Objective. To investigate the hypothesis that whole bacteria might be found in the joints of patients with Chlamydia-associated reactive arthritis.Methods. The presence of 2 plasmid-and 2 chromosome-specific sequences of Chlamydia DNA was investigated by amplification with the polymerase chain reaction, in synovial fluid (SF) samples from 71 patients with various arthropathies.Results. Chlamydia DNA was found in SF samples from 22 patients.Conclusion. Whole chlamydiae are likely present in the SF of patients with Chlamydia-associated reactive arthritis.Reactive arthritis may occur following an infection of the urogenital tract caused by Chlamydia. The presence in joint material of chlamydial antigens, suggestive of elementary and reticulate bodies, has been reported (1-6). An important question is whether whole chlamydiae, or only fragments, are present in the joint. Bacterial remnants, in contrast to live bacteria, would not contain appreciable amounts of undegraded nucleic acids.Studies investigating for the presence of Chlamydia nucleic acids have had variable results. Initial attempts to use the polymerase chain reaction (PCR) (7) to detect Chlamydia DNA were unsuccessful.
A degenerate primer pair was selected to amplify specifically a 260-bp DNA fragment from Clostridium botulinum types A, B, E, F, and G, and five individual probes allowed identification of each toxinotype by hybridization of the PCR products. The 72 strains of different Clostridium species tested and 11 other bacterial species commonly found in food samples gave an amplification product. This assay was able to detect 1 C. botulinum type A or B and 10 C. botulinum type E strains per reaction. With 184 artificially contaminated food samples, after an 18-h enrichment step, the sensitivity was 10 bacteria per g of sample and the correlation with the mouse bioassay reached 95.6%.
The frequency occurrences of K-tuple (overlapping sequences of defined length, K) were computed from the known human genome sequences. The significance of these frequencies for the whole human genome was tested by polymerase chain reaction (PCR). A computer programs based on these results was written to choose primers to amplify DNA target sequences, either of human genes or of human infectious agents. The software also gave nested primer sequences which were used to synthesize non radioactive probes by PCR. We applied these two methods, primer selection and non radioactive probes, to easily and quickly set up very efficient PCR sets to work in the human genome context.
A double PCR procedure is proposed for identification of Clostridium botulinum C and D. This method consists of a first PCR amplification with a degenerate primer pair able to amplify a 340 bp common DNA fragment from botulinum neurotoxin (BoNT) C1 and D genes, followed by two subsequent PCR amplifications with two primer pairs specific for BoNT/C1 and D respectively (198 bp DNA fragment). This method was found to be specific for C. botulinum C and D, amongst 81 strains of C. botulinum and 21 different species of other Clostridium and bacteria tested. The detection limit ranged from 10 to 10(3) bacteria in the reaction volume according to the C. botulinum C and D strains. In 160 naturally contaminated animal and food samples submitted to a 48 h enrichment culture, the double PCR showed an 89.4% correlation rate with the standard mouse bioassay. A clear distinction between botulism type C and D was obtained. The double PCR provides a reliable alternative for detection and identification of C. botulinum C and D in clinical and food samples.
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