# Molecular Techniques and Target Selection for the Identification of _Candida_ spp. in Oral Samples
## 4 DNA Target Selection for the Identification of _Candida_ spp.  
The selection of a suitable molecular target in the diagnosis of any infectious disease is of pivotal importance. Regardless of the molecular technique, an accurate diagnostic strongly depends on the molecular target specificity and its discriminatory capacity. _ITS_ has been selected as the barcode of choice for the identification of fungal species [160]. The same happens for _Candida_ spp., with _ITS1_ and _ITS2_ being widely used. Nonetheless, there are some alternatives available. The _MP65_ gene plays a role in maintaining cell wall integrity, adherence to epithelia, and biofilm formation in C. _albicans_[57, 161]. DNA  
\begin{table}
\begin{tabular}{c c c} \hline
**Molecular Technique** & **Advantages** & **Disadvantages** \\ \hline \multirow{2}{*}{Conventional PCR} & Low cost compared with other PCR-based techniques, low in complexity & Requires an additional amplification detection step \\ \hline \multirow{2}{*}{RT-PCR} & Real-time detection and quantification, no additional step of detection & Expensive equipment \\ \hline \multirow{2}{*}{Nested PCR} & Sequence primers available for different gene targets & Requires more reagents than other PCR-based techniques and an additional set of primers. Prone to contamination \\ \hline \multirow{2}{*}{Multiplex PCR} & \multirow{2}{*}{Detection of multiple gene targets} & Requires an additional amplification detection step \\ \hline \multirow{2}{*}{RFLP} & High specificity, Sequence primers available for different gene targets & High-cost enzymes and storage, requires an additional amplification detection step. \\ \hline \multirow{2}{*}{HRMA} & Low risk of contamination when compared to RFLP or nested PCR & Not capable of distinguishing between some _Candida_ spp. \\ \hline \multirow{2}{*}{MLST} & High discriminatory power, useful for epidemiological studies, and evolution of virulence-associated mechanisms & High cost \\ \hline \multirow{2}{*}{LAMP} & High specificity, high sensitivity, high-speed, low-cost equipment. & Requires attention in optimization and primer design \\ \cline{1-1}  & Several methods for amplification detection & Requires attention in optimization and primer design \\ \hline \multirow{2}{*}{NGS} & High discriminatory power, large dataset allows for additional analysis & High cost, complex results which require specialized analysis \\ \hline \multirow{2}{*}{PNA FISH} & Rapid identification of _Candida_ spp. in blood cultures & Results visualization adds a cost to equipment \\ \hline \multirow{2}{*}{MALDI-TOF MS} & \multirow{2}{*}{High specificity, rapid identification} & High cost of equipment, lack of spectra characterization for comparison \\ \cline{1-1}  & & \\ \end{tabular}
\end{table}
Table 2: Resume of advantages and disadvantages for currently used molecular techniques in the identification of _Candida_ spp.  
topoisomerase II coding gene is used due to its highly conserved regions, separated by species-specific regions [72].  
Other molecular targets related to virulence, pathogenesis, and antifungal resistance can also be useful as a complement in the identification of _Candida_ spp. The ergosterol biosynthetic genes, _ERG3_, _ERG5_, _ERG6_, and _ERG11_, are common targets for the detection of antifungal drug resistance to amphoteric B (AMB) [17, 162]. A study also showed that potential mutations in the _ERG5_ gene confer resistance against AMB [163].  
Several genes are useful for MLST, although their use is not widespread. Sequencing can contribute to improving and simplifying current MLST strategies, as recently described for _C. glabrata_ and _C. albicans_[164, 165]. Additionally, as mentioned in the NGS Section 3.10, sequencing is relevant for metagenomics studies, the identification of drug resistance, and the diagnosis of rare infections. This is crucial for difficult-to-identify or emerging pathogens, such as _C. auris_[166], or for adequate therapeutic directions in drug-resistant species [161].