利用高通量测序快速检测疑似感染患者体内未知病原体

Rapid detection of potential new pathogens in patient using high-throughput sequencing

采用第二代高通量测序技术从复杂的疑似感染患者组织标本中直接快速检测未知病原体。方法:取临床和实验室均不能确诊的病人血液和淋巴组织,进行病毒DNA和RNA的提取,直接用于第二代高通量测序,将测序结果用于生物信息学分析,与本地化的病毒核酸数据库进行比对,寻找潜在的病毒序列。结果:生物信息学分析显示,3901条序列reads与人内源性病毒K113同源;当使用K113特异性引物对经过DnaseI处理的患者和正常个体进行荧光定量RT-PCR时,发现在患者体内K113病毒基因的表达远远高于正常个体,提示该患者可能存在K113病毒的感染。结论:第二代高通量测序可能用于快速检测未知病原体。

To direct detect potential pathogens in suspect patient tissues with next generation sequencing.Methods：DNA and RNA samples were prepared from tissues of a patient who is suspected of infection but the clinical and laboratory diagnosis is not available to confirm the infection.The DNA and RNA （converted into cDNA） samples were directly subjected to next-generation sequencing.The sequencing data were cleaned with fastx-toolkits softwarepackage and then analyzed with BLAST using locally adopted virus nucleotide sequence database.Results：Bioinformatics analysis revealed an endogenous virus K113 which comprise 3901 reads,with the highest coverage of 487 fold.Subsequent PCR analysis with K113 specific primers demonstrated that K113 viral RNA was significant abundant in the patient lymphocytes than in that of the healthy control,which suggesting possible active replication of the endogenous K113 virus.Conclusion：Next generation sequencing is a powerful tool for detection of unknown pathogen infection.