- 期刊
以巢式複合即時PCR技術檢測肺炎黴漿菌抗藥性菌株
Detection of Macrolide Resistance Strain of Mycoplasma Pneumoniae by a Novel Nested Duplex Real-Time PCR Assay
摘要
肺炎黴漿菌（Mycoplasma pneumoniae）為引發原發性非典型肺炎（CAP，community-acquired pneumonia）的主因之一，治療肺炎黴漿菌感染的藥物通常為巨環類抗生素（macrolide antibiotics，ML），此菌株23S rRNA基因某些位點產生單點突變時，會對巨環類抗生素產生不同程度之抗藥性，其中A2063G及A2064G為最常見且會導致用藥無效的突變位點，影響臨床藥物治療之成效。許多文獻顯示各國感染肺炎黴漿菌抗藥性菌株的比例逐年增加，但傳統藥物敏感性檢測方法費工耗時，目前尚無敏感度高、快速的分子檢驗技術可直接檢測臨床檢體是否有抗藥性。因此本研究之目的乃建立快速且高敏感度的巢式複合即時PCR（Nested Duplex Real-time PCR）檢測技術，針對多種抗藥性位點設計高專一性之雙重螢光探針，用以區分抗藥性菌株及非抗藥性菌株。研究結果顯示，此系統不需經過培養，可以直接從臨床檢體中檢測肺炎黴漿菌及抗藥性基因位點，抗藥性菌株及非抗藥性菌株之敏感度皆可達1 copy／μL。我們分析76例已確診為肺炎黴漿菌感染案例，結果顯示巢式複合即時PCR可以檢測出23例抗藥性菌株、53例非抗藥性菌株，而傳統基因定序則僅鑑定出20例抗藥性菌株、49例非抗藥性菌株。我們更進一步檢測2010年6月至2012年5月肺炎黴漿菌感染之臨床檢體共181例，其中16.6%（31／181）為抗藥性菌株，包含1例A2063C、1例A2064G及29例A2063G，且抗藥性菌株數量有上升的趨勢。因此，使用此快速且高敏感度之抗藥性菌株鑑定技術，不但能提供臨床治療參考之訊息，而且可以用來進行台灣地區流行病學之分析調查。
並列摘要
Mycoplasma pneumoniae is a common cause of respiratory tract infections of community-acquired pneumonia. Macrolide antibiotics (ML) are frequently administered to treat mycoplasmal pneumonia. Specific sites of the 23S rRNA mutation contribute to ML resistance in M. pneumoniae, especially mutations at A2063 or A2064, which made an impact on clinical medication.Recent reports indicated that MLs-resistant (MR) strains have increased. However, detecting MR by traditional assay is time consuming. Current molecular assays for resistance-associated mutations are low sensitivity and could not be performed directly from clinical specimen. To this aim, we constructed a novel nested duplex real-time PCR method to detect multiple resistant strains. By using the assay, we can distinguish non-resistant strain and resistant strains with different fluorescent probes simultaneously. The data showed this method has high sensitivity (up to 1 copy/μL) and specificity, and could be performed directly in clinical samples without culturing. Among the 76 specimens with confirmed M. pneumoniae infection, 23 MR strains and 53 non-MR strains were identified by nested duplex real-time PCR, while only 20 MR strains and 49 non-MR strains were detected by Sanger sequencing. Moreover, we analyzed 181 clinical samples with M. pneumoniae infection between June 2010 and May 2012, 16.6% (31/181) of which were identified with drug-resistant strains, including one strain of A2063C and A2064G, and 29 strains of A2063G.The data showed the number of MR strain of M. pneumoniae has the tendency to increase. By using the rapid and high sensitivity assay in our study, appropriate information can be provided for clinical treatment and epidemiology can also be surveyed for drug-resistant strain of M. pneumoniae in Taiwan.
延伸閱讀
- 宋洽興、張淑萍、方鈺評、趙崟呈、李俊毅（2020）。中台灣區域教學醫院之PCR陽性肺炎黴漿菌樣本的分子特徵探討。秀傳醫學雜誌，19(1)，77-88。https://doi.org/10.3966/156104972020061901007
- Liang, C. A. (2017). 內科加護病房碳青黴烯類抗藥性鮑氏不動桿菌肺炎:流行病學調查與不同抗生素治療組合之有效性探討 [master's thesis, Kaohsiung Medical University]. Airiti Library. https://www.airitilibrary.com/Article/Detail?DocID=U0011-1708201721592000
- 陳俊堂（2014）。碳青黴烯類抗生素抗藥性克雷伯氏肺炎桿菌莢膜型分析和抗藥性機制之探討〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2014.10656
- 陳珊民（2021）。新冠肺炎PCR檢測與抗原快篩探討〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU202104244
- Chan, W. K. (2012). Development of real-time PCR and pyrosequencing for detection of macrolide resistance of mycoplasma pneumoniae directly from clinicalspecimens [master's thesis, The University of Hong Kong]. Airiti Library. https://www.airitilibrary.com/Article/Detail?DocID=U0029-1812201200020120