Application of Reverse Transcriptase-PCR-DGGE as a rapid method for routine determination of Vibrio spp. in foods- BIOLOGÍA MOLECULAR- Laura María López
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Application of Reverse Transcriptase-PCR-DGGE as a rapid method for routine determination of Vibrio spp. in foods- BIOLOGÍA MOLECULAR- Laura María López
1. L A U R A M A R Í A L Ó P E Z F R A N C O
B I O L O G Í A M O L E C U L A R / 3 E R S E M E S T R E / U P B
2. INTRODUCTION
Family of Proteobacteria
Gram-negative organisms and
facultative anaerobes, capable of
fermentation.
They contain oxidase and have one or
more flagella, which are generally polar.
Distributed in the environment,
specifically in seawater, estuaries and
freshwater environments,
Contribute to the cycle of organic and
inorganic compounds
Implicated intestinal and extraintestinal
tract infections, related to water and
shellfish
These contamination bacteria can be
controlled and is limited to fish and
fishery products
VIBRIONACEAE
http://redbiobancos.es/Pages/Docs/PNT_Acidos_Nucleicos.pdf
3. INTRODUCTION
PCR DGGE
PCR is an enzymatic process in
which a specific region of DNA is
replicated over and over again to
yield many copies of a particular
sequence
Denaturing gradient gel
electrophoresis (DGGE) use
either a chemical gradient or a
temperature gradient to
separate samples of DNA (or
RNA) to an electrophoresis gel
that contains a denaturing agen
PCR-DGGE techniques, were used for the detection of Vibrio in food
4. OBJECTIVE
The aim of this work was (i) to evaluate the potential
of PCRDGGE and Reverse Transcriptase-PCR
DGGE (Rev-T-PCR-DGGE) as Vibrios detection
technique and (ii) to evaluate effects of food
processing conditions on Vibrios and their detection
by Rev-T-PCR-DGGE and (iii) to apply this
technique in screening food samples and comparing
its efficiency with that of the standard methods.
5. MÉTODOS
CEPAS
Cepas de microorganismos se mantuvieron a
temperaturas específicas, con nutrientes y un
tiempo adecuado para el cultivo. Luego fueron
almacenadas en condiciones ideales
Centrifugación- sedimentación de células
Extracción ARN y DNA para análisis Rev-T-PCR-DGGE y PCR-
DGGE
suspensiones celulares con diferentes poblaciones bacterianas
Determinación DGGE de vibrios en alimentos (calamares) se
descontaminaron y se agregaron varias especies de vibrios
CONTROL
muestra de calamar sin cocinar
TABLA 1
Cultivos de referencia de
cepas bacterianas y sus
fuentes.
9. RESULTADOS
FIGURA 1
Análisis de Rev-T-PCR-DGGE de 16S
rDNA amplificado de 10 cultivos de Vibrio
de referencia.
FIGURA 2
Análisis de Rev-T-PCR-DGGE de 16S rDNA amplificado de
diferentes condiciones de células Vibrios.
(a) muestra de células viables (VC), (b) muestra de células
lesionadas (IVC) y (c) muestra de célula lesionada pre-
enriquecida (PIVC).
11. RESULTADOS
FIGURA 3
Análisis de Rev-T-PCR-DGGE de 16S rDNA
amplificado de células de Vibrio paraheamolyticus
enriquecidas en muestra de mariscos
FIGURA 4
Vibrios asociados con los mariscos según lo
determinado por los métodos estándar y
Rev-TPCR-DGGE.
12. RESULTADOS
TABLA 3
Límites de detección de PCR-DGGE (ADN) y Rev-T-
PCR-DGGE (ADNc) en poblaciones mixtas de
especies de Vibrio.
13. DISCUSION
AUTHOR CONCEPT YES OR NOT
Eiler and
Bertilsson
The DNA amplified from the 16sDNA region of Vibrio
parahaemolyticus and Vibrio harveyi could not be discriminated on
DGGE gel since these two species had a very close evolution. The
sequences of DNA in this region of both species were therefore
absolutely conserved and having similar migratory pattern
Szaboa and
Mackkey
the effect of heating process on conformation of Salmonella
Enteritidis RNA. This process could greatly alter RNA conformation,
and thus inhibit the conversion of RNA to cDNA. Therefore, heat-
treated Salmonella Enteritidis could not be detected by Rev-T-PCR
Tamura et al In this study; V. metschnikovii, Morganella morganii ssp. morganii
and Aeromonas salmonicida from GenBank of National Center for
Biotechnology (NCBI) were used. Those nucleotide sequences were
used to construct phylogenetic tree
Eiler and
Bertilsson
The PCR technique could efficiently differentiate Vibrios with limit of
detection of approximately 200 cells per single population in food
14. CONCLUSIONS
The Rev-T-PCR-DGGE method in the identification of vibrios in foods has a limit of
detection lower than the standard, it is observed that the sensitivity of the method
increases when the sample is pre-enriched and it is represented in DNA band intensity,
which indicates that it has greater sensitivity to identify strains.
RNA and the number of rRNA operons represent an individual property of bacterial
species; the use of these as additional markers in Rev-PCR-DGGE analysis would help to
a greater identification and sensitivity when studying Vibrios