Species-specific RT-PCR amplification of human enteroviruses:: a tool for rapid species identification of uncharacterized enteroviruses
Species-specific RT-PCR amplification of human enteroviruses:: a tool for rapid species identification of uncharacterized enteroviruses
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DOI：
10.1099/vir.0.81179-0
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发表时间：
2006-01-01
影响因子：
3.8
通讯作者：
Pallansch, MA
中科院分区：
文献类型：
--
作者：
Oberste, MS;Maher, K;Pallansch, MA
人类肠道病毒的 65 种血清型主要根据多个基因组区域的系统发育关系分为四个物种：人类肠道病毒 (HEV) A 至 D。肠病毒的 3'-非平移区域在一个物种内高度保守，但在物种之间存在很大差异。根据这些信息，开发了物种特异性 RT-PCR 引物，可用于快速筛选肠道病毒分离株的集合，以识别感兴趣的物种。当针对肠道病毒原型株和已知血清型分离株（总共 193 个分离株）进行测试时，这四个引物对具有 100% 特异性。为了在典型应用中进行评估，使用物种特异性引物筛选 186 种以前未表征的非脊髓灰质炎肠道病毒分离株。 HEV-B引物扩增了68.3%的分离株，而HEV-A和HEV-C引物分别占分离株的9.7%和11.3%；使用 HEV-D 引物未扩增任何分离株。十二个分离株 (6.5%) 通过多于一个引物组扩增，八个分离株 (4.3%) 没有通过四个引物对中的任何一个扩增。通过对 VP1 衣壳基因进行部分测序来鉴定血清型，并且在每种情况下测序均证实物种特异性 PCR 结果是正确的；由超过一种物种特异性引物对扩增的分离株是两种（11个分离株）或三种（1个分离株）病毒物种的混合物。未通过物种特异性引物扩增的八个分离株包含四种新血清型（EV76、EV89、EV90 和 EV91），根据 VP1、3D 和 3' 非翻译区序列，它们似乎是 HEV-A 的独特成员。
The 65 serotypes of human enteroviruses are classified into four species, Human enterovirus (HEV) A to D, based largely on phylogenetic relationships in multiple genome regions. The 3'-non-transiated region of enterovinuses is highly conserved within a species but highly divergent between species. From this information, species-specific RT-PCR primers were developed that can be used to rapidly screen collections of enterovirus isolates to identify species of interest. The four primer pairs were 100 % specific when tested against enterovirus prototype strains and panels of isolates of known serotype (a total of 193 isolates). For evaluation in a typical application, the species-specific primers were used to screen 186 previously uncharacterized non-polio enterovirus isolates. The HEV-B primers amplified 68.3 % of isolates, while the HEV-A and HEV-C primers accounted for 9.7 and 11.3 % of isolates, respectively; no isolates were amplified with the HEV-D primers. Twelve isolates (6.5%) were amplified by more than one primer set and eight isolates (4.3%) were not amplified by any of the four primer pairs. Serotypes were identified by partial sequencing of the VP1 capsid gene, and in every case sequencing confirmed that the species-specific PCR result was correct; the isolates that were amplified by more than one species-specific primer pair were mixtures of two (11 isolates) or three (one isolate) species of viruses. The eight isolates that were not amplified by the species-specific primers comprised four new serotypes (EV76, EV89, EV90 and EV91) that appear to be unique members of HEV-A based on VP1, 3D and 3'-non-translated region sequences.