Clinical identification of common species of dermatophytes by PCR and PCR-RFLP

Abstract

To find a fast and efficient way of identifying seven common dermatophytes in clinical practice, we used the techniques of polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) targeting Topoisomerase II gene. The DNA of 7 dermatophytes, along withCandida albicans, Aspergillus terreus andAspergillus flavus were amplified by consensus primer dPsDl. They were then subjected to a second PCR with primers dPsD2 and species-specific primers PsT and PsME separately. 6 of the products generated by dPsD2 were digested with restriction enzyme Hinc II. DNA fragments of 3390 bp and 2380 bp was amplified by using consensus primer dPsD1 and dPsD2 from the genomic DNA of each dermatophyte species separately. By combining the results of the two species-specific primer sets (PsT and PsME), all species of dermatophyte yielded unique sizes-set of PCR products expect forT. mentagrophytes andT. tonsurans. From the restriction profiles of Hinc II, 6 of the 7 dermatophytoses were diagnosed to species level includingT. mentagrophytes andT. tonsurans. By combining the results of the PCR and PCR-RFLP, the 7 common dermatophytes can be identified to species level. It is conclude that the multiplex PCR and PCR-RFLP identification targeting the DNA topoisomerase II gene is rapid and efficient.

Keywords

dermatophyte / topoisomerase / Nested PCR / Multiplex PCR / RFLP

References

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