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

Establishment and Primary Application of A Novel Resequencing Pathogen Microarray-Based Assay for Detecting Pathogens in Patients with Unexplained Diarrhea

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20～2000拷贝／gL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, a novel resequencing pathogen microarray （RPM）-based multi-pathogen detection assay was developed to simultaneously detect 14 rotaviruses, 7 caliciviruses, 8 astroviruses, 28 enteroviruses, and 16 rare diarrhea viruses in patients with diarrhea syndrome. The specificity of the assay was examined using confirmed virus-positive specimens, and the sensitivity was evaluated by serial ten-fold dilu- tions of in vitro transcribed RNA. RPM assay could detect and differentiate virus types/subtypes at 20- 2000 copies/~tL. The detection threshold of RPM was determined by adjusting the reference concentration, and the detection steps were optimized to type Enterovirus. The nucleic acids of 10 stool samples from pa- tients with unexplained diarrhea were screened, and 6 of them showed positive results. The RPM results were further verified by singleplex PCR followed by sequencing, and no difference was found between the two assays. In conclusion, we have established a high-throughput RPM assay with high specificity and sensitivity, which demonstrates a great potential for the identification of pathogens in patients with unexplained diarrhea and the management of emerging epidemic.