Development and evaluation of loop-mediated isothermal amplification (LAMP) for the rapid diagnosis ofPenicillium marneffeiin archived tissue samples

Abstract

Penicillium marneffei is the etiologic agent of a severe systemic disease in immunocompromised hosts in Southeast Asia. In the present study, a novel method, known as loop-mediated isothermal amplification (LAMP), is described for the rapid and specific detection of the species, using a primer set derived from the internal transcribed spacer (ITS) region of the rRNA gene. Amplification products can be detected macroscopically by visual inspection in vials using SYBR Green I as well as by electrophoresis on agarose gel. The LAMP assay resulted in specific amplification of P. marneffei ITS using pure cultures after a 1-h reaction at 65 degrees C in a water bath; no cross-reactivity with other fungi including other biverticillate penicillia was observed. The detectable DNA limit was two copies. In addition, specific amplification was achieved using paraffin wax-embedded tissue samples from patients with penicilliosis marneffei and tissue samples from bamboo rats. The method provides a powerful tool for rapid diagnostics in the clinical lab, and has potential for use in ecological studies.