Detección de Paenibacillus larvae mediante PCR en tiempo real a partir de esporas provenientes de escamas

FERNANDEZ NJ; QUINTANA S; ALVAREZ BS; MEDICI SK; GENDE LB; EGUARAS MJ

Mar del Plata

Congreso; X Congreso de Microbiología general SAMIGE; 2014

Asociación civil de microbiología general

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.