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Am. J. Trop. Med. Hyg., 70(4), 2004, pp. 351-356
Copyright © 2004 by The American Society of Tropical Medicine and Hygiene

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

JU JIANG, TEIK-CHYE CHAN, JOSEPH J. TEMENAK, GREGORY A. DASCH, WEI-MEI CHING, AND 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

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.

Received September 24, 2003. Accepted for publication December 14, 2003.

Acknowledgments: We gratefully acknowledge Steve Yevich, Kathryn DeCarlo, Won Suh, and Meaghan Pimsler from the Thomas Jefferson High School for Science and Technology (Alexandria, VA) mentorship program for their invaluable technical support and discussions through the progress of this work.

Financial support: This work was supported by U.S. Department of Defense work unit numbers B998.0000.000.A0074 and B998.0000.000.A0310.

Disclaimer: The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large.

Authors’ addresses: Ju Jiang, Rickettsial Diseases Department, Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, Telephone: 301-319-7249, Fax: 301-319-7460, E-mail: JiangJ{at}nmrc.navy.mil. Teik-Chye Chan, Rickettsial Diseases Department Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, Telephone: 301-319-7436, Fax: 301-319-7460, E-mail: ChanC{at}nmrc.navy.mil. Joseph J. Temenak, Bacterial Vaccines and Allergenic Products Branch, Division of Vaccines and Related Products Applications, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Department of Health and Human Services, Rockville, MD 20852, Telephone: 301-827-3070, Fax: 301-827-3532, E-mail: Temenak{at}cber.fda.gov. Gregory A. Dasch, Rickettsial Diseases Section, Viral and Rickettsial Zoonoses Branch, Division of Viral and Rickettsial Diseases, National Centers for Infectious Disease, Centers for Disease Control and Prevention, Mail-stop G-13, 1500 Clifton Road, Atlanta, GA 30033, Telephone: 404-639-4140, Fax: 404-639-4436, E-mail: ged4{at}cdc.gov. Wei-Mei Ching, Rickettsial Diseases Department, Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, Telephone: 301-319-7438, Fax: 301-319-7460, E-mail: ChingW{at}nmrc.navy.mil. Allen L. Richards, Rickettsial Diseases Department, Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, Telephone: 301-319-7668, Fax: 301-319-7460, E-mail: RichardsA{at}nmrc.navy.mil.

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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Copyright © 2004 by the American Society of Tropical Medicine and Hygiene.