ABSTRACT
The method is based on the hybridization of synthetic deoxyribonucleic acid probes to ribosomal ribonucleic acid sequences unique to Listeria. This method was compared to 2 culture methods: the U.S. Food and Drug Administration method for the detection of Listeria in dairy products and seafoods and the U.S. Department of Agriculture, Food Safety and Inspection Service method for Listeria in meats. Six food types with replicate samples containing various concentrations of Listeria were analyzed by the collaborating laboratories. Listeria was detected in 774 samples using the DNAH method and in 772 samples using a culture method. The DNAH and culture methods were in agreement for 668 samples containing Listeria and 306 samples without Listeria. The overall rate of agreement between methods was 82.3%. The method has been adopted first action by AOAC INTERNATIONAL.
Subject(s)
DNA, Bacterial/analysis , Dairy Products/microbiology , Food Microbiology , Listeria/isolation & purification , Meat/microbiology , Nucleic Acid Hybridization/methods , Seafood/microbiology , Animals , Brachyura/microbiology , Cattle , Cheese/microbiology , Colorimetry/statistics & numerical data , Listeria/genetics , Milk/microbiology , Nucleic Acid Hybridization/statistics & numerical data , Sensitivity and Specificity , SwineABSTRACT
A dot-blot hybridization assay was developed to detect B19 DNA using strand-specific RNA probes labelled with digoxigenin. The sensitivity of the assays was evaluated either using 'plus' and 'minus' sense RNA probes in two different hybridization assays, or in two successive reactions of the same assay. The hybridized probes were revealed immunoenzymatically using anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. The enzyme was visualized by colorimetric reaction. Since 'minus' sense RNA probe gave the best results in the dot-blot procedures, we increased the sensitivity of the hybridization assay visualizing the 'minus' sense digoxigenin-labelled RNA probe by chemiluminescent reaction. In these experimental conditions up to 20 fg of target B19 DNA could be visualized. In the search for B19 DNA, 4656 serum samples were analyzed by chemiluminescent reaction of 'minus' sense digoxigenin-labelled RNA probe and for comparison with the digoxigenin-labelled DNA probe. Positive results were confirmed by Southern blotting. Out of 4656 serum samples analyzed, 4648 gave negative results, 1 resulted positive to all the hybridization assays, 6 only using RNA probe and 1 only by DNA probe.
Subject(s)
DNA, Viral/blood , DNA, Viral/genetics , Molecular Probe Techniques , Parvovirus B19, Human/genetics , Parvovirus B19, Human/isolation & purification , RNA Probes , Adult , Blotting, Southern , Child , Colorimetry , Digoxigenin , Evaluation Studies as Topic , Female , Humans , Luminescent Measurements , Male , Molecular Probe Techniques/statistics & numerical data , Nucleic Acid Hybridization/methods , Nucleic Acid Hybridization/statistics & numerical data , Pregnancy , Sensitivity and Specificity , Virology/methods , Virology/statistics & numerical dataABSTRACT
Interleukin-2 receptor (IL-2R) expression on bronchoalveolar lavage (BAL) cells was studied in patients with sarcoidosis using immune cytochemistry and cytometric analysis. A low percentage of alveolar lymphocytes (AL) was found positive for IL-2R, with 7% in patients with impaired lung function and 6% in patients with normal lung function (0.4% in control subjects). Expression of IL-2R on alveolar macrophages (AM) was considerably higher, with 25% in patients with lung function impairment compared with 14% in patients without lung function impairment (1.5% in control subjects). Serum-soluble IL-2R (ssIL-2R) was significantly elevated only in patients with impaired lung function (140.0 pM), but not in patients with normal lung function (52.2 pM; control subjects, 40.0 pM). These elevated levels of ssIL-2R positively correlated with the percentage of IL-2R positive AM (p < 0.001). Immunosuppressive treatment in three patients resulted in a decrease of IL-2R+ AM and in a decrease of ssIL-2R, whereas IL-2R+ AL were unaffected. The positive correlation and the concomitant decrease of IL-2R+ AM and ssIL-2R are consistent with the idea that in sarcoidosis with clinically apparent lung involvement, elevated levels of ssIL-2R may be derived from AM and may thus be a useful indicator of the degree of activation of these cells.
Subject(s)
Lung Diseases/metabolism , Macrophages, Alveolar/metabolism , Receptors, Interleukin-2/metabolism , Sarcoidosis/metabolism , Adult , Aged , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/cytology , Enzyme-Linked Immunosorbent Assay/statistics & numerical data , Female , Humans , Immunohistochemistry , Lung Diseases/epidemiology , Macrophages, Alveolar/chemistry , Male , Middle Aged , Nucleic Acid Hybridization/methods , Nucleic Acid Hybridization/statistics & numerical data , RNA/genetics , Receptors, Interleukin-2/analysis , Regression Analysis , Sarcoidosis/epidemiology , SolubilityABSTRACT
The simplicity of enterotoxigenic Escherichia coli (ETEC) stool blot hybridization, where the total bacterial growth of a fecal inoculum is examined directly for the presence of enterotoxin genes, has been marred by reports of unsatisfactory sensitivity and/or specificity. To assess the accuracy of stool blot hybridization and to study the effect of varying proportions of ETEC among fecal E. coli (ETEC/E. coli) on test performance, a detailed 'blind' study of 166 stool specimens from children with diarrhea was performed. Oligonucleotide probes were found to be superior to polynucleotide probes, having a sensitivity of 80%, a specificity of 99%, a positive predictive value of 89% and a negative predictive value of 97%. The sensitivity was found to be 100% when ETEC/E. coli > 2/12, as compared with 20% when ETEC/E. coli < or = 2/12 (p = 0.001), showing that the proportion of ETEC among fecal E. coli is of paramount importance for test sensitivity.
