摘要
为了建立一种灵敏度高、无需冷链运输、操作简便的猫传染性腹膜炎病毒(FIPV)检测方法,本试验以FIPV的N基因序列为靶基因,进行体外转录制备阳性参考品;筛选特异性引物和探针;对反应体系和条件进行优化;筛选适宜的冻干保护剂并对本试验方法的特异性、灵敏度和重复性进行评估;对冻干体系进行稳定性试验,预测室温条件下的有效期;利用本试验方法和商品试剂盒对临床采集的11份确诊FIPV感染病料进行检测,对比二者检出率;绘制接受者操作特征(ROC)曲线,计算曲线下面积,评估本试验方法的诊断效果,计算Cut off值。结果显示,本试验方法可特异性检出FIPV;最低检测限为43 copies/mL;组内和组间重复试验变异系数分别为0.12%~1.67%和0.03%~1.62%;室温有效期至少可达567 d;本试验方法的检出率为100%,市售试剂盒的检出率为91%;ROC曲线下面积为1,Cut off值为39.40。结果表明,本试验建立的FIPV实时荧光定量PCR(冻干型)检测方法具有灵敏度高,方便运输且易于贮藏的优势,可为FIPV的临床检测和流行病学调查提供技术支持。
To develop a highly sensitive,cold-chain-free,and easy-to-operate detection method for feline infectious peritonitis virus(FIPV),this study targeted the N gene sequence of FIPV to prepare in vitro transcribed positive reference samples.Specific primers and probes were screened,and the reaction system and conditions were optimized.Suitable lyophilization protectants were selected,and the specificity,sensitivity,and repeatability of the method were evaluated.A stability test was conducted on the lyophilized system to predict its shelf life under room temperature conditions.The method was compared to a commercial kit by testing 11 clinically confirmed FIPV samples,and the detection rates were compared.A receiver operating characteristic(ROC)curve was plotted,and the area under the curve(AUC)was calculated to evaluate the diagnostic performance of the method and determine the Cut off value.The results showed that the method specifically detected FIPV,with a minimum detection limit of 43 copies/mL.The intra-assay and inter-assay coefficient of variation ranged from 0.12%to 1.67%and 0.03%to 1.62%,respectively.The shelf life at room temperature was at least 567 days.The detection rate of this method was 100%,compared to 91%for the commercial kit.The AUC of the ROC curve was 1,with a Cut off value of 39.40.These results indicate that the established real-time fluorescent quantitative PCR(lyophilized type)detection method for FIPV is highly sensitive,convenient for transportation,and easy to store,providing technical support for the clinical detection and epidemiological investigation of FIPV.
作者
柯丽婷
宋丹丹
王菲
侯广争
曹鹏程
丁艳磊
刘琪琦
范云鹏
KE Liting;SONG Dandan;WANG Fei;HOU Guangzheng;CAO Pengcheng;DING Yanlei;LIU Qiqi;FAN Yunpeng(College of Veterinary Medicine,Northwest A&F University,Yangling 712100,China;College of Life Science and Technology,Shandong Normal University,Ji'nan 250399,China;Bioinformatics Center of Academy of Military Medical Sciences,Beijing 100850,China)
出处
《中国兽医杂志》
CAS
北大核心
2024年第10期61-67,共7页
Chinese Journal of Veterinary Medicine
基金
国家重点研发计划(2019YFC1200603)。
