Cloning and sequencing of a part of the heat shock protein 65 gene (hsp65)of "Troaheryma whippelii" and its use for detection of "T-whippelii" in clinical specimens by PCR

Using broad-spectrum primers, we have amplified, cloned, and sequenced a 62 0-bp fragment of the "Tropheryma whippelii" heat shock protein 65 gene (hsp 65) from the heart valve of a patient with Whipple's endocarditis. The dedu ced amino acid sequence shows high similarity to those from actinobacteria, confirming that "T. whippelii" is indeed a member of this phylum. Based on the nucleotide sequence, we have developed a "T. whippelii"-specific semin ested PCR Seventeen patients shown to be positive by 16S ribosomal DNA (rDN A) PCR and/or internal transcribed spacer (ITS) PCR were also positive by h sp65 PCR All 33 control specimens from patients without Whipple's disease a nd negative for "T. whippelii" by both seminested 16S rDNA and ITS PCR rema ined negative. All amplicons digested with XhoI revealed an identical restr iction pattern. Eight of the 17 hsp65 amplicons representing all three prev iously described ITS types were sequenced. Three of the amplicons showed sl ight differences, but none of the mutations detected affected the amino aci d sequence of the corresponding protein. We conclude that the hsp65 gene is a suitable target for the specific detection of "T. whippelii." Its produc t represents a putative antigen for a future serodiagnostic assay.