Development of detection procedure for macrolide-resistant Treponema pallidum with peptide nucleic acid-mediated loop-mediated isothermal amplification assay

• Project/Area Number: 18K08446
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 54030:Infectious disease medicine-related
• Research Institution: Saitama Medical University
• Principal Investigator: Tarumoto Norihito 埼玉医科大学, 医学部, 准教授 (00746993)
• Co-Investigator(Kenkyū-buntansha): 前田 卓哉 埼玉医科大学, 医学部, 教授 (20383763) ; 早川 智 日本大学, 医学部, 教授 (30238084) ; 中山 周一 国立感染症研究所, 細菌第一部, 主任研究官 (80280767)
• Project Period (FY): 2018-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2020: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2019: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2018: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: 梅毒トレポネーマ / マクロライド / 23S rRNA / LAMP / peptide nucleic acid / マクロライド耐性 / PNAプローブ / 梅毒 / PNA-LAMP法 / マクロライド系薬耐性 / PNA

Outline of Final Research Achievements

We designed loop-mediated isothermal amplification (LAMP) primers and peptide nucleic acid (PNA) probes to detect 2058/2059th mutation in 23S rRNA Treponema pallidum. The assay showed specificity against 42 pathogens. In the presence of probe, LAMP amplified up to 10 copies/reaction using plasmids harbouring the complementary mutant sequences (A2058G or A2059G), whereas amplification was completely blocked for the wild-type sequence up to a concentration of 1000 copies/reaction. For the 66 PCR-positive clinical specimens, the overall detection rate via LAMP was 93.9 % (62/66). Amplification was successful for all 53 mutant samples and was incomplete for all nine WT samples by the PNA-mediated LAMP assays. In conclusion, we constructed a method for rapidly identifying Treponema pallidum and confirming macrolide resistance by using loop-mediated isothermal amplification (LAMP) with peptide nucleic acids (PNAs).

Academic Significance and Societal Importance of the Research Achievements

近年、梅毒罹患者が急激に増加しており、マクロライド耐性を示す23S ribosomal RNA のA2058G、A2059G変異株が流行しているため、培養法によらないマクロライド耐性のスクリーニング検査法の確立は急務の課題である。
LAMP法は特別な遺伝子抽出ステップを必要とせず、結果を目視で判定できることから、検査体制が整備されない医療機関における梅毒検査を実現できると考えられ、本研究の成果は、抗菌薬選択を含めた迅速な病原体診断を可能とするPoint Of Care Testing (POCT) として、抗菌薬適正使用の推進の一助となることが期待される。

Research Products

• [Journal Article] A novel peptide nucleic acid- and loop-mediated isothermal amplification assay for the detection of mutations in the 23S rRNA gene of Treponema pallidum (2020)
  • Author(s): Tarumoto Norihito、Imai Kazuo、Nakayama Shu-ichi、Itoda Ichiro、Sakai Jun、Murakami Takashi、Maesaki Shigefumi、Hayakawa Satoshi、Ohnishi Makoto、Maeda Takuya
  • Journal Title: Journal of Medical Microbiology Volume: 69 Issue: 12 Pages: 1339-1345
  • DOI: 10.1099/jmm.0.001275
  • Peer Reviewed
• [Presentation] PNA-LAMP法を用いたマクロライド耐性 梅毒トレポネーマの検出に関する検討 (2019)
  • Author(s): 樽本憲人、中山周一、井戸田一朗、前﨑繁文、早川智、大西真、前田卓哉
  • Organizer: 日本性感染症学会第32回学術大会
• [Presentation] A novel detection procedure for mutations in the 23S rRNA gene of Treponema pallidum with peptide nucleic acid-mediated loop-mediated isothermal amplification assay (2019)
  • Author(s): Norihito Tarumoto, Kazuo Imai, Jun Sakai, Kazuhisa Misawa, Shuichi Nakayama, Shigefumi Maesaki, Makoto Ohnishi, Takuya Maeda
  • Organizer: the 29th European Congress of Clinical Microbiology & Infectious Diseases
  • Int'l Joint Research