Wang Jun's team at the Institute of Microbiology established a new method for pathogen detection of targeting RNA, mtNGS and mtTGS

Pathogenic infections remain a huge threat to human health around the world, placing a heavy burden on clinical diagnosis and treatment. Clinically, traditional 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 pathogenic microorganisms that are difficult to culture; 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 number of samples tested is small and the microbial content of samples is low, such as throat swab samples, bronchoalveolar lavage fluid samples, blood samples and cerebrospinal fluid samples. At the same time, because host cells and nucleotides tend to account for a large proportion of these samples (usually > 90% of the host components), the sequencing efficiency of microbial identification is greatly reduced.

Currently, mNGS mainly targets the DNA of pathogenic microorganisms and uses second-generation sequencing techniques to produce shorter sequences, so some pathogenic microorganisms with RNA as the main genetic material are ignored. Even though there have been relatively few studies of RNA-based analysis or macro-transcriptomes, they have shown that detection of targeted RNA is effective in revealing functionally active members/genes; moreover, although the bacterial genome has only a few copies of rDNA, the amount of rRNA molecules in active cells is extremely high, and the proportion of preemptive but dormant microbes that can theoretically be avoided is the proportion of sequencing readings.

Three-generation sequencing, represented by Oxford Nanopore Technology sequencing technology, has the advantages of rapid preparation library and ultra-long reading length, which can effectively improve the detection of pathogens in clinical samples. At the same time, ONT direct RNA sequencing technology also provides the possibility of unbiased detection of RNA pathogens in clinical samples. So far, the Research Group of Wang Jun of the Institute of Microbiology of the Chinese Academy of Sciences, in cooperation with the Department of Respiratory and Critical Care Medicine of the PLA General Hospital, the Pearl River Medical College of Southern Medical University and the Third Hospital of Peking University, collected alveolar lavage fluid (BALF), cerebrospinal fluid (CSF) and blood (Blood) samples, and established RNA/cDNA targeted sequencing (mtNGS) to reduce the proportion of host nucleotides in clinical samples. And by combining with Oxford Nanopore Technology (ONT) (mtTGS) to reduce sequencing time.

Studies have shown that compared with mNGS, mtNGS improves the proportion of microbial readings, promotes the identification of bacterial pathogens, and can detect fungal, viral and antibiotic resistance genes, which is basically consistent with traditional clinical test results. In addition, ONT-based mtTGS further improves the identification of pathogenic microorganisms and also speeds up the time to diagnosis due to its long read length advantage. Tests using ONT's direct RNA sequencing and targeted sequencing show that ONT shows important potential for pathogen detection, but further development is needed. Therefore, this study suggests that pathogen detection in clinical samples targeting RNA has higher potential, especially when combined with the development of ONT and technological updates.

The above research results have been published in the journal Advanced Science in October 2021, zhao na, assistant researcher of the Key Laboratory of Pathogenic Immunity of the Institute of Microbiology, Chinese Academy of Sciences, cao jiabao, doctoral student, Xu Jiayue, assistant researcher, Dr. Liu Beibei of the Third Hospital of Peking University, Liu Bin, doctoral student of the Department of Respiratory and Critical Care Medicine of the Eighth Medical Center of the PLA General Hospital, Dr. Chen Dingqiang of the Pearl River School of Medicine of Southern Medical University as the first author, and Wang Jun, researcher of the Institute of Microbiology of the Chinese Academy of Sciences. Professor Zhou Hongwei of Zhujiang Medical College of Southern Medical University and Xie Lixin, Director of the Department of Respiratory and Critical Care Medicine of the Eighth Medical Center of the PLA General Hospital, are the corresponding authors. The research has been supported by the National Natural Science Foundation of China, the Pilot Project of the Chinese Academy of Sciences and the Key Research and Development Program of the Ministry of Science and Technology.

Thesis Link:

https://onlinelibrary.wiley.com/doi/10.1002/advs.202102593

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