ABSTRACT
Two monoclonal antibody-mediated immunomagnetic separation PCR kits (AnDiaTec ParaTub-S IMS-PCR-ELISA and ParaTub-SL IMS-real time PCR) were developed and evaluated for the automated high-throughput detection of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) in bulk milk of naturally infected dairy herds and made commercially available. M. paratuberculosis are first isolated from milk by high-throughput immunomagnetic bead separation using a completely automated magnetic particle pipetting robot within 45 min and released subsequently for analysis directly into PCR amplification mixtures for real time PCR or for PCR-ELISA. The threshold detection level and specificity of the tests were evaluated first with different M. paratuberculosis pure cultures and artificially contaminated (spiked) bulk milk samples. Both experiments proved a good detection limit, specificity and reliability of the tests that consistently detected 20 or less M. paratuberculosis organisms from cattle, deer and mufflon in 1 ml milk. Experiments with more than 200 bulk milk samples that were tested in parallel with the PCR methods and with the cultural method in a second evaluation study demonstrated that both PCR tests are superior to culture and sufficiently sensitive to detect single shedders in pooled milk samples. The experiments proved that the newly developed tests are sensitive, specific and fast, and thus for the first time allow the standardized large-scale routine M. paratuberculosis screening of bulk milk samples at acceptable costs.
Subject(s)
Cattle Diseases/diagnosis , Immunomagnetic Separation/methods , Milk/microbiology , Mycobacterium avium subsp. paratuberculosis/isolation & purification , Paratuberculosis/diagnosis , Polymerase Chain Reaction/methods , Animals , Cattle , Cattle Diseases/microbiology , Colony Count, Microbial , Female , Food Microbiology , Paratuberculosis/microbiology , Sensitivity and SpecificityABSTRACT
OBJECTIVE: Most of the PCR-based assays for the detection of adenoviruses require gel electrophoresis for resolution, which is time- and work-consuming and interpretation difficulties often occur. Therefore our objective was to develop and evaluate a PCR-ELISA for broad detection of adenoviruses, that is easy to perform and leads to an increase of specificity in virus detection. METHODS: A total of 64 fecal specimens from patients with acute gastroenteritis, previously tested negative for other viruses than adenovirus and for bacterial pathogens, were tested for adenovirus in parallel with an Antigen-ELISA, virus isolation in cell culture, with a previously published hybridization-based PCR and our newly developed semi-nested PCR-ELISA. RESULTS: The new PCR-ELISA turned out to be the best method with 26 samples positive for adenovirus. Of these, 22 became positive by Ag-ELISA, 19 by virus isolation in cell culture and only 14 by the previously published hybridization-based PCR. CONCLUSION: The semi-nested PCR-ELISA is a sensitive, reliable and rapid method for the detection of enteric adenoviruses from fecal samples.
Subject(s)
Adenovirus Infections, Human/diagnosis , Adenoviruses, Human/isolation & purification , Enzyme-Linked Immunosorbent Assay/methods , Face/virology , Polymerase Chain Reaction/methods , Antigens, Viral/analysis , Child , Child, Preschool , DNA, Viral/analysis , DNA, Viral/chemistry , Humans , Infant , Molecular Sequence Data , Nucleic Acid Hybridization , Sensitivity and Specificity , Sequence Analysis, DNA , Virus CultivationABSTRACT
A commercially available PCR kit (AnDiaTec Salmonella sp. PCR-ELISA) was developed and evaluated for the detection of Salmonella sp. in food samples. The test is based on PCR amplification and hybridization of the amplified DNA to a microtiter plate followed by the detection of PCR product in the manner of an enzyme-linked immunosorbent assay test. The sensitivity and specificity were evaluated first with Salmonella pure cultures and artificially contaminated food samples, including food types for which an inhibition of the PCR reaction was expected. Both experiments proved a very good sensitivity, specificity, and reliability of the test with a very broad range of food types. In a second evaluation study, more than 1,100 food samples of different types were tested in parallel with the PCR method and with the International Standardization Organization 6579 bacteriological reference method. The results of this evaluation study and the results from other experiments on dilutions of artificially contaminated food samples led to the establishment of a positive-negative cutoff value (optical density at 450 nm of more than 0.9) with respect to the conventional bacteriological method. Using this positive-negative cutoff, 98% agreement to the bacteriological method was obtained.
Subject(s)
DNA, Bacterial/analysis , Enzyme-Linked Immunosorbent Assay/methods , Food Contamination/analysis , Food Microbiology , Polymerase Chain Reaction/methods , Salmonella/isolation & purification , Evaluation Studies as Topic , Food Analysis , Gene Amplification , Reference Values , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
We report the case of a 9-year-old girl in Germany with acute meningoencephalitis associated with rotavirus gastrointestinal infection. Sequence analysis revealed a genetic relationship of the strain to rotaviruses with subgroup II specificity. Reverse transcription-PCR was positive for rotavirus.
Subject(s)
Meningoencephalitis/diagnosis , Rotavirus Infections/diagnosis , Rotavirus/isolation & purification , Antiviral Agents/administration & dosage , Female , Follow-Up Studies , Germany , Humans , Infant , Meningoencephalitis/drug therapy , RNA, Viral/analysis , Rare Diseases , Reverse Transcriptase Polymerase Chain Reaction , Risk Assessment , Rotavirus Infections/drug therapy , Severity of Illness Index , Treatment OutcomeABSTRACT
Enterovirus 71 (EV71) is mainly known as a cause of hand-foot-and-mouth disease (HFMD) but sometimes associated with neurological disease, even as fatal brainstem encephalitis. In Europe, EV71 infections are extremely rare, in contrast to the worldwide situation. This is the first report of molecular characterization of an EV71 strain isolated in Europe that had caused neurological disease. The german strain is closest related to sublineage B2 strains isolated in the United States, which where mainly associated with neurological disease. Phylogenetic analysis also showed that the strain must have been imported to Germany several years ago, and continues to circulate since then.
