Molecular identification of Penicillium marneffei using rolling circle amplification

Penicillium marneffei is the aetiological agent of a severe systemic disease in immunocompromised hosts in Southeast Asia. In the present study, we evaluated an identification method based on rolling circle amplification (RCA) enabling rapid and specific detection of single nucleotide differences. Three padlock probes were designed on the basis of the internal transcribed spacers 1 and 2 (ITS) of the rRNA operon. One of these (PmPL1) allowed specific amplification of P. marneffei DNA within one working day using a newly conceived protocol, while no cross-reactivity was observed with other fungi including related biverticillate penicillia. Amplification products can be detected by electrophoresis on agarose gel. The method provides a powerful tool for a rapid specific identification of P. marneffei in the clinical laboratory and has potential for ecological studies.