Wang Jun's team from the Institute of Microbiology, Chinese Academy of Sciences establishes new methods for the detection of pathogens targeting RNA, mtNGS and mtTGS

Pathogen infection is still a huge problem threatening human health all over the world, and it has brought a heavy burden to clinical diagnosis and treatment
.

Traditional clinical pathogen identification is mainly based on culture and biochemical detection, but with the continuous advancement of next-generation sequencing technology, metagenomic sequencing (mNGS) has greatly improved the efficiency of pathogen detection and helped to identify difficult-to-cultivable pathogens.
Pathogenic microorganisms; in addition, the identification of antibiotic-resistant microorganisms through sequencing also provides a basis for improving treatment options
.

However, mNGS still faces many obstacles in clinical application, especially when the total amount of test samples is small and the sample's microbial content is low, such as throat swab samples, bronchoalveolar lavage fluid samples, blood samples, and cerebrospinal fluid samples
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At the same time, since host cells and nucleotides often account for a large proportion of these samples (usually >90% of host components), the sequencing efficiency of microbial identification is greatly reduced
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At present, mNGS mainly targets the DNA of pathogenic microorganisms and uses second-generation sequencing technology to generate shorter sequences.
Therefore, some pathogenic microorganisms whose main genetic material is RNA are ignored
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Even though RNA-based analysis or metatranscriptome research is relatively rare, they all show that targeted RNA detection can effectively reveal functionally active members/genes; in addition, although the bacterial genome has only a few rDNA copies, the content of rRNA molecules is active But it is extremely high in cells.
In theory, targeted RNA can avoid the predominant but dormant microorganisms accounting for the proportion of sequencing reads
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The third-generation sequencing represented by Oxford Nanopore Technology's sequencing technology has the advantages of rapid library preparation and ultra-long read length, which can effectively improve the detection of pathogens in clinical samples
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At the same time, ONT direct RNA sequencing technology also provides the possibility for unbiased detection of RNA pathogens in clinical samples
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So far, Wang Jun’s research team from the Institute of Microbiology of the Chinese Academy of Sciences has cooperated with the Department of Respiratory and Critical Care Medicine of PLA General Hospital, Zhujiang Medical University of Southern Medical University and Peking University Third Hospital to collect alveolar lavage fluid (BALF), cerebrospinal fluid (CSF) and For blood samples, RNA/cDNA targeted sequencing (mtNGS) was established to reduce the proportion of host nucleotides in clinical samples, and combined with Oxford Nanopore Technology (ONT) (mtTGS) to shorten sequencing time
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Studies have shown that compared with mNGS, mtNGS increases the ratio of microbial readings, facilitates the identification of bacterial pathogens, and can detect fungi, viruses, and antibiotic resistance genes, which are basically the same as traditional clinical test results
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In addition, the ONT-based mtTGS further improves the identification of pathogenic microorganisms due to its longer read length, and also speeds up the diagnosis time
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Tests using ONT's direct RNA sequencing and targeted sequencing show that ONT shows important pathogen detection potential, but further development is still needed
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Therefore, this study shows that RNA-targeted pathogen detection in clinical samples has a higher potential, especially when combined with the development and technological update of ONT
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The above research results were published in the journal Advanced Science in October 2021.
Assistant researcher Zhao Na, Ph.
D student Cao Jiabao, Ph.
D.
student Cao Jiabao, Assistant Researcher Xu Jiayue, Peking University Third Hospital, Peking University Third Hospital, Dr.
Liu Beibei, Key Laboratory of Pathogen Immunity, Chinese Academy of Sciences Liu Bin, PhD student in the Department of Respiratory and Critical Care Medicine, the Eighth Medical Center of PLA General Hospital; Dr.
Chen Dingqiang from Zhujiang Medical College of Southern Medical University as the first author; Researcher Wang Jun from Institute of Microbiology, Chinese Academy of Sciences; Professor Zhou Hongwei from Zhujiang Medical College of Southern Medical University; PLA General Hospital Xie Lixin, director of the Respiratory and Critical Care Medicine Department of the Eighth Medical Center, is the corresponding author
.

The research was supported by the National Natural Science Foundation of China, the pilot project of the Chinese Academy of Sciences and the key research and development plan of the Ministry of Science and Technology
.


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wiley.
com/doi/10.
1002/advs.
202102593