Direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased high-throughput sequencing approach.
Direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased high-throughput sequencing approach.
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DOI：
10.1371/journal.pone.0004219
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发表时间：
2009
期刊：
影响因子：
3.7
通讯作者：
Nakaya T
中科院分区：
文献类型：
--
作者：
Nakamura S;Yang CS;Sakon N;Ueda M;Tougan T;Yamashita A;Goto N;Takahashi K;Yasunaga T;Ikuta K;Mizutani T;Okamoto Y;Tagami M;Morita R;Maeda N;Kawai J;Hayashizaki Y;Nagai Y;Horii T;Iida T;Nakaya T
随着2003年的严重急性呼吸综合征疫情和对禽流感病毒大流行的重新关注，需要新的监测系统来更早地发现新出现的传染病。我们应用“下一代”并行测序平台对2005年至2007年日本大阪季节性流感病毒感染和诺沃克病毒暴发期间采集的鼻咽和粪便样本进行病毒检测。从0.1~0.2 5ml的鼻咽抽提物(N = 3)和粪便标本(N = 5)中扩增出10µg以上的DNA。对这8个样本的无偏高通量测序在一次7.5小时的运行中产生了15,298-32,335(平均24,738)个读数。在鼻咽样本中，虽然无法进行全基因组分析，因为大多数(>；90%)的读数来自宿主基因组，但检测到20-460个流感读数，这足以进行亚型鉴定。在粪便样本中，通过离心法去除细菌和宿主细胞，从而获得484-15,260个诺沃克病毒序列读数(覆盖了整个基因组的78-98%)，除了一个通过RT-PCR检测不到的样本。这些结果表明，我们的无偏高通量测序方法对于在没有事先遗传信息的情况下直接检测致病病毒是有用的。尽管其成本和技术可用性使该系统不太可能很快成为全球诊断标准，但该系统可能有助于更早地发现新出现的病毒和生物恐怖主义，而传统程序很难发现这些病毒和生物恐怖主义。
With the severe acute respiratory syndrome epidemic of 2003 and renewed attention on avian influenza viral pandemics, new surveillance systems are needed for the earlier detection of emerging infectious diseases. We applied a “next-generation” parallel sequencing platform for viral detection in nasopharyngeal and fecal samples collected during seasonal influenza virus (Flu) infections and norovirus outbreaks from 2005 to 2007 in Osaka, Japan. Random RT-PCR was performed to amplify RNA extracted from 0.1–0.25 ml of nasopharyngeal aspirates (N = 3) and fecal specimens (N = 5), and more than 10 µg of cDNA was synthesized. Unbiased high-throughput sequencing of these 8 samples yielded 15,298–32,335 (average 24,738) reads in a single 7.5 h run. In nasopharyngeal samples, although whole genome analysis was not available because the majority (>90%) of reads were host genome–derived, 20–460 Flu-reads were detected, which was sufficient for subtype identification. In fecal samples, bacteria and host cells were removed by centrifugation, resulting in gain of 484–15,260 reads of norovirus sequence (78–98% of the whole genome was covered), except for one specimen that was under-detectable by RT-PCR. These results suggest that our unbiased high-throughput sequencing approach is useful for directly detecting pathogenic viruses without advance genetic information. Although its cost and technological availability make it unlikely that this system will very soon be the diagnostic standard worldwide, this system could be useful for the earlier discovery of novel emerging viruses and bioterrorism, which are difficult to detect with conventional procedures.
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影响因子：
11.8
作者：
Nakamura S;Maeda N;Miron IM;Yoh M;Izutsu K;Kataoka C;Honda T;Yasunaga T;Nakaya T;Kawai J;Hayashizaki Y;Horii T;Iida T
通讯作者：
Iida T
影响因子：
14.9
作者：
Pruesse E;Quast C;Knittel K;Fuchs BM;Ludwig W;Peplies J;Glöckner FO
通讯作者：
Glöckner FO
影响因子：
56.9
作者：
Poinar, HN;Schwarz, C;Schuster, SC
通讯作者：
Schuster, SC
影响因子：
9.4
作者：
Quan, Phenix-Lan;Palacios, Gustavo;Lipkin, W. Ian
通讯作者：
Lipkin, W. Ian
影响因子：
15.8
作者：
Lopman B;Zambon M;Brown DW
通讯作者：
Brown DW