Molecular Properties of Poliovirus Isolates: Nucleotide Sequence Analysis, Typing by PCR and Real-Time RT-PCR
Molecular Properties of Poliovirus Isolates: Nucleotide Sequence Analysis, Typing by PCR and Real-Time RT-PCR
复制标题
DOI：
10.1007/978-1-4939-3292-4_9
复制
发表时间：
2016-01-01
期刊：
影响因子：
--
通讯作者：
Kew, Olen M.
中科院分区：
文献类型：
--
作者：
Burns, Cara C.;Kilpatrick, David R.;Kew, Olen M.
病毒学监测对于世界卫生组织根除脊髓灰质炎倡议的成功至关重要。分子方法已用于检测组织培养分离物中的脊髓灰质炎病毒，这些分离物来源于通过监测急性弛缓性麻痹获得的粪便样本。本章介绍了如何使用实时 PCR 检测来识别脊髓灰质炎病毒并进行血清分型。特别是，使用简并的、含肌苷的泛脊髓灰质炎病毒 (panPV) PCR 引物组来区分脊髓灰质炎病毒和 NPEV。脊髓灰质炎病毒之间高度的核苷酸序列多样性对基于核酸的试剂的系统设计提出了挑战。为了适应脊髓灰质炎病毒基因组的广泛变异性和快速进化，模板上的简并密码子位置与引物和 TaqMan (TM) 探针上的混合碱基或脱氧肌苷残基相匹配。其他分析可区分 Sabin 疫苗株和非 Sabin 株。本章还介绍了使用通用脊髓灰质炎病毒特异性引物以及简并引物和含肌苷引物对脊髓灰质炎病毒分离株进行常规 VP1 测序。这些引物与非简并血清型特异性 Sabin 引物一起也可用于对混合物中的单个脊髓灰质炎病毒进行测序。
Virologic surveillance is essential to the success of the World Health Organization initiative to eradicate poliomyelitis. Molecular methods have been used to detect polioviruses in tissue culture isolates derived from stool samples obtained through surveillance for acute flaccid paralysis. This chapter describes the use of realtime PCR assays to identify and serotype polioviruses. In particular, a degenerate, inosine-containing, panpoliovirus (panPV) PCR primer set is used to distinguish polioviruses from NPEVs. The high degree of nucleotide sequence diversity among polioviruses presents a challenge to the systematic design of nucleic acid-based reagents. To accommodate the wide variability and rapid evolution of poliovirus genomes, degenerate codon positions on the template were matched to mixed-base or deoxyinosine residues on both the primers and the TaqMan (TM) probes. Additional assays distinguish between Sabin vaccine strains and non-Sabin strains. This chapter also describes the use of generic poliovirus specifi c primers, along with degenerate and inosine-containing primers, for routine VP1 sequencing of poliovirus isolates. These primers, along with nondegenerate serotype-specifi c Sabin primers, can also be used to sequence individual polioviruses in mixtures.