Development of a 5′-Nuclease Real-Time PCR Assay Targeting fliP for the Rapid Identification of Burkholderia mallei in Clinical Samples

BACKGROUND: Burkholderia mallei is a potential biological agent that causes glanders or farcy in solipeds, a disease notifiable to the Office International des Epizooties (OIE). The number of reported outbreaks has increased steadily during the last decade, but diagnosis is hampered by the low bacterial load in infected tissues and excretions. METHODS: We developed a B. mallei-specific 5'-nuclease real-time PCR assay that targets the fliP gene of B. mallei and includes an internal amplification control. Specificity was assessed with 19 B. mallei strains, 27 Burkholderia pseudomallei strains, other Burkholderia strains of 29 species, and clinically relevant non-Burkholderia organisms. RESULTS: Amplification products were observed in all B. mallei strains but in no other bacteria. The linear range of the B. mallei real-time PCR covered concentrations from 240 pg to 70 fg of bacterial DNA/reaction. The detection limit was 60 fg of B. mallei DNA. The clinical applicability of the assay was demonstrated by use of organ samples from diseased horses of a recent outbreak that was reported to the OIE by the United Arab Emirates in 2004. CONCLUSIONS: Compared with conventional PCR, our rapid 5'-nuclease real-time PCR assay for the specific identification of B. mallei has a lower risk of carryover contamination and eliminates the need for post-PCR manipulations. This real-time PCR assay also shortens the turnaround time for results and has the potential for automation.