Novel primer sequences were designed and the seminested PCR was capable of detecting a larger number of Candida species by using annealing temperatures of either 60 °C or 65 °C in the second round of the seminested PCR.

At the Universiti Putra Malaysia (Serdang, Selangor, Malaysia) scientists designed species-specific reverse primers (SSRPs) of 10 Candida species by retrieving at least three internal transcribed spacer (ITS) gene sequences of each species. DNA amplification was performed in a MasterCycler gradient thermocycler. To test the specificity of the method, 27 Candida clinical isolates, 10 Candida American Type Culture Collection (ATCC; Manassas, VA, USA) strains, six Aspergillus ATCC strains, one Cryptococcus neoformans clinical isolate, and one human genomic DNA from an apparently healthy human volunteer were tested.

After optimizing the PCR conditions on the MasterCycler thermocycler (Eppendorf; Hamburg, Germany), the targeted 10 Candida species showed an expected approximate size of PCR amplicons after the second round of PCR with their respective SSRPs. A similar observation was seen when tested on the 27 Candida clinical isolates of various species in the analytical specificity testing. The limit of detection for all Candida species ranged from 0.26 pg to 0.46 pg, except for Candida guilliermondii and Candida kefyr where a 10-fold increase in DNA amount was required.

The authors concluded that the advantages of the high sensitivity feature of seminested PCR were successfully applied, while the drawback usually faced, especially on the limited capability of detecting more species, was overcome. Its usage is favorable in invasive types of fungal infection cases since early rapid detection can be done. This helps to lessen the rate of drug resistance emergence and the precarious use of costly and toxic antifungal drugs for prophylaxis and treatment purposes. The study was published on December 10, 2011, in the journal Diagnostic Microbiology and Infectious Disease.

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Universiti Putra Malaysia
American Type Culture Collection
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