INVESTIGADORES
FERNANDEZ Natalia Jorgelina
congresos y reuniones científicas
Título:
REAL-TIME PCR DETECTION OF Paenibacillus larvae DNA FROM SPORES OF SCALE SAMPLES
Autor/es:
NATALIA J. FERNÁNDEZ ; SILVINA QUINTANA ; BRENDA S. ÁLVAREZ; SANDRA K. MEDICI ; LIESEL B. GENDE; MARTÍN J. EGUARAS
Lugar:
Mar del Plata
Reunión:
Congreso; X CONGRESO ARGENTINO DE MICROBIOLOGIA GENERAL SAMIGE; 2014
Institución organizadora:
Sociedad Argentina de Microbilogía General
Resumen:
The Gram-positive bacterium Paenibacillus larvae is a major pathogen
of Apis mellifera, causing the
disease known as American foulbrood. This condition affects the larval stage
and causes, as a final step desiccation of the larvae leaving only a scale,
which contains millions of bacterial spores. Spores of the microorganism
initiate the infectious stage and are the major vectors for the spread of the
disease. The delay in diagnosis causes the collapse of infected hives,
therefore the development of a fast and reliable method of detection will be of
great help to prevent the spread of the disease. The objective of this work was
to develop a real-time PCR methodology for the detection of P. larvae DNA from spores from scales
samples. Methodology: Validation of real-time
PCR reactions for the detection of P.
larvae DNA was performed with DNA extracted from pure cultures from
Arthropods Laboratory strain collection, with primers that amplify a 380 bp fragment
of the bacterial 16S rRNA sequence. A methodology for DNA extraction from scales
was optimized by using the commercial kit Multisource Genomic DNA Miniprep
AxyPrep. To verify the success of DNA extraction from the samples and lack of
inhibition in the PCR reactions DNA amplifications of A. mellifera beta actin gene were performed. In those suitable
samples (beta actin Ct values < 35) PCR reactions for detection of P. larvae were carried out using EvaGreen
as fluorescent intercalating dye, in a final volume of 20 µl. Detection of the
amplified product was monitored on a Rotor Gene Q thermocycler. Results: It was possible to apply a real-time PCR
method for the detection of P. larvae
DNA from bacterial isolates and also in scale samples. Thus, the real-time PCR
in a few hours could determine the presence or absence of P. larvae DNA in isolates and scale samples. This is the first
report of P. larvae detection by real-time
PCR directly from scales.