一种基于新型再测序芯片的不明原因腹泻样品检测方法的建立和初步应用

发布时间：2018-10-26 20:40

【摘要】：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。
[Abstract]:In this study, a novel multi-pathogen detection method for diarrhea syndrome based on a novel resequencing chip (Resequencing Pathogen Microarray,RPM was developed. It can detect 14 rotaviruses, 7 calix viruses, 8 stellate viruses and 28 enterovirus simultaneously. There are 16 rare diarrhea viruses. The specificity of the method was evaluated by using a validated positive virus sample as a template. The sensitivity of RPM was tested by cloning plasmid and in vitro transcription RNA gradient dilution solution. RPM could still detect and distinguish similar virus subtypes at 20 ~ 2 000 copies / 渭 L level. The positive detection threshold was optimized by adjusting the concentration of reference material, and the detection procedure of enterovirus was improved to complete the typing. Ten unexplained diarrhea samples were screened and 6 diarrhea virus positive samples were detected. The single PCR was sequenced according to the RPM results, and the RPM was consistent with the sequencing results. In this study, a high throughput, high specificity and high sensitivity RPM method was established, which has great application value in the diagnosis of unknown diarrhea cases and the treatment of sudden outbreaks.
【作者单位】：中国疾病预防控制中心病毒病预防控制所;
【基金】：传染病重大专项(2013ZX10004-001,2012ZX10004-215,2013ZX10004-202,2013ZX10004804-007)
【分类号】：R373

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