Development of an Efficient Entire-Capsid-Coding-Region Amplification Method for Direct Detection of Poliovirus from Stool Extracts
Development of an Efficient Entire-Capsid-Coding-Region Amplification Method for Direct Detection of Poliovirus from Stool Extracts
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DOI：
10.1128/jcm.02384-14
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发表时间：
2015-01-01
影响因子：
9.4
通讯作者：
Shimizu, Hiroyuki
中科院分区：
文献类型：
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作者：
Arita, Minetaro;Kilpatrick, David R.;Shimizu, Hiroyuki
自1988年以来，实验室诊断在全球根除脊髓灰质炎行动中发挥了关键作用，通过使用细胞培养作为检测脊髓灰质炎病毒的高度敏感系统，从粪便标本中分离和鉴定脊髓灰质炎病毒（PV）。在本研究中，我们的目的是发展一种直接从粪便提取物中检测PV的分子方法，该方法具有与细胞培养相媲美的高效率。我们开发了一种方法来有效地扩增整个衣壳编码区的人类肠道病毒（EV），包括PV。从少至50个拷贝的PV基因组获得整个衣壳编码区（3.9kb）的cDNA。用改进的PV特异性实时荧光定量PCR系统和VP 1编码区的核苷酸序列分析从cDNA中检测PV。为了进行检测验证，我们分析了84份细胞培养物中PV阳性的粪便提取物，并使用该方法结合PV特异性提取方法从100%的提取物（84/84份样本）中检测到PV基因组。在2/4的粪便提取物样品中可以检测到PV，这些粪便提取物样品在细胞培养中为PV阴性。在PV阳性样本中，也检出了高频率的EV C种病毒（27% [23/86份样本]）。这种方法将是有用的直接检测PV粪便提取物，而不使用细胞培养。
Laboratory diagnosis has played a critical role in the Global Polio Eradication Initiative since 1988, by isolating and identifying poliovirus (PV) from stool specimens by using cell culture as a highly sensitive system to detect PV. In the present study, we aimed to develop a molecular method to detect PV directly from stool extracts, with a high efficiency comparable to that of cell culture. We developed a method to efficiently amplify the entire capsid coding region of human enteroviruses (EVs) including PV. cDNAs of the entire capsid coding region (3.9 kb) were obtained from as few as 50 copies of PV genomes. PV was detected from the cDNAs with an improved PV-specific real-time reverse transcription-PCR system and nucleotide sequence analysis of the VP1 coding region. For assay validation, we analyzed 84 stool extracts that were positive for PV in cell culture and detected PV genomes from 100% of the extracts (84/84 samples) with this method in combination with a PV-specific extraction method. PV could be detected in 2/4 stool extract samples that were negative for PV in cell culture. In PV-positive samples, EV species C viruses were also detected with high frequency (27% [23/86 samples]). This method would be useful for direct detection of PV from stool extracts without using cell culture.