ZCLINICALLY PRACTICAL SEMINESTED PCR FOR BURKHOLDERIA-PSEUDOMALLEI QUANTITATED BY ENZYME-IMMUNOASSAY WITH AND WITHOUT SOLUTION HYBRIDIZATION

Diagnosis of melioidosis, an infectious disease caused by Burkholderia pseudomallei (formerly Pseudomonas pseudomallei), is made initially b y antibody testing, which is not always sensitive or specific, We have developed two seminested PCR protocols combined with enzyme immunoass ay (EIA) to detect the conserved ribosomal regulatory region of B. pse udomallei. Both PCRs used one biotinylated primer for capturing PCR pr oducts on EIA plates. One system, termed solution hybridization EIA (S HEIA), hybridized PCR products with a digoxigenin-labeled probe in sol ution, Another system, termed primer-labeled EIA (PLEIA), used a digox igenin-labeled nested primer to generate products that were directly d etected without hybridization, To prevent amplicon contamination, pre- PCR uracil DNA glycosylase treatment or post-PCR UV irradiation was in corporated into each system, By a rapid method of blood sample prepara tion for PCR, these systems had sensitivities of 75 bacteria per mi fo r SHEIA and 300 bacteria per mi for PLEIA. No nonspecific amplificatio n of other bacterial DNAs was detected, This seminested PCR coupled wi th SHEIA or PLEIA fulfills all the requirements for a diagnostic test to be used in developing countries where B. pseudomallei is endemic.