Detection of Bacillus anthracis spores in soil by PCR

Abstract

A polymerase chain reaction (PCR)-based protocol was developed for rapid detection of Bacillus anthracis spores in various types of soil. Eleven soil samples representing miscellaneous vegetation types were mixed with anthrax spores and incubated overnight. In no case could the spores of the mixtures be detected directly by PCR. The PCR was based on primers derived from the capsule-encoding cap genes of the pXO2 plasmid. Therefore, the focus of the study became to develop a suitable method for preparation of DNA that would allow rapid detection of the anthrax spores. Various published techniques were investigated, but none enabled detection of the spores from a majority of the samples. A successful protocol was eventually developed based on a combination of a published preparative technique and a form of nested PCR. First, amplification by PCR was performed with a degenerate primer and then samples were diluted and subjected to a second round of amplification using the B. anthracis-specific cap primers. The protocol is rapid and resulted in DNA of sufficient purity from all of the 11 investigated soil samples to permit detection of B. anthracis spores.