Probe directed at a segment of Rickettsia rickettsii rRNA amplified with polymerase chain reaction.
In an effort to explore a sensitive taxon-specific detection system for bacteria, we sequenced the 16S rRNA from two strains of Rickettsia rickettsii, five other rickettsiae, and Coxiella burnetti to find a probe site unique to R. rickettsii. We then synthesized a 16-mer that hybridized only to the rRNA of R. rickettsii. Using a primer complementary to a sequence found only in rickettsial rRNA, we then generated a cDNA. We amplified the probe site in a 180-base segment of the cDNA by using the cDNA primer and a second primer also unique to rickettsiae in a polymerase chain reaction. The segments of rRNA from each of the rickettsiae were amplified 10(6)- to 10(7)-fold, and the R. rickettsii probe hybridized only to the amplified segment from R. rickettsii. The rRNAs from Staphylococcus aureus, C. burnetii, and Neisseria meningitidis were not amplified and did not hybridize with the probe. The approach detailed below may prove clinically useful in the direct detection of pathogens that are difficult to cultivate.
Wilson
K H
KH
Division of Infectious Diseases, Duke University, Durham, North Carolina.
Blitchington
R
R
Shah
P
P
McDonald
G
G
Gilmore
R D
RD
Mallavia
L P
LP
D001483:Base Sequence; D003381:Coxiella / Q000235:genetics; D005784:Gene Amplification; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000235:genetics; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012335:RNA, Ribosomal / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D012281:Rickettsia / Q000235:genetics; D012283:Rickettsia prowazekii / Q000235:genetics; D012284:Rickettsia rickettsii / Q000235:genetics* / Q000302:isolation & purification; D012286:Rickettsia typhi / Q000235:genetics; D013211:Staphylococcus aureus / Q000235:genetics
D015345:Oligonucleotide Probes; D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D012367:RNA, Viral