ABSTRACT
This study evaluates five cryptic plasmid-derived DNA probes in a 4-h slot-blot hybridization assay for the detection of Chlamydia trachomatis in cultures and clinical specimens. The probes, consisting of either the entire cloned 7.5 kbp cryptic plasmid pSE8 or one of four Hin dIII/Eco RI fragments measuring 710, 1055, 710, and 500 bp, respectively, were labelled with Photoprobe biotin. The probe was detected using a streptavidin-alkaline phosphatase conjugate followed by addition of BCIP and NBT. The sensitivity of the assay, using 25 ng of probe DNA, ranged from 50 pg (with the entire plasmid as probe) to 5 ng (with the 500 bp fragment as probe). A total of 103 reference strains of Chlamydia trachomatis and other bacteria were tested for reactivity with the probes. All 18 reference strains of C. trachomatis hybridized with the probes. None of the DNA from the heterologous organisms tested was found to hybridize with any of the probes. A total of 174 samples taken from patients visiting the STD clinic at the University Hospital, University of Seville were used in the study. The overall sensitivity of the assay, using the 710 bp biotinylated probe was 94.5% compared to culture while the specificity was 97.5%. Positive and negative predictive values were 96.5% and 97.5%, respectively. It appears that the plasmid-derived probes used in this study could serve as useful tools for the rapid and specific detection of Chlamydia trachomatis in cell cultures and clinical specimens.
Subject(s)
Chlamydia Infections/diagnosis , Chlamydia trachomatis/isolation & purification , DNA Probes , Plasmids/genetics , Bacteria , Blotting, Southern , Cells, Cultured , Cloning, Molecular , DNA, Bacterial , Evaluation Studies as Topic , Female , Gene Library , Humans , Male , Restriction Mapping , Sensitivity and Specificity , Sequence Homology, Nucleic AcidABSTRACT
The digestive glands of many marine molluscs are rich sources of arylsulfatase enzymes which may function in the catabolism of sulfated polysaccharides in the diets of herbivorous species. Arylsulfatases, partially purified from the hepatopancreas of the red abalone, Haliotis rufescens, were investigated with respect to heterogeneity, catalytic requirements, and timing of induction during development. Four hepatopancreatic enzymes were purified from adult animals using a combination of hydrophobic interaction and anion-exchange chromatography. Zymograms of the four partially-purified enzymes produced by electrophoresis under nondenaturing conditions revealed a fifth, relatively more basic isozyme. All four partially-purified enzymes appear to be monomeric, with molecular weights of approximately 43,000 Da each, as measured by gel filtration. The affinities for p-nitrocatechol sulfate, pH optima, and strengths of inhibition by anions displayed by these enzymes are similar to the values reported for other molluscan arylsulfatases. Three of the four enzymes have Km values between 0.8 and 2.0 mM for p-nitrocatechol sulfate; the remaining enzyme (A2) has a Km of 6.7 mM. All four enzymes have pH and temperature optima of 5.5 and 45 degrees C, respectively. Three of the four enzymes have-t 1/2 (50 degrees C) values of 3.5 min; the enzyme A4 has a t 1/2 (50 degrees C) of 8.5 min. A monoclonal antibody directed against form A1b does not cross react with any of the other hepatopancreatic arylsulfatases when assayed by Western blot, confirming the structural heterogeneity of the adult enzymes. Total arylsulfatase activity increases in a biphasic manner during early abalone development, with the first increase occurring early in larval maturation.(ABSTRACT TRUNCATED AT 250 WORDS)
