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DEVELOPMENT OF A QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION ASSAY SPECIFIC FOR ORIENTIA TSUTSUGAMUSHI

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  • 1 Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland; Division of Vaccines and Related Products Applications, Food and Drug Administration, Rockville, Maryland; Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; Department of Preventive Medicine and Biometrics, Uniformed Services University of the Health Sciences, Bethesda, Maryland

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/μL of mouse blood, 14,448-86,012 copies/μL of mouse liver/spleen homogenate, and 3-21 copies/μL of monkey blood.

Author Notes

Reprint requests: Allen L. Richards, Rickettsial Diseases Department Naval Medical Research Center 503 Robert Grant Avenue Silver Spring, MD 20910-7500.
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