Study of Rapid Diagnostic Method for Bone and soft Tissue Infection by Post-Sonication Next-Generation Sequencing and Rapid PCR

2021 Fiscal Year Final Research Report

• Project/Area Number: 20K18013
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 56020:Orthopedics-related
• Research Institution: Kansai Medical University
• Principal Investigator: TOYAMA Takeyasu 関西医科大学, 医学部, 助教 (50829306)
• Project Period (FY): 2020-04-01 – 2022-03-31
• Keywords: 骨軟部組織感染症 / 迅速 PCR / 次世代シーケンサー / 超音波処理法 / 骨髄炎 / 化膿性腱鞘炎 / 全自動遺伝子抽出装置

Outline of Final Research Achievements

The next-generation sequencer (NGS) is useful for the detection of uncultured microbes. However, the quality of NGS data depends on the amount of microbial DNA, and accurate diagnosis with NGS sometimes becomes difficult such as in the field of orthopedics. The purpose of this research was to verify the ability and limit of qualitative and quantitative diagnoses with rapid PCR in bacterial infection by NGS with automatic extraction device with DNA concentration extraction mechanism.The single measurement value of the intraclass correlation coefficient of Cq by magLEAD method was higher than the Qiagen method. Our findings show that the concentrated extraction of DNA by an automated device can contribute to stable results and an increase in the detection limit as a clinical genetic diagnostic tool for infection.

Free Research Field

整形外科 (手の外科)

Academic Significance and Societal Importance of the Research Achievements

現在課題として挙げられる遺伝子抽出問題に関し、 DNA抽出の一般的方法である QIAamp DNA mini kit (Qiagen)(QIAGEN法)を用いたものでは、時間と手間を要し、全自動機器 magLEADでは、作業効率の改善および精度の向上が確認できた。本研究は、骨軟部組織感染症における超音波処理法の有用性の成果報告に至った。迅速 PCRの検討は、既存の特異的領域 PCRに加え、変異遺伝子を多く含む 16sDNAの定量 PCRを行えるようプロトコール構築を行った。nanoporeシーケンサーはコントロールを用いたプロトコール検討を行ない、整形外科骨軟部組織感染症 NGSへ発展できる。