摘要
利用牛轮状病毒(BRV)阳性血清(中和效价1∶256)为一抗,FITC标记的山羊抗牛Ig G为二抗,建立了一种检测牛轮状病毒的间接免疫荧光试验方法,并对其工作浓度和孵育时间进行优化,获得了最佳条件。采用该方法对48份临床疑似牛轮状病毒样本进行检测,并与RT-PCR、病毒分离这2种病原学检测结果进行比较。结果显示,一抗1∶300稀释、孵育120 min,二抗1∶500稀释、孵育120 min为最佳工作条件;对BCoV、IBRV、BVDV、BRSV、BPIV3无交叉反应;病毒滴度为101.25TCID50/0.1 mL时仍有特异性绿色荧光,且不同时刻在相同条件试验时,荧光强度不受影响;临床样本检测结果显示,IFA方法相对于病毒分离方法具有更高的敏感性,与RT-PCR检测结果的符合率为100%。表明,建立的间接免疫荧光方法具有较好的特异性、敏感性、重复性和准确性,可用于临床牛轮状病毒感染的检测。
Using the positive serum of bovine rotavirus(with a neutralization titer of 1:256)as the primary antibody and the FITC-labeled goat anti-bovine IgG as the secondary antibody,an indirect immunofluorescence assay method for detecting bovine rotavirus was established.The working concentrations and incubation times were optimized,and the optimal conditions were obtained.This method was used to detect bovine rotavirus in 48 clinical suspected samples,and the results were compared with those of two etiological detection methods,RT-PCR and virus isolation.The results showed that the optimal working conditions were a 1:300 dilution of the primary antibody and an incubation of 120 minutes,a 1:500 dilution of the secondary antibody and an incubation of 120 minutes;there was no cross-reaction with BCoV,IBRV,BVDV,BRSV,and BPIV3;when the virus titer was 101.25 TCIDso/0.1 mL,there was still specific green fluorescence,and the fluorescence intensity was not affected by different times under the same conditions,the clinical sample detection results indicated that the IFA method had higher sensitivity than the virus isolation method,and the coincidence rate with the RT-PCR detection results was 100%.The indirect immunofluorescence method established in this study has good specificity,sensitivity,repeatability,and accuracy,and can be used for the detection of clinical bovine rotavirus infection.
作者
于文博
迟田英
郑辉
李博文
吴发兴
崔进
代飞燕
YU Wenbo;CHI Tianying;ZHENG Hui;LI Bowen;WU Faxing;CUI Jin;DAI Feiyan(College of Veterinary Medicine,Yunnan Agriculture University,Kunming 650201,China;China A nimal Health and Epidemiology Center,Qingdao 266032,China)
出处
《中国兽医科学》
北大核心
2025年第7期891-897,共7页
Chinese Veterinary Science
基金
云南省高原畜禽消化道营养与健康创新团队项目(202305AS350024)
云南省个旧市牛产业科技特派团项目(202304BI090007)
石林县农民院士科技服务站项目(A3012024017)。
关键词
牛
轮状病毒
间接免疫荧光法
bovine
rotavirus
indirect immunofluorescence
