Development of a Quantitative Real-Time Polymerase Chain Reaction Assay Specific for Orientia Tsutsugamushi
NAVAL MEDICAL RESEARCH CENTER SILVER SPRING MD
Pagination or Media Count:
Two specific and sensitive polymerase chain reaction PCR assays were developed to detect and quantitate Orientia tsutsugamushi, the agent of scrub typhus, using a portion of the 47-kD outer membrane protein antigen high temperature requirement A gene as the target. A selected 47-kD protein gene primer pair amplified a 118-basepair fragment from all 26 strains of O. tsutsugamushi evaluated, but it did not produce amplicons when 17 Rickettsia and 18 less-related bacterial nucleic acid extracts were tested. Similar agent specificity for the real-time PCR assay, which used the same primers and a 31-basepair fluorescent probe, was demonstrated. This sensitive and quantitative assay determination of the content of O. tsutsugamushi nucleic acid used a plasmid containing the entire 47-kD gene from the Kato strain as a standard. Enumeration of the copies of O. tsutsugamushi DNA extracted from infected tissues from mice and monkeys following experimental infection with Orientia showed 27 5,552 copies L of mouse blood, 14,448 86,012 copies L of mouse liverspleen homogenate, and 3 21 copies L of monkey blood.