Rapid identification of clinical important Aspergillus using real-time PCR combined with melting curve analysis

Abstract

Abstract:

Objective Real-time PCR combined with melting curve analysis were carried out for rapid identification of clinical important Aspergillus to the species level.Method (1) PCR amplification of fungal ITS region, and then sequence alignment were done to identificate Aspergillus isolates correctly and design species specfic primers and probes. (2) Five species of clinical important Aspergillus were identified to species level according to different melting temperatures (Tm) of species specific probes in a biprobe real-time PCR.Results (1) The Tm value of A.terrus specific probe were 64.5℃ and 55.2℃, and that of A.flavus,A.fumigatus,A.nidulans and A.niger specific probe was 57.4℃,61.4℃,65.8℃,and 67.7℃ respectively.Phialophora verrucosa and Rhizopus microspores had cross reactions with A.fumigatus specific probe.(2)The detection limit of A.fumigatus was 56.8 fg, and that of A.flavus 1.11×10³ fg,A.niger 13.7 fg,A.terrus 1.23×10² fg and A.nidulans 7.80×10² fg.(3)The Tm value fluctuation was within 0.5℃ in repeated reactions.Conclusion The real-time PCR is time-saving and with high sensitivity, specificity and repeatablity. Therefore, it is helpful for diagnosis of invasive aspergillosis and the use of antifungal drugs.

Key words: real-time PCR, Aspergillus, melting curve

CLC Number:

R 379.6

SHI Jun-yan, GUO Li-na, XU Ying-chun, WANG Yao, WANG He, LIU Wen-jing. Rapid identification of clinical important Aspergillus using real-time PCR combined with melting curve analysis[J]. Chinese Journal of Mycology, 2012, 7(6): 330-334,338.

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