One-Step Multiplex PCR for Simultaneous Detection and Identification of Eight Medically Important <i>Candida</i>Species

Recently, the incidence of Candida infections has substantially increased. Conventional identification methods for Candida species are technically difficult to conduct and cannot accurately distinguish each species. The purpose of the present study was to design primers to identify and detect simultaneously eight medically important Candida species using one-step multiplex PCR. PCR primers were designed based on partial sequences of intergenic spacer (IGS) and internal transcribed spacer (ITS) genes of eight medically important Candida species. These primers were able to distinguish each Candida species and did not display cross-reactivity with representative Candida species other than the eight Candida species. Moreover, our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.

The Effectiveness of a Portable Fluorescence Spectrophotometer for Early Detection of Oral Cancer

Purpose: To investigate whether photodynamic diagnosis (PDD) using a portable fluorescence spectrophotometer (FC-1) can easily and objectively discriminate between normal and tumor cells at the dental chairside, and to further compare it with PDD that requires speculum examination by focusing on protoporphyrin IX (PPIX). Methods: Three cell lines (2 human oral squamous cell carcinoma-derived cell lines, HSC-2 and HSC-3 cells, and oral keratinocytes, HOK cells) were cultured. 5-Aminolevulinic acid hydrochloride (5-ALA) and deferoxamine mesylate (DFO) were mixed in DMEM, and the mixture was set to Control (DMEM only) and PDD (5-ALA+DFO) groups. And then, a fluorescence was measured under room temperature (RT) and 37°C (Incubation) by using FC-1. In this study, the two conditions were combined with the Control and PDD groups to form the Control/RT, Control/Incubate, PDD/RT, and PDD/Incubate groups. Additionally, the amount of singlet oxygen (1O2) generated by irradiation with 405 nm LED was measured using electron spin resonance spectroscopy to detect PPIX in the cell supernatant after 24 hours. Results: In HSC-2 and HSC-3, the fluorescence intensity values increased significantly at 2 hours between the Control/RT and PDD/RT groups. In addition, there was a significant difference between HSC-2 and HSC-3 compared to HOK. In all cell lines, the fluorescence intensity values of the PDD/Incubate group were significantly higher than those of the PDD/Control group. The amount of 1O2 generated by 405 nm LED irradiation was higher in the cell supernatants of all cell lines in the order of Control/RT < Control/Incubate < PDD/RT < PDD/Incubate group, and HSC-3 in the PDD/Incubate group showed a significant increase compared to HOK. Conclusion: It is suggested that PDD using FC-1 can clearly distinguish between normal cells and tumor cells in vitro studies using cell lines at 2 hours under 37°C, and it can detect not only intracellular PPIX, but also extracellular PPIX.