Specific identification of Penicillium marneffei by a polymerase chain reaction/hybridization technique

Penicillium marneffei has been described recently as a cause of an emerging mycotic infection in HIV-infected patients. A PCR/hybridization assay was developed to rapidly identify this pathogen. The nucleotide sequence of the 631-bp region of 18S ribosomal DNA of P marneffei was determined using the standard dideoxy chain termination method. An oligonucleotide probe was designed on the basis of the analysed sequences of P marneffei and 18S rDNA sequences of other fungi in the GenBank database. A 631-bp PCR product was amplified using primers RRF1 and RRH1 from P marneffei and seven other fungi, Penicillium spp., Aspergillus fumigatus, A. flavus, Histoplasma capsulatum, Cryptococcus neoformans, Candida albicans and C. krusei. A 15 oligonucleotide segment (Pm3) which was specific for P marneffei was synthesized and used as a probe. Only the PCR products of P marneffei isolates hybridized with the Pm3 oligonucteotide probe. The sensitivity of the assay was approximately 0.5 pg/μl and 0.1 pg/μl of DNA by PCR and Southern hybridization, respectively. The usefulness of this method as a diagnostic tool will require further studies.