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Title: REVERSE TRANSCRIPTASE PCR AMPLIFICATION OF RICKETTSIA TYPHI FROM INFECTED MAMMALIAN CELLS AND INSECT VECTORS

Author
item HIGGINS, JAMES
item RADULOVIC, SUZANA - UNIV MD MED SCHOOL
item NODEN, BRUCE - UNIV MD MED SCHOOL
item TROYER, JILL - UNIV MD MED SCHOOL
item AZAD, ABDU - UNIV MD MED SCHOOL

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/6/1998
Publication Date: N/A
Citation: N/A

Interpretive Summary: Rickettsia typhi is the bacterium responsible for causing endemic, or murine, typhus, and is vectored by fleas, including the common cat flea, Ctenocephalides felis. Because it is an obligate intracellular parasite, it is necessary to propagate R. typhi in cell culture; however, this is an expensive, laborious, and hazardous task, an easy, rapid method for isolating ribonucleic acid (RNA, indicative of viable, metabolizing bacteria) would aid in studies of the pathogenesis of R. typhi. Existing methods for isolating RNA from these cell cultures involve the use of corrosive, dangerous reagents, and can take several hours to perform. The authors developed a quick and easy protocol for isolating RNA from both cultured cells, and vector fleas, containing R. typhi. This RNA was then used as substrate in a reverse-transcriptase polymerase chain reaction (RT-PCR) assay. The RT-PCR assay was able to detect transcripts of R. typhi genes for immunologically important surface antigens. The authors accomplished the following: first, they developed an inexpensive, quick, and easy method for isolating RNA from cultured cells in which R. typhi was being propagated. Secondly, they developed an RT-PCR protocol which is successful in detecting the transcripts of genes coding for R. typhi proteins. Some of these proteins are important in eliciting immune responses in infected people and animals. And third, they demonstrated the assay can detect transcripts from fleas containing R. typhi. This indicates the the R. typhi in the fleas is alive and capable of infecting people, and not dead.

Technical Abstract: We developed a reverse transcriptase PCR assay to detect expression of 120-and 17- kDa antigen genes in Rickettsia typhi. Infected Vero cell and flea RNAs were reverse transcribed by using random hexamers. The cDNA was amplified by using high concentrations of primer and template in an inexpensive, nonradioactive assay.