Use of oligonucleotide microarrays for rapid detection and serotyping of acute respiratory disease-associated adenoviruses
Use of oligonucleotide microarrays for rapid detection and serotyping of acute respiratory disease-associated adenoviruses
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DOI：
10.1128/jcm.42.7.3232-3239.2004
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发表时间：
2004-07-01
影响因子：
9.4
通讯作者：
Stenger, DA
中科院分区：
文献类型：
--
作者：
Lin, BC;Vora, GJ;Stenger, DA
停止对军事受训人员进行腺病毒疫苗接种计划导致了最近几次急性呼吸道疾病（ARD）的爆发。在没有接种疫苗的情况下，快速检测方法对于及时实施预防军事训练设施内腺病毒传播的措施是必要的。为此，我们将荧光实时多重PCR检测与靶向E1 A、纤维蛋白和六邻体基因的四组简并PCR引物与能够鉴定与成人呼吸道感染相关的最常见腺病毒血清型的长寡核苷酸微阵列相结合（血清型3、4、7、16和21）和腺病毒亚群C（血清型6）的代表性成员，其是儿童ARD的常见原因并且通常持续到成年。原型株的分析表明，腺病毒亚群B-1、B-2、C-1和E的代表性成员具有独特的杂交模式，因此可以确定血清型。基于微阵列的灵敏度评估显示，对于腺病毒血清型4（Ad 4）和Ad 7细胞培养裂解物、临床鼻洗液和咽拭子以及来自临床样本的纯化DNA，检测限较低（1至100个基因组拷贝）。当从编码的临床样品中检测到腺病毒时，通过这种方法获得的结果与通过更成熟的腺病毒鉴定方法以及通过荧光抗体染色的细胞培养获得的结果表现出极好的一致性。最后，该方法的实用性进一步得到其检测腺病毒合并感染、污染和潜在重组事件的能力的支持。综上所述，结果证明了开发的简单快速诊断方法的有效性，该方法可在1.5至4.0 h内完成对实验室或临床样本中的ARD相关腺病毒血清型的明确鉴定。
The cessation of the adenovirus vaccination program for military trainees has resulted in several recent acute respiratory disease (ARD) outbreaks. In the absence of vaccination, rapid detection methods are necessary for the timely implementation of measures to prevent adenovirus transmission within military training facilities. To this end, we have combined a fluorogenic real-time multiplex PCR assay with four sets of degenerate PCR primers that target the E1A, fiber, and hexon genes with a long oligonucleotide microarray capable of identifying the most common adenovirus serotypes associated with adult respiratory tract infections (serotypes 3, 4, 7, 16, and 21) and a representative member of adenovirus subgroup C (serotype 6) that is a common cause of childhood ARD and that often persists into adulthood. Analyses with prototype strains demonstrated unique hybridization patterns for representative members of adenovirus subgroups B-1, B-2, C-1, and E, thus allowing serotype determination. Microarray-based sensitivity assessments revealed lower detection limits (between I and 100 genomic copies) for adenovirus serotype 4 (Ad4) and Ad7 cell culture lysates, clinical nasal washes, and throat swabs and purified DNA from clinical samples. When adenovirus was detected from coded clinical samples, the results obtained by this approach demonstrated an excellent concordance with those obtained by the more established method of adenovirus identification as well as by cell culture with fluorescent-antibody staining. Finally, the utility of this method was further supported by its ability to detect adenoviral coinfections, contamination, and, potentially, recombination events. Taken together, the results demonstrate the usefulness of the simple and rapid diagnostic method developed for the unequivocal identification of ARD-associated adenoviral serotypes from laboratory or clinical samples that can be completed in 1.5 to 4.0 h.