Development of a quantitative real-time polymerase chain reaction assay specific for Orientia tsutsugamushi

Am J Trop Med Hyg. 2004 Apr;70(4):351-6.


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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Antigens, Bacterial / blood*
  • Bacterial Outer Membrane Proteins / chemistry
  • Bacterial Outer Membrane Proteins / genetics
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • Macaca fascicularis
  • Mice
  • Orientia tsutsugamushi / genetics*
  • Polymerase Chain Reaction / methods*
  • Scrub Typhus / microbiology
  • Sensitivity and Specificity


  • Antigens, Bacterial
  • Bacterial Outer Membrane Proteins
  • DNA, Bacterial