title: Development of a quantitative real-time polymerase chain reaction assay specific for Orientia tsutsugamushi.
abstract: 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 27-5552 copies/microL of mouse blood, 14448-86012 copies/microL of mouse liver/spleen homogenate, and 3-21 copies/microL of monkey blood.
authors.affiliation: Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland 20910-7500, USA. JiangJ@nmrc.navy.mil
authors.forename: Ju
authors.initials: J
authors.lastname: Jiang
authors.forename: Teik-Chye
authors.initials: TC
authors.lastname: Chan
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Temenak
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Dasch
authors.forename: Wei-Mei
authors.initials: WM
authors.lastname: Ching
authors.forename: Allen L
authors.initials: AL
authors.lastname: Richards
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000097:blood*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D008252:Macaca fascicularis; D051379:Mice; D012285:Orientia tsutsugamushi / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012612:Scrub Typhus / Q000382:microbiology; D012680:Sensitivity and Specificity
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial