The development of a GeXP-based multiplex reverse transcription-PCR assay for simultaneous detection of sixteen human respiratory virus types/subtypes
The development of a GeXP-based multiplex reverse transcription-PCR assay for simultaneous detection of sixteen human respiratory virus types/subtypes
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DOI：
10.1186/1471-2334-12-189
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发表时间：
2012-08-14
影响因子：
3.7
通讯作者：
Ma, Xue-Jun
中科院分区：
文献类型：
--
作者：
Li, Jin;Mao, Nai-Ying;Ma, Xue-Jun
背景：现有的标准非分子诊断方法，例如病毒培养和免疫荧光（DFA），耗时、费力或灵敏度有限。几种多重分子测定的成本很高。因此，需要开发一种快速、灵敏的呼吸道病毒病原体诊断方法。方法：开发了一种基于GeXP的多重RT-PCR测定（GeXP测定）来同时检测16种不同的呼吸道病毒类型/亚型。使用十七组嵌合引物启动RT-PCR，并使用一对通用引物进行随后的RT-PCR循环。使用每种病毒类型/亚型的阳性对照检查 GeXP 测定的特异性。通过对所有 RNA 病毒的体外转录 RNA 和含有 Adv 和 HBoV 靶序列的质粒进行连续十倍稀释的测定来评估灵敏度。使用 126 个临床样本进一步评估 GeXP 检测，并与 Luminex xTAG RVP 快速检测进行比较。结果：GeXP 检测对单一病毒的灵敏度为 20-200 个拷贝，当所有 16 个预混合病毒靶点都存在时，灵敏度为 1000 个拷贝。使用 GeXP 检测对 126 份临床标本进行的分析表明，109/126 (88.51%) 的标本中 GeXP 检测和 RVP Fast 检测完全一致。 GeXP 检测对 HRV 和 PIV3 的检测比 RVP Fast 检测更灵敏，而对 HMPV、Adv、RSVB 和 HBoV 的检测灵敏度稍差。 GeXP法检测12个样本的整个过程在2.5小时内完成。结论：综上所述，GeXP法是一种快速、经济、灵敏、特异、高通量的呼吸道病毒感染检测方法。
Background: Existing standard non-molecular diagnostic methods such as viral culture and immunofluorescent (DFA) are time-consuming, labor intensive or limited sensitivity. Several multiplex molecular assays are costly. Therefore, there is a need for the development of a rapid and sensitive diagnosis of respiratory viral pathogens.Methods: A GeXP-based multiplex RT-PCR assay (GeXP assay) was developed to detect simultaneously sixteen different respiratory virus types/subtypes. Seventeen sets of chimeric primers were used to initiate the RT-PCR, and one pair of universal primers was used for the subsequent cycles of the RT-PCR. The specificity of the GeXP assay was examined with positive controls for each virus type/subtype. The sensitivity was evaluated by performing the assay on serial ten-fold dilutions of in vitro-transcribed RNA of all RNA viruses and the plasmids containing the Adv and HBoV target sequence. GeXP assay was further evaluated using 126 clinical specimens and compared with Luminex xTAG RVP Fast assay.Results: The GeXP assay achieved a sensitivity of 20-200 copies for a single virus and 1000 copies when all of the 16 pre-mixed viral targets were present. Analyses of 126 clinical specimens using the GeXP assay demonstrated that GeXP assay and the RVP Fast assay were in complete agreement for 109/126 (88.51%) of the specimens. GeXP assay was more sensitive than the RVP Fast assay for the detection of HRV and PIV3, and slightly less sensitive for the detection of HMPV, Adv, RSVB and HBoV. The whole process of the GeXP assay for the detection of 12 samples was completed within 2.5 hours.Conclusions: In conclusion, the GeXP assay is a rapid, cost-effective, sensitive, specific and high throughput method for the detection of respiratory virus infections.