Wang Xuan, Fu Yong-Feng, Wang Rui-Ying, Li Li, Cao Ya-Hui, Chen Yan-Qiong, Zhao Hua-Zhen, Zhang Qiang-Qiang, Wu Ji-Qin, Weng Xin-Hua, Cheng Xun-Jia, Zhu Li-Ping
Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China.
Department of Medical Microbiology and Parasitology, Shanghai Medical College of Fudan University, Shanghai, China.
PLoS One. 2014 May 16;9(5):e98110. doi: 10.1371/journal.pone.0098110. eCollection 2014.
Multilocus PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) is a new strategy for pathogen identification, but information about its application in fungal identification remains sparse.
One-hundred and twelve strains and isolates of clinically important fungi and Prototheca species were subjected to both rRNA gene sequencing and PCR/ESI-MS. Three regions of the rRNA gene were used as targets for sequencing: the 5' end of the large subunit rRNA gene (D1/D2 region), and the internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions). Microbial identification (Micro ID), acquired by combining results of phenotypic methods and rRNA gene sequencing, was used to evaluate the results of PCR/ESI-MS.
For identification of yeasts and filamentous fungi, combined sequencing of the three regions had the best performance (species-level identification rate of 93.8% and 81.8% respectively). The highest species-level identification rate was achieved by sequencing of D1/D2 for yeasts (92.2%) and ITS2 for filamentous fungi (75.8%). The two Prototheca species could be identified to species level by D1/D2 sequencing but not by ITS1 or ITS2. For the 102 strains and isolates within the coverage of PCR/ESI-MS identification, 87.3% (89/102) achieved species-level identification, 100% (89/89) of which were concordant to Micro ID on species/complex level. The species-level identification rates for yeasts and filamentous fungi were 93.9% (62/66) and 75% (27/36) respectively.
rRNA gene sequencing provides accurate identification information, with the best results obtained by a combination of ITS1, ITS2 and D1/D2 sequencing. Our preliminary data indicated that PCR/ESI-MS method also provides a rapid and accurate identification for many clinical relevant fungi.
多位点聚合酶链反应结合电喷雾电离质谱法（PCR/ESI-MS）是一种新的病原体鉴定策略，但关于其在真菌鉴定中的应用信息仍然稀少。
对112株具有临床重要性的真菌和原壁菌属菌株及分离株进行rRNA基因测序和PCR/ESI-MS检测。rRNA基因的三个区域用作测序靶点：大亚基rRNA基因的5'端（D1/D2区域）以及内部转录间隔区1和2（ITS1和ITS2区域）。通过结合表型方法和rRNA基因测序结果获得的微生物鉴定（Micro ID）用于评估PCR/ESI-MS的结果。
对于酵母和丝状真菌的鉴定，三个区域的联合测序表现最佳（种水平鉴定率分别为93.8%和81.8%）。酵母通过D1/D2测序（92.2%）以及丝状真菌通过ITS2测序（75.8%）实现了最高的种水平鉴定率。两种原壁菌属物种可通过D1/D2测序鉴定到种水平，但不能通过ITS1或ITS2鉴定。对于PCR/ESI-MS鉴定范围内的102株菌株及分离株，87.3%（89/102）实现了种水平鉴定，其中100%（89/89）在种/复合体水平上与Micro ID一致。酵母和丝状真菌的种水平鉴定率分别为93.9%（62/66）和75%（27/36）。
rRNA基因测序提供了准确的鉴定信息，通过ITS1、ITS2和D1/D2测序相结合可获得最佳结果。我们的初步数据表明，PCR/ESI-MS方法也为许多临床相关真菌提供了快速准确的鉴定。
