11
2020-12
2020年11月09日，NATURE MEDICINE 雜志（IF:36.13）發(fā)表《Rapid pathogen detection by metagenomic next - generation sequencing of infected body fluids》研究文章，探討新一代宏基因組測(cè)序?qū)Ω腥倔w液快速進(jìn)行病原檢測(cè)的應(yīng)用，該研究入組160名急性病患者，并收集了182種體液進(jìn)行mNGS檢測(cè)，并與培養(yǎng)法、PCR、納米孔測(cè)序等方法進(jìn)行微生物學(xué)測(cè)試的對(duì)比。
我們使用體液中的無(wú)細(xì)胞DNA進(jìn)行了mNGS測(cè)試，以鑒定病原體。使用兩種測(cè)序平臺(tái)對(duì)160名急性病患者的182種體液的mNGS測(cè)試性能進(jìn)行了評(píng)估，并與使用培養(yǎng)物、16S細(xì)菌PCR和/或28S-內(nèi)部轉(zhuǎn)錄核糖體基因間隔子（28S-ITS）真菌PCR的微生物學(xué)測(cè)試進(jìn)行對(duì)比。通過(guò)Illumina測(cè)序，細(xì)菌的檢測(cè)靈敏度和檢測(cè)特異性分別為79％和91％，真菌為91％和89％；通過(guò)納米孔測(cè)序，細(xì)菌分別為75％和81％，真菌為91％和100％。在12例培養(yǎng)/ PCR陰性體液但最終被確診為感染的患者中，有7例（58％）是mNGS陽(yáng)性。實(shí)時(shí)計(jì)算分析可通過(guò)中值50分鐘測(cè)序和6小時(shí)樣品到應(yīng)答時(shí)間的納米孔測(cè)序?qū)崿F(xiàn)病原體鑒定?？焖俚膍NGS測(cè)試是診斷體液未知感染的潛在工具。
圖a：mNGS體液分析工作流程示意圖
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.
（臨床金標(biāo)準(zhǔn)由培養(yǎng)物、細(xì)菌16SPCR和/或真菌28S-ITSPCR的共同結(jié)果，而復(fù)核標(biāo)準(zhǔn)還包括與Sanger的驗(yàn)證性數(shù)字PCR測(cè)序和臨床診斷。 對(duì)于< 6? h的納米孔測(cè)序，核酸提取需40~60? min，mNGS文庫(kù)制備需2~ 2.5?h，納米孔1D 建庫(kù)需1?h，納米孔測(cè)序分析需1?h。）
圖b：研究中包括的182例體液樣本的分析工作流程
b, Analysis workflow for the 182?body fluid samples included in the study. 170?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.
（在準(zhǔn)確性評(píng)估中包括170個(gè)樣本，從臨床診斷感染的患者中收集的12個(gè)樣本，但對(duì)陰性微生物樣本進(jìn)行了mNGS檢測(cè)。餅圖顯示研究中分析的體液樣本類(lèi)型。）
圖c：相對(duì)于培養(yǎng)的mNGS測(cè)試的時(shí)機(jī)
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.
（基于培養(yǎng)的病原體鑒定可能需要幾天到幾周，而使用納米孔或Illumina測(cè)序平臺(tái)進(jìn)行mNGS測(cè)試的總體運(yùn)行時(shí)間為5-24小時(shí)。）
參考文獻(xiàn)：[1]. Rapid pathogen detection by metagenomic next-generation sequencing of infected body fluids.[J]. Nature medicine,2020.
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