Microplate Hybridization Method Research Articles

Escherichia albertii sp. nov., a diarrhoeagenic species isolated from stool specimens of Bangladeshi children.

The taxonomic position of a group of five D-sorbitol- and lactose-negative enterobacterial isolates recovered from diarrhoeal stools of children at the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), was investigated by DNA-DNA hybridization, phenotypic characterization and 16S rDNA sequencing. These strains were originally identified as 'Hafnia alvei-like' with the API 20E system but, in fact, show more phenotypic and genotypic resemblance to members of the genus Escherichia. By 16S rDNA sequencing, one representative strain of the ICDDR,B group was shown to be closely affiliated to the genera Escherichia and Shigella. Using the fluorimetric microplate hybridization method, the diarrhoeagenic ICDDR,B isolates were found to constitute a homogeneous taxon (> or = 82% internal DNA relatedness), with the closest affiliation to the type strains of Escherichia coli (55-64%) and Shigella flexneri (54-60%). The DNA-DNA hybridization levels were much lower with members of other described Escherichia species (16-45%) and with the type strain of H. alvei (9-17%). The G + C content of the ICDDR,B strains ranged from 50.5 to 50.7 mol%. Together with the diagnostic characteristics reported previously, including the presence of the eaeA gene of enteropathogenic E. coli and of the E. coli and Shigella-specific phoE gene, it is concluded that the ICDDR,B strains represent a novel taxon in the genus Escherichia, for which the name Escherichia albertii sp. nov. is proposed. Its type strain is Albert 19982(T) (= LMG 20976(T) = CCUG 46494(T)).

Development and evaluation of a PCR–microplate capture hybridization method for direct detection of verotoxigenic Escherichia coli strains in artificially contaminated food samples

For the purpose of detecting, directly in food, veroxigenic Escherichia coli, a microplate hybridization method for the detection of PCR products from the SLT I and SLT II genes, was developed and evaluated. Two pairs of primers and two probes, specific for the SLT I gene and for the SLT II gene, were designed and tested. For the strains containing both genes, two PCR products of different molecular weights were obtained, whereas when only one gene was present only one fragment resulted from PCR. The use of the biotin-labeled probes allowed the immobilization of the PCR products in the microtiter plate wells and by this means their detection was possible using an ELISA-based technique. Forty artificially contaminated and fifty naturally contaminated food samples were analyzed by using the PCR–microplate hybridization technique developed in this study. All the artificially contaminated food samples were positive, independently of the number of cells inoculated before the enrichment step, whereas the naturally contaminated food samples were all negative.

Detection of varicella-zoster virus DNA in tear fluid and saliva of patients with Ramsay Hunt syndrome

Ramsay Hunt syndrome develops when the varicella-zoster virus (VZV) is reactivated. In the present study, we examined the secretion kinetics of VZV DNA in the tear fluid, submandibular gland saliva and parotid gland saliva of 15 patients with Ramsay Hunt syndrome. The presence of VZV DNA was detected using PCR and a microplate hybridization method. Hybridization signals were measured using the fluorescence density of an enzymatic reaction product using fluoroscan and a system involving streptavidin-conjugated beta-galactosidase. The results were converted into numerical values and used to estimate the number of virus DNA copies. VZV DNA was detected in the tear fluid, submandibular gland saliva and parotid gland saliva of the Ramsay Hunt syndrome patients. The rate of VZV DNA detection in the submandibular gland saliva was 72%, and the detection rate in the parotid gland saliva was 57%. The detection rate in the tear fluid was 27%, which is significantly lower than other two detection rates. Regarding the submandibular gland saliva and the parotid gland saliva, the VZV DNA was detected in samples collected at a comparatively early stage of onset. In the tear fluid, the detection rate increased significantly in samples collected 2 weeks after onset or later. Thus, differences in the detection rate were observed depending on the type of secretory gland and the timing of the sample collection. The VZV DNA in the tear fluid is thought to derive from the ganglion trigeminale. The increase and decrease in the number of VZV DNA copies detected in samples collected at different times is considered to substantiate VZV reactivation in Ramsay Hunt syndrome.

Detection of beer spoilage bacteria Megasphaera and Pectinatus by polymerase chain reaction and colorimetric microplate hybridization

Anaerobic bacteria of the genera Megasphaera and Pectinatus cause beer spoilage by producing off flavours and turbidity. Detection of these organisms is complicated by the strict anaerobic conditions and lengthy incubation times required for their cultivation, consequently there is a need for more rapid detection methods. A polymerase chain reaction (PCR) method and a colorimetric microplate hybridization assay were developed for the rapid and specific detection of Megasphaera cerevisiae and Pectinatus spp. A biotinylated primer pair was designed for the amplification of a 403 base pair (bp) fragment of the M. cerevisiae 16S rRNA gene and a primer pair from literature was used for the amplification of an 816 bp fragment of Pectinatus 16S rRNA gene. Amplified PCR products were analyzed by the colorimetric microplate hybridization method in which a biotinylated PCR product was captured by streptavidin and hybridized with a digoxigenin-labelled oligonucleotide probe. In the final step an enzyme-linked antibody and a colorimetric reaction were utilized. A simple and rapid sample treatment was set up for the PCR detection of contaminants in beer. Detection of M. cerevisiae (≥5·10 3 colony forming units [cfu]/100 ml) and Pectinatus frisingensis (≥5·10 5 cfu/100 ml) in beer was successful, but the sensitivity of the assay still needs to be improved for direct detection of the small amounts of bacteria present in beer.

Detection of enterovirus 70 in acute hemorrhagic conjunctivitis by PCR-stringent microplate hybridization method

The surveillance of acute hemorrhagic conjunctivitis (AHC) caused by enterovirus 70 (EV70) is insufficient because of the difficulties of virus isolation using conventional methods. In this study, reverse transcription-polymerase chain reaction (RT-PCR) and stringent microplate hybridization (SMH) methods were assessed for the detection of EV70 in conjunctival swabs collected from the patients of AHC which broke out in Miyazaki Prefecture in 1990 and Okinawa Prefecture in 1994. Furthermore, with the use of the SMH method, we compared the genetic homogeneity of EV70 detected from the samples collected in 1990 and 1994 to the J670/71 strain isolated approximately 25 years ago. A similar investigation was performed between recent coxsackievirus A24 variant (CA24v) and EH24/70 strain, a standard strain of CA24v that was isolated approximately 25 years ago. As a result, RT-PCR products were detected in 26 of 34 conjunctival swabs collected in Miyazaki Prefecture in 1990. Likewise, RT-PCR products were detected in 9 of 10 conjunctival swabs collected in Okinawa Prefecture in 1994, and these RT-PCR products were all identified as EV70 by SMH method. Such a high level of virus recovery from specimen shows the evidence of usefulness of these techniques. Recent EV70 showed a reaction of about 60% to a probe synthesized with J670/71. Likewise, recent CA24v showed a reaction of about 30% to a probe synthesized with EH24/70. These finding are highly suggestive of the progress of viral mutation.

Identification of bifidobacteria from dairy products and evaluation of a microplate hybridization method

Sixteen strains of Bifidobacterium isolated from 15 dairy products such as yogurt, cultured milk, butter and cheese were characterized on the basis of phenotypic characteristics and DNA similarities were examined by a microplate hybridization method. Three of the strains were identified as Bifidodobacterium longum, one strain was identified as Bifidobacterium bifidm, and one strain was assigned to the species Bifidobacterium breve on the basis of phenotypic characteristics, and this identification was confirmed by the analysis of DNA similarities. The remaining 11 strains could not be identified by examining their phenotypic characteristics and, contrary to the product label information, these strains were identified as Bifudidobacterium animalis on the basis of DNA similarities. The applicability of the colorimetric hybridization method in micro dilution wells to genetic identification of Bifidobacterium species was also studied.

Distribution of Aeromonas species in the intestinal tracts of river fish

Aeromonas isolates were obtained from fish intestines, water, and sediments from an urban river and identified by the DNA-DNA microplate hybridization method. The isolates were Aeromonas veronii (22%), Aeromonas caviae (18%), Aeromonas hydrophila (13%), Aeromonas sobria (8%), Aeromonas jandaei (7%), and other Aeromonas spp. (33%). Aeromonas species occurred at high densities with high incidences, regardless of season. The results strongly suggest that aeromonads are indigenous in fish intestines, water, and sediments of rivers and have the potential to be predominant in aquatic environments.

Detection of dengue viral RNA by microplate hybridization.

Dengue virus infection is a major public health problem throughout tropical countries. In endemic areas, dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) are common complications resulting in death. However, serological confirmation of dengue-related illness is often complicated and time-consuming. Detection of dengue viruses in clinical or field samples usually depends on virus isolation in susceptible cell lines or in mosquitoes, followed by viral protein identification using polyclonal or monoclonal antibodies. The increasing incidence of dengue virus infections has prompted increased efforts to develop rapid and reliable diagnostic techniques. A simple microplate hybridization method was developed for identification of viral RNA. Microplate hybridization is simpler than enzyme-linked immunosorbent assay and has several advantages over the conventional dot-blot hybridization method: (1) radioisotopes are not necessary; (2) synthetic oligonucleotide for the probe is not needed; (3) the time required for washing of the solid phase is greatly reduced; and (4) baking is eliminated. The results show that this procedure is sensitive, rapid and easy to perform.

Identification of Aeromonas Species Isolated from Freshwater Fish with the Microplate Hybridization Method

Aeromonas isolates were obtained from the intestinal tracts of six species of cultured freshwater fish and identified on the basis of their genotypic and phenotypic characters. The microplate hybridization method could differentiate type strains of Aeromonas species and related bacteria. DNA-DNA hybridization analysis showed that 65 aeromonad isolates were 72 to 100% related with either Aeromonas caviae, Aeromonas hydrophila, Aeromonas jandaei, Aeromonas sobria, or Aeromonas veronii. As many as 48% of the genotypically identified A. caviae, A. hydrophila, and A. sobria isolates differed from the type strains of corresponding species in one to three phenotypic characters. These results strongly suggest that not all aeromonad isolates from freshwater fish could be identified correctly on the basis of only the phenotypic characters, indicating the usefulness of the microplate hybridization method for the identification of aeromonads.

Clinical application of microplate DNA-DNA hybridization procedure for rapid diagnosis of mycobacterial infections

Setting: As an alternative to biochemical analysis, microplate DNA-DNA hybridization was applied for rapid diagnosis of mycobacterial infection. Objective: To assess how rapidly and correctly the microplate hybridization method can progress from clinical sample to final species identification of mycobacteria. Design: Clinical samples (pooled sputa or bronchial lavage fluid) were obtained from patients. Depending on the estimated bacterial amounts, genetic identification was performed either directly or following primary culture. Extracted DNA labeled by photobiotin was hybridized in microdilution wells with type-strain DNAs from 4 species ( Mycobacterium tuberculosis, M. avium, M. intracellulare, and M. kansasii), then identified on the basis of genetic relatedness, which was quantitated by colorimetric detection. Results: With samples containing more than 10 8 colony-forming units [CFU](5 cases), species identification was successfully performed on the day of sample preparation. With samples of not more than 10 7 CFU (14 cases), although 4–21 days' primary culture were necessary, species were also correctly identified by the microplate method. Furthermore, M. avium and M. intracellulare were distinctly identified. All the results precisely corresponded to those of biochemical analysis, which took 4–12 weeks to complete identification. Conclusion: We consider that microplate DNA-DNA hybridization is a dependable technique for rapid diagnosis of mycobacterial infection.

Identification of O/129-Sensitive <i>Aeromonas</i> by the Microplate Hybridization Method

Identification of O/129-Sensitive <i>Aeromonas</i> by the Microplate Hybridization Method

Identification of Plesiomonas shigelloides Isolated from Freshwater Fish with the Microplate Hybridization Method

A total of 74 strains were isolated from the intestines of seven freshwater fish and 10 water samples, and identified to the species level by the conventional method based on morphological, physiological and biochemical properties, and DNA-DNA hybridization method. Achromopeptidase treatment was found to be useful for the recovery of DNAs from Plesiomonas shigelloides cells because the amount of DNA obtained with this treatment was 3.1–7.2 times greater than with lysozyme. All the strains were identified as P. shigelloides on the basis of the mol% G + C contents of DNAs and homology values against the type strain of this microorganism, along with morphological, physiological, and biochemical properties. The P. shigelloides was detected in 29 out of 51 fish specimens and four out of 10 water samples. Black bass, Japanese eel, ayu, and tilapia harbored high densities of P. shigelloides, ranging from 104–108 CFU/g, with high frequencies of occurrence (73–100%). These results strongly suggested that the microplate hybridization method is an excellent procedure for the ecological study of P. shigelloides.

Evaluation of the microplate hybridization method for rapid identification ofLegionella species

Rapid genetic identification of 23 Legionella species was performed using microdilution plate hybridization. When an isolate appeared to be a strain of the Legionella species, its DNA was quickly extracted, labelled with photobiotin and distributed to microdilution wells where 23 different DNAs had previously been immobilized. After hybridization for 90 min at 45 degrees C, the microdilution well which emitted the maximum fluorescence was determined. The criterion for interpreting results of this genetic identification method is given.