Use of PCR - DGGE for the discrimination of Candida species isolated from natural habitats by: Ana María Escobar Robledo

1. ANA MARIA ESCOBAR

2. Introduction: Candida
■ Candida is a genus of yeasts and is the most common
cause of fungal infections worldwide
■ The conventional techniques based on
microbiological characterization cannot differentiate
between some of the yeasts species especially that
presented in the fermentation process, therefore the
molecular techniques based on PCR amplification
could be an alternative tool for the yeast’s
identification
■ Six candidas strains were isolated from natural
environments

3. Introduction: PCR and
DGGE
■ PCR: Polymerase chain reaction amplify a single copy
or a few copies of a segment of DNA across several
orders of magnitude
- In this case we are using 600bp.
■ DGGE: (Denaturing Gradient Gel Electrophoresis)
Technique used to separate PCR amplification of
rRNA gene, comprising few nucleotide changes hat
obtained directly from natural environments.
■ The use of PCR together with DGGE based on 26s
rRNA gene sequences has been used to monitor and
characterize yeast communities in complex
ecosystems such as soil, oil, wine and food
fermentations, wastewater and human oral cavity

4. AIM
■ Search another technique for a correct identification of
candida species
■ Demonstrated that DGGE technique using NL1-GC/LS2
primers could use for the rapid discrimination of yeast strains
belonging to the same genera.

5. Materials
and Methods
Soil samples, yeast
isolation and DNA
extraction
Were collected from
different locations
It was a Clayey with an
organic matter
content of 1.3%
electricity conductivity
26s rRNA gene
amplification
Extract genomic DNA
from yeast
Were used to amplify
the region of the
D1/D2 domain of
26sRNA gene

6. Materials
and Methods
PCR – DGGE and
the use of
primers
The D1/D2 domain of the
26S rRNA gene was
amplified by a nested PCR
The first PCR was conducted
with the primers NL1 and
NL4
First PCR was diluted and further
amplified with a second PCR,
using the GC-clamp primer NL1
and reverse primer LS2
Analysis of the
DGGE profile
The GC clamp products were
separated and sequenced,
using DGGE, with a Bio-Rad
DCode universal mutation
detection system
GenBank
accession
number
The 26S rRNA nucleotide gene
sequences of yeasts reported
in this study were deposited in
the DDBJ, EMBL and GenBank

7. Fig.1

8. Fig.2

9. D
i
s
c
u
s
s
i
o
n
“Results showed that by using the NL1-GC-LS2 primer pair, DGGE technique could
detect different Candida species.”
- P. Mohammadi, A. Hamidkhani, E. Asgarani
"The used primers, NL1-GC-LS2, have been proven for their suitability for DGGE
analysis of yeast populations in milk.”
- L. Cocolin, D. Aggio, M. Manzano, C. Cantoni, G. Comi
“The use of the D1/D2 domain is generally accepted as the main tool for yeast taxonomy allowing
for identification of new ascomycetous and basidiomycetous yeasts previously not recognized as a
novel through use of conventional identification techniques.”
- C.P. Kurtzman

10. Conclusion
The study confirmed
the methods that can
be used for the
identification of yeast
isolates
1
The methods
(amplification) were
used correctly
2
DGGE could be use for
the discrimination of
yeast strains
3

11. MAPA
http://www.tratamientocandidiasis.com/pruebas-para-detectar-la-candidiasis/
https://miherbolario.com/articulos/salud/6/candida-albicans-un-hongo-oportunista