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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序?qū)Ω腥倔w液快速進(jìn)行病原檢測的應(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，并與培養(yǎng)法、PCR、納米孔測序等方法進(jìn)行微生物學(xué)測試的對比。

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.

我們使用體液中的無細(xì)胞DNA進(jìn)行了mNGS測試，以鑒定病原體。使用兩種測序平臺(tái)對160名急性病患者的182種體液的mNGS測試性能進(jìn)行了評估，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)測試進(jìn)行對比。通過Illumina測序，細(xì)菌的檢測靈敏度和檢測特異性分別為79％和91％，真菌為91％和89％；通過納米孔測序，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陽性。實(shí)時(shí)計(jì)算分析可通過中值50分鐘測序和6小時(shí)樣品到應(yīng)答時(shí)間的納米孔測序?qū)崿F(xiàn)病原體鑒定?？焖俚膍NGS測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測序和臨床診斷。對于< 6? h的納米孔測序，核酸提取需40~60? min，mNGS文庫制備需2~ 2.5?h，納米孔1D 建庫需1?h，納米孔測序分析需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，但對陰性微生物樣本進(jìn)行了mNGS檢測。餅圖顯示研究中分析的體液樣本類型。）

圖c：相對于培養(yǎng)的mNGS測試的時(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測序平臺(tái)進(jìn)行mNGS測試的總體運(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.

關(guān)于精科醫(yī)學(xué)

廣州精科醫(yī)學(xué)檢驗(yàn)所有限公司是國家基因檢測技術(shù)應(yīng)用示范中心、國家高新技術(shù)企業(yè)，主承擔(dān)重大遺傳性疾病基因篩查服務(wù)及試劑研發(fā)生產(chǎn)建設(shè)項(xiàng)目，是廣東省重大遺傳性疾病基因篩查產(chǎn)業(yè)技術(shù)創(chuàng) 新聯(lián)盟（廣東省科學(xué)技術(shù)廳）的牽頭創(chuàng)辦單位。公司致力于推進(jìn)基因檢測技術(shù)在出生缺陷三級防控篩查、腫瘤精準(zhǔn)防治、病原微生物等重要健康領(lǐng)域的生態(tài)化應(yīng)用項(xiàng)目開發(fā)和推廣。

作為一家集科研轉(zhuǎn)化、專業(yè)臨床檢驗(yàn)和大健康服務(wù)于一體的高新生物技術(shù)企業(yè)，公司擁有以中國科學(xué)院魏于全院士領(lǐng)銜，分子病理學(xué)專家/博士研究生導(dǎo)師李宏教授為醫(yī)學(xué)總監(jiān)，中國轉(zhuǎn)化醫(yī)學(xué)聯(lián)盟為載體的核心技術(shù)團(tuán)隊(duì)，與各大高校、科研機(jī)構(gòu)及醫(yī)院深入展開教學(xué)、科研及臨床合作。

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