Highly Sensitive Direct Detection and Quantification of Burkholderia pseudomallei Bacteria in Environmental Soil Samples by Using Real-Time PCR

<jats:title>ABSTRACT</jats:title><jats:p>The soil bacterium and potential biothreat agent<jats:named-content content-type="genus-species">Burkholderia pseudomallei</jats:named-content>causes the infectious disease melioidosis, which is naturally acquired through environmental contact with the bacterium. Environmental detection of<jats:named-content content-type="genus-species">B. pseudomallei</jats:named-content>represents the basis for the development of a geographical risk map for humans and livestock. The aim of the present study was to develop a highly sensitive, culture-independent, DNA-based method that allows direct quantification of<jats:named-content content-type="genus-species">B. pseudomallei</jats:named-content>from soil. We established a protocol for<jats:named-content content-type="genus-species">B. pseudomallei</jats:named-content>soil DNA isolation, purification, and quantification by quantitative PCR (qPCR) targeting a type three secretion system 1 single-copy gene. This assay was validated using 40 soil samples from Northeast Thailand that underwent parallel bacteriological culture. All 26 samples that were<jats:named-content content-type="genus-species">B. pseudomallei</jats:named-content>positive by direct culture were<jats:named-content content-type="genus-species">B. pseudomallei</jats:named-content>qPCR positive, with a median of 1.84 × 10<jats:sup>4</jats:sup>genome equivalents (range, 3.65 × 10<jats:sup>2</jats:sup>to 7.85 × 10<jats:sup>5</jats:sup>) per gram of soil, assuming complete recovery of DNA. This was 10.6-fold (geometric mean; range, 1.1- to 151.3-fold) higher than the bacterial count defined by direct culture. Moreover, the qPCR detected<jats:named-content content-type="genus-species">B. pseudomallei</jats:named-content>in seven samples (median, 36.9 genome equivalents per g of soil; range, 9.4 to 47.3) which were negative by direct culture. These seven positive results were reproduced using a nested PCR targeting a second, independent<jats:named-content content-type="genus-species">B. pseudomallei</jats:named-content>-specific sequence. Two samples were direct culture and qPCR negative but nested PCR positive. Five samples were negative by both PCR methods and culture. In conclusion, our PCR-based system provides a highly specific and sensitive tool for the quantitative environmental surveillance of<jats:named-content content-type="genus-species">B. pseudomallei</jats:named-content>.</jats:p>