Analysis and Detection of Chlamydial DNA

Abstract

Elementary bodies of lymphogranuloma venereum (LGV) strains of Chlamydia trachomatis contain, in addition to the genomic DNA, a 6.7 kb plasmid. The plasmid from serovar L2 (434-B) was cloned at the BamHI site of pBR327 into Escherichia coli and a restriction cleavage map of this pLGV125 recombinant plasmid determined. All 15 C. trachomatis serovars contained DNA sequences that hybridized with pLGV125. When total DNA from L2 elementary bodies was used as a probe in Southern blotting and spot hybridization, serovars L1, L2 and L3 exhibited significant homology. The detection level of homologous DNA was 100 pg and LGV DNA was detectable in infected cells when total L2 probe was used in the nucleic acid hybridization test. These DNA probes may be useful as investigative and diagnostic reagents for C. trachomatis.

Detection of Chlamydia trachomatis in Clinical Specimens by Nucleic Acid

“…Purification of C. trachomutis DNA from elementary bodies and characterization of the cloned L2 plasmid have been described previously (Hyypia et al, 1984). For the hybridization experiments, the hybrid plasmid was digested with BamHI (Boehringer-Mannheim) and the L2 plasmid insert was recovered from agarose gels by use of a DEAE-cellulose membrane (Dretzen et al, 1981).…”

“…We recently described a spot hybridization test for detection of chlamydial DNA (Hyypia et al, 1984). In the present report we have further evaluated the sensitivity and specificity of the test, and used it to detect C. trachomatis DNA in clinical specimens.…”

Detection of <i>Chlamydia trachomatis</i> in Pregnant Women by the Papanicolaou Technique, Enzyme Immunoassay and Polymerase Chain Reaction

“…It has been reported that the DNA hybridization tests have a detection limit of 1-10 pg, 21,50 whereas in PCR assays, when DNA is purified, there is a detection limit of 100 ng. 13 These results indicate that the sensitivity values obtained are more than adequate to demonstrate the presence of chlamydial plasmid DNA in clinical samples.…”

“…These last characteristics of PCR are not true of the other methods of diagnosis. 20,21,59 It has been proposed that detection of microorganisms based on recognition of the chlamydial plasmid is more sensitive than detection based on recognition of chromosomal DNA due to the presence of multicopies. For this reason, in this study we used oligonucleotides that recognize a 200-bp plasmid sequence 61 and used as the control the pLGV125 plasmid 21 (Figure 2A).…”

“…The reaction was carried out at a final volume of 15 µL, containing 1 µL of sample, 5 mM MgCl 2 , 250 µM of dATP, dCTP, dGTP and dTTP, 0.5 µM of each of the primers and 0.5 U of Taq DNA polymerase (Promega Corporation, Madison, Wisconsin, U.S.A.). The positive control consisted of 4 ng/µL of the plasmid pLGV125, 21 which contains the complete plasmid (6,700 bp). This control plasmid was obtained from the L2 (434-L) strain of C trachomatis.…”