Rapid detection of pathogenic virus genome sequence from throat and nasal swab samples using an exhaustive gene amplification method
Rapid detection of pathogenic virus genome sequence from throat and nasal swab samples using an exhaustive gene amplification method
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DOI：
10.1007/s12024-019-00128-z
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发表时间：
2019-09-01
影响因子：
1.8
通讯作者：
Nakahara, Hiroaki
中科院分区：
文献类型：
--
作者：
Hosokawa-Muto, Junji;Sakai, Hiroki;Nakahara, Hiroaki
迅速查明病原体对于应对生物犯罪和生物恐怖主义的出现十分重要，有助于进行适当的隔离和治疗。由于采用穷举基因扩增技术的病毒基因组序列快速测定系统（RDV法）可用于快速鉴定，我们研究了该方法是否可应用于法医样品。为了检测疑似病毒感染的猫中的致病性病毒，将液体拭子样本应用于RDV方法。进行以下步骤：病毒增殖、病毒基因组提取、第一文库扩增、文库片段化、使用非特异性引物组扩增第二文库以及扩增子直接测序。为了确认通过该方法检测的病毒，我们使用病毒特异性引物进行常规PCR。我们从拭子样本中检测到致病性病毒基因组序列，并确认感染了这些病毒。此外，我们直接从鼻拭子样本中检测到病毒基因组序列，而无需病毒繁殖步骤。RDV方法很少用于法医分析。该方法在一般实验室设备上是可行的，可用于法医学标本中病原病毒的快速检测和鉴定。该方法也适用于细菌和真菌的检测。
Rapid identification of pathogenic agents is important in response to the emergence of biocrime and bioterrorism, to facilitate appropriate confinement and treatment. As the rapid determination system of viral genome sequences (RDV method) using exhaustive gene amplification is useful for rapid identification, we examined whether this method could be applied to forensic samples. To detect pathogenic virus in a cat with suspected viral infections, fluid swab samples were applied to the RDV method. The following steps were performed: viral propagation, extraction of the viral genome, amplification of the first library, fragmentation of the library, amplification of the second library using non-specific primer sets, and direct sequencing of the amplicon. To confirm the viruses detected by this method, we performed conventional PCR using virus-specific primers. We detected pathogenic virus genome sequences from the swab samples and confirmed infection with these viruses. In addition, we directly detected a viral genome sequence from the nasal swab sample without the viral propagation step. The RDV method is infrequently used in forensic analysis. This method is practicable with equipment existing in a normal laboratory and is useful for rapid detection and identification of pathogenic viruses in forensic samples. This method would also be applicable to the detection of bacteria and fungi.