中国比较医学杂志Issue(6)：63-68,6.DOI:10.3969/j.issn.1671.7856.2014.006.014
树鼩呼肠孤病毒 RT-nPCR 检测方法的建立及初步应用
Establishment and application of a RT-nPCR assay for detection of orthoreovirus in tree shrews
摘要
Abstract
Objective To establish a reverse transcription nested polymerase chain reaction ( RT-nPCR ) assay for detection of tree shrews orthoreovirus (TRV).Methods Three strains of TRV were respectively isolated from fresh feces of three tree shrews that came from the same field at different times .We designed and synthesized two pairs of MRV L1 gene nested primers and established the system of RT-nPCR.The TRV RNA was extracted and reversely transcribed to cDNA as a template for nested-PCR amplification.The developed RT-nPCR was optimized.The specificity and sensitivity were tested.Finally, the RT-nPCR was used to detect TRV in 25 tree shrew samples.Results Taking the genomic RNA of TRV as template, the RT-nPCR was able to amplify a specific fragment band targeting the L 1 gene, while there were no target bands in the normal cell control , ( Wa strain rotavirus , hepatitis A virus , and herpes simplex virus ) .The RNA of TRV was diluted by 1:10 to 1:109 .Each dilution sample was analyzed by the RT-nPCR.The minimum detectable concentration of RNA was 0.01 pg/μL.The results of RT-nPCR detection showed that 4 of the 15 tree shrews were TRV-positive in the survival group , and 10 of 10 tree shrews were TRV-positive in the death group . Conclusions The RT-nRCR assay established in this study is accurate , specific and sensitive .Therefore, it can be used for routine detection of TRV in quality assurance testing .关键词
树鼩/哺乳动物呼肠孤病毒/反转录巢式聚合酶链式反应/巢式引物Key words
Tree Shrew/Mammalian orthoreovirus/RT-nPCR/Nested primers分类
医药卫生引用本文复制引用
李晓飞,殷安国,张媛,罗军,孙晓梅,代解杰..树鼩呼肠孤病毒 RT-nPCR 检测方法的建立及初步应用[J].中国比较医学杂志,2014,(6):63-68,6.基金项目
国家科技支撑计划（2011BAI15B01-21；2012BAI39B01；2014BAI01B01），云南省科技创新平台建设 （）
