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.
作者
黄宗文
王吉
马宏
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)
出处
《实验动物科学》
2020年第4期21-26,共6页
Laboratory Animal Science
作者简介
黄宗文(1986-),男,硕士研究生,研究方向:病毒学.E-mail:hzwcns@163.com;通信作者:马宏(1974-),女,副研究员,研究方向:免疫学.E-mail:04656@bit.edu.cn;通信作者:王吉(1974-),女,研究员,研究方向:实验动物病毒学.E-mail:wj_nd_jds@sina.com。
