Detection of Bacillus anthracis DNA by LightCycler PCR

Abstract

Anthrax is a zoonotic disease that is also well recognized as a potential agent of bioterrorism. Routine culture and biochemical testing methods are useful for the identification of Bacillus anthracis, but a definitive identification may take 24 to 48 h or longer and may require that specimens be referred to another laboratory. Virulent isolates of B. anthracis contain two plasmids (pX01 and pX02) with unique targets that allow the rapid and specific identification of B. anthracis by PCR. We developed a rapid-cycle real-time PCR detection assay for B. anthracis that utilizes the LightCycler instrument (LightCycler Bacillus anthracis kit; Roche Applied Science, Indianapolis, Ind.). PCR primers and probes were designed to identify gene sequences specific for both the protective antigen (plasmid pX01) and the encapsulation B protein (plasmid pX02). The assays (amplification and probe confirmation) can be completed in less than 1 h. The gene encoding the protective antigen (pagA) was detected in 29 of 29 virulent B. anthracis strains, and the gene encoding the capsular protein B (capB) was detected in 28 of 29 of the same strains. Three avirulent strains containing only pX01 or pX02, and therefore only pagA or pagB genes, could be detected and differentiated from virulent strains. The assays were specific for B. anthracis: the results were negative for 57 bacterial strains representing a broad range of organisms, including Bacillus species other than anthracis (n = 31) and other non-Bacillus species (n = 26). The analytical sensitivity demonstrated with target DNA cloned into control plasmids was 1 copy per microl of sample. The LightCycler Bacillus anthracis assay appears to be a suitable method for rapid identification of cultured isolates of B. anthracis. Additional clinical studies are required to determine the usefulness of this test for the rapid identification of B. anthracis directly from human specimens.

FIG. 1.

(A) Representative LightCycler assay for detection of the pagA gene by using FRET probes. Shown are the results from three samples. The solid line indicates a DNA preparation from a B. anthracis strain with a relatively high concentration of target DNA, the dotted line indicates a positive plasmid control, and the dashed line indicates a DNA preparation from another B. anthracis strain with a relatively low concentration of target DNA. (B) Confirmatory analysis (quality control step) by evaluation of melting curves. In this case, the negative derivative of fluorescence, −d(F2/F1), over the derivative of time, dT, is compared to temperature. F1 refers to the F1 channel of the LightCycler that detects the 530-nm light emitted by the fluorescein fluorophore. F2 refers to the F2 channel of the LightCycler that detects the 640-nm light emitted by the LC (LightCycler)-Red640 acceptor fluorophore. The same melting curve occurs for the two samples and the positive control shown in panel A, indicating that DNA with identical sequences has been amplified for each of the three samples.