摘要
为建立一种可定量检测鲤浮肿病毒(carp edema virus,CEV)的方法,以CEV P4a基因作为靶基因设计并合成引物和探针,经优化反应体系和退火温度,建立了与实时荧光PCR有良好线性关系的微滴式数字PCR(droplet digital PCR,ddPCR)方法,并进行了灵敏度、特异性、重复性验证。最后,将建立的方法应用于临床样品检测。结果显示:当引物、探针浓度分别为1.10、0.30μmol/L,退火温度为53℃时,扩增效率最高,且阴阳性微滴分布界限最为明显,检测限低至0.38 copies/μL;CEV核酸拷贝数浓度为0~7.48×10^(4)copies/μL时,ddPCR与实时荧光PCR检测的线性关系较好(R^(2)=0.9929),变异系数较低(2.61%);与其他10种常见水生动物疫病病原的阳性核酸均无交叉反应;在155份临床样品检测中,检出阳性样品2份,与实时荧光PCR的检出率相当;在8份能力验证样品检测中,检出阳性样品3份,与能力验证“满意”结果一致。结果表明,本研究建立的CEV ddPCR方法灵敏度高、特异性强、重复性好,为CEV定量检测和低病毒载量样品检测提供了技术支持。
In order to quantificationally detect carp edema virus(CEV),a droplet digital PCR(ddPCR)with a good linear relationship to real-time fluorescence PCR was established through optimizing reaction system and annealing temperature of primers and probes that were designed targeting CEV P4a gene,followed by validation for its sensitivity,specificity and repeatability,and subsequently it was applied to clinical sample testing.The results showed that the highest effi ciency of amplifi cation was observed,with the most distinct separation between positive and negative droplets,and the lowest detection limit of 0.38 copies/μL,when the concentrations of the primers and probe were 1.10 and 0.30μmol/L,respectively,and the annealing temperature reached 53℃;good linear relationship(R^(2)=1110.9929)was presented between ddPCR and real-time fluorescence PCR,with low coefficient of variation(2.61%),when the concentration of CEV nucleic acids ranged from 0 to 7.48×10^(4)copies/μL;no cross-reactivity was found with positive nucleic acids of 10 common aquatic animal pathogens;2 of 155 clinical positive samples were detected,with a detection rate comparable to that of real-time fluorescence PCR;3 of 8 samples for proficiency testing were positive,which was consistent with the“satisfactory”result of the proficiency test.In conclusion,the established CEV ddPCR assay was highly sensitive,specific and reproducible,technically supporting future quantitative detection of CEV and testing of samples with low viral loads.
作者
林仰孝
叶毅飞
卢伟昌
刘雪怡
钟鸣
殷进方
龙海鹰
钟群芳
谢柱彬
李敏
张险朋
Lin Yangxiao;Ye Yifei;Lu Weichang;Liu Xueyi;Zhong Ming;Yin Jinfang;Long Haiying;Zhong Qunfang;Xie Zhubin;Li Min;Zhang Xianpeng(Hongmei Township Center for Agricultural Technology Service,Dongguan 523160,Guangdong,China;Dongguan Key Laboratory of Zoonosis,Dongguan Center for Animal Disease Prevention and Control,Dongguan 523086,Guangdong,China;Dongkeng Township Center for Agricultural Technology Service,Dongguan 523451,Guangdong,China;Dongguan Agricultural Science Research Center,Dongguan 523086,Guangdong,China)
出处
《中国动物检疫》
2025年第12期110-119,共10页
China Animal Health Inspection
基金
东莞市2021年度省乡村振兴战略专项(20211800400112)。
