摘要
本试验根据GenBank中登录的禽白血病病毒(ALV)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314 bp,建立了适合ALV快速检测的套式PCR方法(nested-PCR)。采用该方法对ALV毒株进行了检测,试验结果表明,能扩增到314 bp的条带,禽流感病毒(H9亚型)、新城疫病毒、传染性法氏囊病病毒、减蛋综合征病毒、禽网状内皮增生病病毒、禽呼肠孤病毒、马立克氏病病毒的扩增结果均为阴性。该方法第1次扩增的敏感性是100pg,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。所建立的套式PCR方法具有敏感性高、重复性好、特异性强等优点,可用于禽白血病病毒(ALV)的临床诊断和分子流行病学调查等。
According to the sequence of RNA polymerase gene of avian leukosis virus subgroup J（ALV-J） strain published in GenBank,two pairs of primers were designed and synthesized.The outer primers amplified a fragment of 478 bp in length,and the inner primers amplification fragment size was 314 bp in length,a nested PCR assay for rapid detection of ALV was established.A specific 314 bp fragment was amplified from RNA templates of ALV strain,but no bands were amplified with templates extracted respectively from avian influenza virus（AIV） subtype H9,Newcastle disease virus（NDV）,infectious bursal disease virus（IBDV）,egg drop syndrome virus（EDSV）,reticuloendotheliosis virus（REV）,avian reovirus（ARV）,Marek＇s disease virus（MDV）.Sensitivity of the 1st and 2nd amplifications by the nested PCR assay was 100 pg and 1 fg,respectively.The sensitivity of the 2nd amplifications was increased by 10 5 times.The results showed that the nested PCR was specific,sensitive,rapid,and accurate,and could be used as a routine assay for the detection of ALV.This method had good reproducibility,specificity and sensitivity,and might detect low content ALV 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 ALV.
出处
《中国畜牧兽医》
CAS
北大核心
2012年第12期60-63,共4页
China Animal Husbandry & Veterinary Medicine
