GENOTYPE-SPECIFIC INVITRO AMPLIFICATION OF SEQUENCES OF THE WILD TYPE-3 POLIOVIRUSES FROM MEXICO AND GUATEMALA
GENOTYPE-SPECIFIC INVITRO AMPLIFICATION OF SEQUENCES OF THE WILD TYPE-3 POLIOVIRUSES FROM MEXICO AND GUATEMALA
复制标题
DOI：
10.1016/0168-1702(92)90124-r
复制
发表时间：
1992-08-01
期刊：
影响因子：
5
通讯作者：
KEW, OM
中科院分区：
文献类型：
--
作者：
YANG, CF;DE, LN;KEW, OM
野生脊髓灰质炎病毒独立基因型之间广泛的核苷酸序列异质性允许系统地设计基因型特异性分子试剂。我们为墨西哥和危地马拉最近流行的野生脊髓灰质炎病毒3型基因型准备了两组特异性聚合酶链反应（PCR）引物。1989年在墨西哥分离的具有代表性的野生3型病毒的核苷酸序列与相应的Sabin 3 （Leon 12 a1b）序列在VP1区900个位点中的167个位点存在差异。根据序列数据，设计野生病毒特异性引物对，以与Sabin 3模板互补高错配区（> 33%）。引物结合位点沿基因组间隔，因此预测的扩增产物（142 bp和163 bp）可以很容易地从我们的Sabin菌株特异性引物（Sabin 1:97 bp; Sabin 2:1 71 bp; Sabin 3:53 bp）产生的产物中电泳分离出来。在13年期间（1977-1990年）从墨西哥和危地马拉获得的所有野生3型脊髓灰质炎病毒分离物的rna可作为142 bp和163 bp产物扩增的有效模板。来自疫苗相关脊髓灰质炎病毒和大多数异源野生脊髓灰质炎病毒的基因组模板在同等反应条件下无活性。扩增产生一个具有广泛反应的引物对的114 bp产物，与5'-非编码区高度保守的序列相匹配，为脊髓灰质炎病毒（或肠病毒）rna样本中的存在提供了阳性对照。通过在含有大量疫苗相关rna（高达10(6)倍）的化学过量反应中设置引物，获得了墨西哥-危地马拉野生3型脊髓灰质炎病毒序列的选择性扩增。我们使用了野生基因型特异性PCR引物集，以促进临床和环境样本中存在的野生脊髓灰质炎病毒的鉴定。
The extensive nucleotide sequence heterogeneity among independent genotypes of wild polioviruses permits the systematic design of genotype-specific molecular reagents. We have prepared two sets of polymerase chain reaction (PCR) primer pairs specific for the genotype of wild poliovirus type 3 recently endemic to Mexico and Guatemala. Nucleotide sequences of a representative wild type 3 virus isolated in Mexico in 1989 differed from the corresponding Sabin 3 (Leon 12 a1b) sequences at 167 of 900 positions within the VP1 region. From the sequence data, wild virus-specific primer pairs were designed to complement regions of high mismatch (> 33%) with Sabin 3 templates. Primer binding sites were spaced along the genome so that the predicted amplification products (142 bp and 163 bp) could be easily resolved electrophoretically from the products generated with our Sabin strain-specific primers (Sabin 1: 97 bp; Sabin 2: 71 bp; Sabin 3: 53 bp). RNAs of all wild type 3 poliovirus isolates from Mexico and Guatemala obtained over a 13-year period (1977-1990) served as efficient templates for amplification of the 142-bp and 163-bp products. Genomic templates derived from vaccine-related polioviruses and most heterologous wild polioviruses were inactive under equivalent reaction conditions. Amplifications generating a 114-bp product with a broadly reacting primer pair, matching highly conserved sequences in the 5'-noncoding region, provided a positive control for the presence in samples of poliovirus (or enterovirus) RNAs. Selective amplification of wild Mexico-Guatemala type 3 poliovirus Sequences was obtained with either primer set in reactions containing large stoichiometric excesses (up to 10(6)-fold) of vaccine-related RNAs. We have used wild genotype-specific PCR primer sets to facilitate identification of wild polioviruses present in both clinical and environmental samples.