南省588份临床疑似感染猪传染性胃肠炎病毒的血清样品进行检测，抗体阳性率为64.97%。更多还原

【Abstract】 Porcine transmissible gastroenteritis virus(TGEV) is a highly contagious pathogen that can cause severe diarrhea in pigs, especially piglets, leading to significant economic losses. Aiming to establish a highly specific and sensitive indirect ELISA detection method for TGEV serum antibody to provide technical support for monitoring the prevalence of porcine transmissible gastroenteritis virus in pig farms and evaluating antibody levels after immunization, in this study, using inactivated whole virus of the TGEV HN-2012 strain as the solid-phase coating antigen, a highly specific indirect ELISA detection method was constructed by systematically optimizing the reaction parameters, and the optimal reaction conditions were determined. The results showed that the cut-off value for positive/negative of the established indirect ELISA detection method was 0.4 392, the optimal reaction conditions were as follows: the inactivated antigen(virus titer before inactivation: 10-7.0 TCID50/ml) diluted 1∶8 with carbonate buffer, 50 μL/well, incubated at 37 ℃ for 3 h, and blocked with 2.5% of BSA at 37 ℃ for 2 h; serum samples diluted 1∶64, incubated at 37 ℃ for 45 min; enzyme-labeled antibody diluted 1∶6,000, incubated at 37° C for 60 min; TMB color development for 10 min. This detection method exhibited good specificity, with no crossreactivity with positive sera from other common porcine viruses; high sensitivity, as hyperimmune positive serum diluted 1∶102 4 still yielded positive results; and good intra-and inter-batch reproducibility, with coefficients of variation less than 10%. Using the established ELISA method, 100 pig serum samples suspected of TGEV infection were tested, and the concordance rate between the detection results and the serum neutralization test was 98%. Detection of 588 clinical serum samples from Henan province suspected of porcine transmissible gastroenteritis virus infection showed an antibody positive rate of 64.97%.更多还原