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PLoS One. 2014 May 16;9(5):e98110. doi: 10.1371/journal.pone.0098110. eCollection 2014.

Identification of clinically relevant fungi and prototheca species by rRNA gene sequencing and multilocus PCR coupled with electrospray ionization mass spectrometry.

Author information

1
Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China.
2
Department of Medical Microbiology and Parasitology, Shanghai Medical College of Fudan University, Shanghai, China.
3
Mycology Lab, Department of Dermatology, Huashan Hospital, Fudan University, Shanghai, China.

Abstract

BACKGROUND:

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.

METHODS:

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.

RESULTS:

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.

CONCLUSIONS:

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.

PMID:
24835205
PMCID:
PMC4024029
DOI:
10.1371/journal.pone.0098110
[Indexed for MEDLINE]
Free PMC Article

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