Validation of an rpoB gene PCR assay for detection of Tropheryma whipplei: 10 years' experience in a National Reference Laboratory

Abstract

The performance of a real-time PCR assay targeting the Tropheryma whipplei rpoB gene was evaluated using test strains and 1,236 clinical specimens in a national reference laboratory. The novel rpoB-PCR assay proved to be specific, revealed improved analytical sensitivity, and substantially accelerated detection of T. whipplei DNA in clinical specimens.

Fig 1

Assessment of the sensitivity of the rpoB gene real-time PCR assay. Using serial dilutions of a suspension of T. whipplei strain Twist ATCC VR-1528 (1 to 8, corresponding to 1,740,000 to 0.174 microorganisms per 5 μl), the sensitivity of the real-time PCR assay was determined at 17.4 microorganisms per 5-μl suspension (dilution 6) used for DNA extraction (3,480 microorganisms/ml). All PCRs were performed in triplicate. Images show dilutions of T. whipplei stained with SYTO 9. At higher resolution (upper left image), single T. whipplei cells are visible but most bacteria form inconclusive clusters. Therefore, images at lower resolution were used for quantification through digital image analysis using daime software as described previously (13, 14). Representative images of dilutions 1 to 3 are shown (3 upper right images). Bars, 5 μm.