仙台病毒荧光定量PCR检测方法的建立及初步应用  

Establishment of a Fluorescent Quantitative PCR Detection Method for Sendai Virus and its Preliminary Application

作　　者：黄宗文 王吉[2] 马宏[1] HUANG Zongwen;WANG Ji;MA Hong(School of Life Science,Beijing Institute of Technology,Beijing 100081,China;National Institutes for food and drug Control,Beijing 102629,China)

机构地区：[1]北京理工大学,北京100081 [2]中国食品药品检定研究院,北京102629

出　　处：《实验动物科学》2020年第4期21-26,共6页Laboratory Animal Science

摘　　要：目的建立敏感特异的仙台病毒(SeV)荧光定量PCR检测方法,并初步应用。方法将不同株SeV病毒序列进行比对,选择对保守区域设计合成引物。人工合成SeV基因组12181~12480 DNA序列,转入质粒中,作为仙台病毒质粒标准品,建立SYBR染料法荧光定量PCR方法,并对样本进行SeV测定。结果建立了特异性的检测SeV的SYBR荧光定量PCR方法,该方法对SeV最低检测限度为10 copies/μL。将所建立的实时荧光定量PCR方法用于40只SPF小鼠和58只裸鼹鼠的肺组织样本的检测,检测结果为仙台病毒核酸阴性;检测4只成年屏障环境饲养黄鼠肺组织和5只清洁级小鼠肺组织,检测结果为仙台病毒核酸阳性率100%。结论该研究建立的SYBR Green染料法荧光定量PCR方法能特异敏感地检测仙台病毒。Objective To establish a fluorescent quantitative PCR(Q-PCR)method for the Sendai virus(SeV).Method A fluorescent quantitative PCR(Q-PCR)method for SeV was developed based on a pair of primers that in accordance with the published sequence of SeV L gene.The specificity,sensitivity,repeatability and stability of the method were verified.The method was used to detect 40 SPF mice,58 naked mole rats,4 Spermophilus dauricus and 5 clean mice.Result The assay could specifically detect SeV and had good sensitivity,the minimum detectable amount of Sendai virus was 10 copies/μL.The 40 SPF mice and 58 naked mole rats were negative by this Q-PCR assay,4 Spermophilus dauricus and 5 clean mice were all positive.Conclusion The developed Q-PCR method is good in linearity,specificity,sensitivity,and can be used for the rapid quantitative detection of SeV samples.It provides technical reference for the monitoring of SeV and improvement of related standards.

关 键 词：仙台病毒 荧光定量PCR 初步应用 

分 类 号：S852.65[农业科学—基础兽医学]