摘要
根据GenBank中登录的禽呼肠病毒(ARV)基因组序列,设计合成了2对引物,外部引物的扩增片段大小为478 bp,内部引物的扩增片段大小为247 bp,建立了适合ARV快速检测的套式RT-PCR方法(RT-nested-PCR)。采用该方法对REV毒株进行了检测,均能扩增到247 bp的条带,而禽流感病毒(H9亚型)、新城疫病毒、传染性腔上囊病病毒、减蛋综合征病毒、禽白血病病毒、禽网状内皮组织增生病病毒、马立克病病毒和鸭瘟病毒的扩增结果均为阴性。该方法第1次扩增的敏感性是100 pg,第2次扩增的敏感性是1 fg,第2次比第1次扩增的敏感性高105倍。该检测方法表明,所建立的逆转录套式RT-PCR方法比普通RT-PCR方法敏感性高,具有重复性好、特异性强、敏感性高等优点,可用于禽呼肠病毒的临床诊断、病料检测和分子流行病学调查等。
According to the sequence of RNA polymerase gene of avian reovirus（ARV） strain published in GenBank,two pairs of primers were designed and synthesized. The outer primers amplified a fragment of 478bp in length, and the inner primers amplification fragment size was 247 bp in length, A nested RT-PCR assay for rapid detection of ARV was established. A specific 247 bp fragment was amplified from RNA templates of ARV strain,but no bands were amplified with templates extracted respectively from avian influenza virus（AIV）subtype Hg, Newcastle disease virus（NDV）, infectious bursal disease virus（IBDV）,egg drop syndrome virus（EDSV）,subgroup J ALV （ALV-J）, Reticuloendotheliosis（REV）, Marek＇S disease virus（MDV）, cluck plague virus（DPV） . Sensitivity of the 1st and 2nd amplifications by the nested RTPCR assay was 100 pg and 1 fg, respectively. The sensitivity of the 2 nd amplifications was increased by 10s times. The results showed that the nested RT-PCR was specific, sensitive,rapid,and accurate,and could be used as a routine assay for the detection of ARV. This method had good reproducibility, specificity and sensitivity,and might detect low content ARV accurately and rapidly. This method could be used as a method for the diagnosis and detection of clinical cases, and for molecular epidemiological investigation of ARV.
出处
《动物医学进展》
CSCD
北大核心
2011年第8期21-24,共4页
Progress In Veterinary Medicine
