摘要
以脊髓灰质炎(简称脊灰)病毒Sabin株Ⅰ、Ⅱ、Ⅲ型2500～2600碱基区域(病毒VP1氨基末端)为目的基因片段,人工合成了寡核苷酸引物和探针。以Digoxigenin-11-dUTP代替^(32)P-dATP,建立了Digoxige-11-dUTP末端标记寡核苷酸探针,以及在PCR过程中Digoxigenin-11-dUTP直接掺入扩增产物制备探针的方法,并将探针应用于分离的脊灰野毒株和疫苗相关株的鉴别诊断。Digoxigenin末端标记探针和掺入PCR探针均具有型特异性,用于鉴别我国部分地区分离的脊灰毒株的结果表明,Ⅰ型分离株中90％为野毒株,Ⅱ、Ⅲ型分离株均为疫苗相关株,说明我国近年引起脊灰暴发的仍以Ⅰ型野毒株为主。探针可检测至少10~2～10~3TCID_(50)/ml样品。与同位素标记探针敏感性一致。在PCR过程中直接掺入Digoxigenin标记探针,不需纯化,大大简化了操作步骤,提高了标记效率。Digoxigenin标记探针敏感、特异,对人体无害,不受同位素半衰期影响,可回收反复使用;操作简单,出结果快,易于判断,适合在我国推广使用。
In this report we describe a non-radioactive hybridization system of Digoxigenin-terminal labeling oligonucleotide probes and Digoxigenin-freedom labeling probes during PCR amplification. The oligonucleotide probes and primes are designed for complementarity to 2500-2600 nucleotide sequence of poliovirus specific Sabin strains,that differ among wild stra ins but are largely conserved among vaccine related isolates.The hybridization using Digoxigenin-labeling probes is as sensitives but much easier and safer than using isotopic labeling probes. It has been used in the laboratory surveillance of poliomyelitis cases in part area of China. Among the isolated polioviruses 90% of type I strains are wild ge-notype all of type Ⅱ and type Ⅲ are vaccine-related strains.
出处
《病毒学报》
CAS
CSCD
北大核心
1993年第2期179-184,共6页
Chinese Journal of Virology
基金
国家自然科学基金
