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2020-12

重磅文章|mNGS在快速病原体检测中的应用(Nat Med, IF:36.13)
发布时间:2020-12-11 17:04:16作者:精科医学 来源:精科医学

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2020年11月09日,NATURE MEDICINE 杂志(IF:36.13)发表《Rapid pathogen detection by metagenomic next - generation sequencing of infected body fluids》研究文章,探讨新一代宏基因组测序对感染体液快速进行病原检测的应用,该研究入组160名急性病患者,并收集了182种体液进行mNGS检测,并与培养法、PCR、纳米孔测序等方法进行微生物学测试的对比。



We developed a metagenomic next-generation sequencing (mNGS) test using cell-free DNA from body fluids to identify pathogens. The performance of mNGS testing of 182 body fluids from 160 patients with acute illness was evaluated using two sequencing platforms in comparison to microbiological testing using culture, 16S bacterial PCR and/or 28S–internal transcribed ribosomal gene spacer (28S–ITS) fungal PCR. Test sensitivity and specificity of detection were 79 and 91% for bacteria and 91 and 89% for fungi, respectively, by Illumina sequencing; and 75 and 81% for bacteria and 91 and 100% for fungi, respec- tively, by nanopore sequencing. In a case series of 12 patients with culture/PCR-negative body fluids but for whom an infectious diagnosis was ultimately established, seven (58%) were mNGS positive. Real-time computational analysis enabled pathogen identification by nanopore sequencing in a median 50-min sequencing and 6-h sample-to-answer time. Rapid mNGS testing is a promising tool for diagnosis of unknown infections from body fluids. 

我们使用体液中的无细胞DNA进行了mNGS测试,以鉴定病原体。使用两种测序平台对160名急性病患者的182种体液的mNGS测试性能进行了评估,并与使用培养物、16S细菌PCR和/或28S-内部转录核糖体基因间隔子(28S-ITS)真菌PCR的微生物学测试进行对比。通过Illumina测序,细菌的检测灵敏度和检测特异性分别为79%和91%,真菌为91%和89%;通过纳米孔测序,细菌分别为75%和81%,真菌为91%和100%。在12例培养/ PCR阴性体液但最终被确诊为感染的患者中,有7例(58%)是mNGS阳性。实时计算分析可通过中值50分钟测序和6小时样品到应答时间的纳米孔测序实现病原体鉴定。快速的mNGS测试是诊断体液未知感染的潜在工具。


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图a:mNGS体液分析工作流程示意图

a, Schematic of mNGS body fluid analysis workflow. The clinical gold standard consisted of aggregated results from cultures, bacterial 16S PCR and/or fungal 28S–ITS PCR, while the composite standard also included confirmatory digital PCR with Sanger sequencing and clinical adjudication. For nanopore sequencing in <6 h, 40–60 min are needed for nucleic acid extraction, 2–2.5 h for mNGS library preparation, 1 h for nanopore 1D library preparation and 1 h for nanopore sequencing and analysis. 

临床金标准由培养物、细菌16SPCR和/或真菌28S-ITSPCR的共同结果,而复核标准还包括与Sanger的验证性数字PCR测序和临床诊断。 对于< 6  h的纳米孔测序,核酸提取需40~60  min,mNGS文库制备需2~ 2.5 h,纳米孔1D 建库需1 h,纳米孔测序分析需1 h。)


图b:研究中包括的182例体液样本的分析工作流程

b, Analysis workflow for the 182 body fluid samples included in the study170 samples were included in the accuracy assessment while 12 samples collected from patients with a clinical diagnosis of infection but negative microbiological testing were included for mNGS analysis. The pie chart displays the body fluid sample types analyzed in the study. 

(在准确性评估中包括170个样本,从临床诊断感染的患者中收集的12个样本,但对微生物样本进行了mNGS检测。饼图显示研究中分析的体液样本类型。)


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c:相对于培养的mNGS测试的时机

c, Timing for mNGS testing relative to culture. Whereas culture-based pathogen identification can take days to weeks, mNGS testing using nanopore or Illumina sequencing platforms has an overall turnaround time of 5–24 h.

(基于培养的病原体鉴定可能需要几天到几周,而使用纳米孔或Illumina测序平台进行mNGS测试的总体运行时间为5-24小时。)


参考文献:[1]. Rapid pathogen detection by metagenomic next-generation sequencing of infected body fluids.[J]. Nature medicine,2020.


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