Group-specific identification of polioviruses by PCR using primers containing mixed-base or deoxyinosine residues at positions of codon degeneracy
Group-specific identification of polioviruses by PCR using primers containing mixed-base or deoxyinosine residues at positions of codon degeneracy
复制标题
DOI：
10.1128/jcm.34.12.2990-2996.1996
复制
发表时间：
1996-12-01
影响因子：
9.4
通讯作者：
Kew, OM
中科院分区：
文献类型：
--
作者：
Kilpatrick, DR;Nottay, B;Kew, OM
我们开发了一种使用 PCR 区分脊髓灰质炎病毒和非脊髓灰质炎肠道病毒的方法。设计了一对泛脊髓灰质炎病毒 PCR 引物来匹配编码 VP1 内氨基酸序列的区间，这些氨基酸序列在脊髓灰质炎病毒中高度保守。起始引物与仅在脊髓灰质炎病毒中发现的 7 个氨基酸序列的密码子杂交；第二个引物与被认为与细胞受体相互作用的域的密码子相匹配。泛脊髓灰质炎病毒 PCR 引物含有混合碱基和脱氧肌苷残基，以补偿目标密码子的高度简并性。来自所有三种血清型的 48 种疫苗相关病毒分离株和 110 种野生脊髓灰质炎病毒分离株的所有 RNA 均可作为 79 bp 产物扩增的有效模板。在同等反应条件下，49 种非脊髓灰质炎肠道病毒参考毒株的基因组序列均未扩增。当扩增产物通过溴化乙锭荧光可视化时，脊髓灰质炎病毒检测的灵敏度低至 100 fg（相当于 25,000 个基因组拷贝或 25 至 250 PFU）。这些简并 PCR 引物应有助于检测所有脊髓灰质炎病毒，包括那些无法获得基因型特异性试剂的野生脊髓灰质炎病毒分离株。
We have developed a method for differentiating polioviruses from nonpolio enteroviruses using PCR. A pair of panpoliovirus PCR primers were designed to match intervals encoding amino acid sequences within VP1 that are strongly conserved among polioviruses. The initiating primer hybridizes with codons of a 7-amino-acid sequence that has been found only in polioviruses; the second primer matches codons of a domain thought to interact with the cell receptor. The panpoliovirus PCR primers contain mixed-base and deoxyinosine residues to compensate for the high degeneracy of the targeted codons. All RNAs from 48 vaccine-related and 110 wild poliovirus isolates of all three serotypes served as efficient templates for amplification of the 79-bp product. None of the genomic sequences of 49 nonpolio enterovirus reference strains were amplified under equivalent reaction conditions. Sensitivities of poliovirus detection were as low as 100 fg (equivalent to similar to 25,000 genomic copies or 25 to 250 PFU) when the amplified products were visualized by ethidium bromide fluorescence. These degenerate PCR primers should aid in the detection of all polioviruses, including those wild poliovirus isolates for which genotype-specific reagents are unavailable.