State Key Laboratory for Molecular Virology and Genetic Engineering, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changbai Rd 155, Beijing, Changping District 102206, China.
BMC Infect Dis. 2012 Aug 14;12:189. doi: 10.1186/1471-2334-12-189.
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.
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.
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.
In conclusion, the GeXP assay is a rapid, cost-effective, sensitive, specific and high throughput method for the detection of respiratory virus infections.
现有的标准非分子诊断方法，如病毒培养和免疫荧光（DFA），既费时又费力，或灵敏度有限。几种多重分子检测方法成本较高。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。
建立了基于 GeXP 的多重 RT-PCR 检测方法（GeXP 检测法），用于同时检测 16 种不同的呼吸道病毒类型/亚型。使用 17 组嵌合引物启动 RT-PCR，随后的 RT-PCR 循环使用一对通用引物。使用每种病毒类型/亚型的阳性对照来检测 GeXP 检测法的特异性。通过对所有 RNA 病毒的体外转录 RNA 进行十倍连续稀释以及含有 Adv 和 HBoV 靶序列的质粒进行检测，评估 GeXP 检测法的灵敏度。进一步用 126 份临床标本评估 GeXP 检测法，并与 Luminex xTAG RVP Fast 检测法进行比较。
当单一病毒的检测灵敏度为 20-200 拷贝时，当所有 16 种预先混合的病毒靶标都存在时，检测灵敏度为 1000 拷贝。使用 GeXP 检测法对 126 份临床标本进行分析，结果表明 GeXP 检测法和 RVP Fast 检测法在 126 份标本中的 109/126（88.51%）完全一致。与 RVP Fast 检测法相比，GeXP 检测法对 HRV 和 PIV3 的检测更敏感，对 HMPV、Adv、RSVB 和 HBoV 的检测略低。使用 GeXP 检测法检测 12 个样本的整个过程在 2.5 小时内完成。
总之，GeXP 检测法是一种快速、经济高效、灵敏、特异和高通量的呼吸道病毒感染检测方法。
