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

JU JIANG 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

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TEIK-CHYE CHAN 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

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JOSEPH J. TEMENAK 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

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GREGORY A. DASCH 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

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WEI-MEI CHING 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

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ALLEN L. RICHARDS 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

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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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