Introduction:

Bacillus anthracis is the most employed biological warfare agent in the world. However, in biological defense laboratories, many times it is convenient to use other bacteria similar to Bacillus anthracis but less dangerous or non-pathogenic. One of the main surrogates of Bacillus anthracis is Bacillus thuringiensis, due to its high homology with B. anthracis and its null pathogenicity for humans.

Objective:

The objective of the present study is to develope and validate a real-time PCR for the rapid identification of Bacillus thuringiensis DNA, biological agent very often employed in the training of the Operative Units of CBRN sampling of the Armed Forces.

Methods:

The identification of Bacillus thuringiensis has been performed by the amplification and detection with a hydrolysis probe of a 69 base pairs fragment of the cry1A gene, which is specific for this bacterium. After optimizing the amplification conditions by testing three different hybridization / extension temperatures, the validation of the new developed method was carried out.

Results:

The new developed real-time PCR showed an efficiency of 93%, as well as a high linearity (regression coefficient R20.9993). The limit of detection at 95% probability was 13 genome equivalents per reaction. Both the inclusiveness and the exclusivity of the method were 100%.

Conclusions:

The molecular method developed at the Molecular Biology Laboratory of INTA allows the rapid identification of Bacillus thuringiensis DNA, surrogate of Bacillus anthracis, with a high analytical sensitivity and specificity.