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本研究建立了一种基于新型再测序芯片(Resequencing Pathogen Microarray,RPM)的腹泻症候群多病原检测方法,能同时检测14种轮状病毒,7种杯状病毒,8种星状病毒,28种肠道病毒,16种少见致泻病毒。选取已验证的阳性病毒样本为模板来评估该方法的特异性。用克隆质粒和体外转录的RNA梯度稀释液来检验RPM的灵敏度,在20~2 000拷贝/μL水平时RPM仍能检测和分辨出相近的病毒亚型。通过调整参考品的浓度优化了阳性判定阈值,并改进了肠道病毒的检测流程以完成分型。对10份不明原因腹泻样品进行了筛查,从中检出6份腹泻病毒阳性样本。根据RPM结果对样品进行了单重PCR并且测序,RPM与测序结果一致。本研究建立了一种高通量,高特异性,灵敏度较高的RPM方法,对于不明原因腹泻病例的诊断和突发疫情的处理有巨大的应用价值。
In this study, we established a multi-pathogen detection method for diarrhea syndrome based on a novel Resequencing Pathogen Microarray (RPM), which can simultaneously detect 14 rotavirus, 7 calicivirus, 8 astrovirus and 28 kinds of intestine Road virus, 16 rare diarrhea virus. A validated positive virus sample was selected as a template to evaluate the specificity of the method. The sensitivity of RPMs was tested using cloning plasmids and in vitro transcribed RNA gradient dilutions, and the RPMs were able to detect and resolve similar virus subtypes at 20-2 000 copies / μL. The positive determination threshold was optimized by adjusting the reference concentration and the enterovirus detection procedure was modified to complete the typing. Ten samples of unexplained diarrhea were screened and six samples of positive diarrhea were detected. Samples were subjected to single-copy PCR and sequencing based on the RPM results, which were consistent with the sequencing results. This study established a high-throughput, high specificity, high sensitivity RPM method for the diagnosis of unexplained diarrhea cases and the treatment of sudden outbreaks of great value.