Microplate Hybridization Research Articles

A prolonged outbreak of Norwalk-like calicivirus (NLV) gastroenteritis in a rehabilitation centre due to environmental contamination.

An outbreak of Norwalk-like calicivirus (NLV) gastroenteritis occurred in a rehabilitation centre in southern Finland between December 1999 and February 2000. An epidemiological investigation was conducted to determine the source and extent of the outbreak. More than 300 guests and staff members became ill during the outbreak. No food or activity in the centre could be linked epidemiologically to illness. NLV genogroup II was detected by RT-PCR in stool samples of symptomatic guests and employees. All strains reacted similarly with the microplate hybridization probe panel and showed the same nucleotide sequence, indicating that they represented the same NLV strain. Food and water samples were negative for NLV, whereas NLV was detected in three environmental specimens. The strains from patients and environment were identical based on microplate hybridization probes, suggesting that environmental contamination may have been important for the spread of calicivirus and the protracted course of the outbreak.

Relative relatedness of 11 mycobacteria species by microplate hybridization methods considering delta Tm

Intra-species variance within Mycobacterium xenopi, Mycobacterium gordonae or Mycobacterium szulgai has been reported in identification employing chemotaxonomic characteristics, 16 S rRNA gene sequences or relative relatedness (relative color index) of genomic DNA-DNA Hybridization. Genomic DNA-DNA reassociation at the constant temperature was found to be unreliable for classification of mycobacterial species. However, nonspecific DNA reassociation could be avoided by hybridization at 56 degrees C after 45 degrees C overnight, and this technique was named delta DDH method in the preceding paper. The present report shows relative relatedness (relative color index) of genomic DNA in delta DDH method among mycobacterial species. Relative relatedness was below 70% among BCG, M. kansasii, M. simiae, M. asiaticum, M. szulgai, M. gordonae, M. xenopi and M. nonchromogenicum. The results satisfied the criteria for bacterial classification, which was proposed by the International Committee for Systematic Bacteriology in 1987. In regard to Mycobacterium avium complex, relative relatedness between M. avium and M. intracellulare were approximately 75%. It appeared that M. avium and M. intracellulare could be classified into one species. It has been recognized, moreover, that there are intermediate strains between M. avium and M. intracellulare. Previously, numerical classification raised a concept of Mycobacterium avium-intracellulare-scrofulaceum complex. The present study revealed that relative relatedness of M. avium and of M. intracellulare to M. scrofulaceum were around 75%, while the percentiles of M. scrofulaceum relative to M. avium and that to M. intracellulare were both less than 50%. The relative relatedness of M. ulcerans against M. marinum was nearly 65%, whereas the relative relatedness of M. marinum against M. ulcerans was approximately 90%. The data may be partly explained by the horizontal gene transfer mechanism.

Gene analysis of free fetal DNA in maternal plasma.

To investigate the feasibility of using free fetal DNA from maternal plasma as the source of fetal material in non-invasive prenatal diagnosis, SRY gene of free DNA in maternal blood of 65 samples were analyzed by using primer extension preamplication (PEP) and probe microplate hybridization techniques. The results showed that the detection rate of SRY gene in maternal blood from women carrying male fetuses detected by probe microplate hybridization alone and probe microplate hybridization with PEP were 76.09% (35/46) and 95.65% (44/46) respectively, and there was a significant difference between them. The non-detection rate of SRY gene in blood samples from women carrying female fetus was 100% (19/19). It is indicated that probe microplate hybridization was an effective method in detecting trace fetal DNA from maternal plasma and the sensitivity could be substantially improved by combined use of the two techniques. Analysis of fetal DNA in maternal plasma can serve as an alternative for non-invasive prenatal diagnosis.

The detection of bovine leukemia virus proviral DNA by PCR–ELISA

A sensitive non-radioactive microplate hybridization assay for the detection of proviral DNA of bovine leukemia virus (BLV)-specific polymerase chain reaction (PCR) product is described. The PCR products are labeled by adding digoxigenin-dUTP to the nested PCR reaction and are captured by a microtitre plate coated with oligonucleotide probe, which is complementary to the inner region of the amplification product. Captured products are reacted with an anti-DIG Fab fragment conjugated to peroxidase, and detected using a colorimetric reaction. The PCR-enzyme linked immunosorbent assay (ELISA), detecting as low as 10 −4 ng of proviral DNA in a background of 1 μg of BLV-negative DNA, was up to 100-fold more sensitive than ethidium bromide staining, and showed equal sensitivity to Southern blot hybridization. Using this method it was possible to monitor the presence of proviral DNA in four sheep infected experimentally with BLV, over a 10 months postinfection period, as well as in 29 cattle infected naturally. The test is rapid and highly sensitive and is a useful additional tool for the detection of BLV-infected animals.

Rapid detection of Campylobacter fetus by polymerase chain reaction combined with non-radioactive hybridization using an oligonucleotide covalently bound to microwells

Campylobacter fetus is recognized as a human and animal pathogen. The isolation and differentiation of C. fetus in diagnostic laboratories is hindered by its relatively slow growth and lack of distinguishing biochemical characteristics. We cloned and sequenced a 1581-bp DNA fragment, IG02, isolated from a C. fetus genomic library. This fragment was used as a probe on DNAs extracted from C. fetus strains and otherCampylobacter species: IG02 hybridized only with DNAs from C. fetus strains. A PCR-based test was developed for the detection of C. fetus. A pair of oligonucleotide primers was designed to amplify a 141-bp fragment of IG02. The amplified product was analysed by a non-radioactive sandwich hybridization in microtiter plate using a capture oligonucleotide and a biotin-labelled oligonucleotide for the detection. The combination of PCR and non-radioactive microplate hybridization is a convenient method for the rapid detection of C. fetus.

Restricted usage of T-cell receptor alpha-chain variable region (TCRAV) and T-cell receptor beta-chain variable region (TCRBV) repertoires after human allogeneic haematopoietic transplantation.

We analysed T-cell receptor alpha-chain variable region (TCRAV) and T-cell receptor beta-chain variable region (TCRBV) repertoires in peripheral blood mononuclear cells (PBMCs) from 34 recipients of allogeneic bone marrow transplantation (allo-BMT), seven of allogeneic peripheral blood stem cell transplantation and 19 of autologous peripheral blood stem cell transplantation using the quantitative microplate hybridization assay. TCR usage skewed at an early period (6-7 weeks) after BMT. The change was more apparent in allogeneic recipients than in autologous recipients. In particular, a predominant increase was detected in the frequency of VA1-4 (26%, 11 of 41 recipients), VA3-1 (32%) and VB24-1 (28%). Interestingly, acidic amino acid residues frequently followed the arginine residue in complementarity-determining region 3 of BV24S1. We further examined the extent of skew using samples obtained at serial time points after transplantation. The normalization of skewed repertoires occurred over a long period of time (> 8 years). There was a significant difference in the rate of normalization of skewed TCR repertoires between adult and child recipients (P < 0.05). The results suggest that these T cells may have expanded in response to allogeneic antigens, such as miHA (minor histocompatibility antigen), and that altered repertoires are eventually normalized by T-cell regeneration via a thymic-dependent pathway in children.

Detection of Legionella pneumophila DNA in urine and serum samples from patients with pneumonia

albicans strains adhere better to epithelial cells of the oral cavity or the esophagus, but it is accepted that esophageal candidiasis represents a form of invasive candidiasis which requires higher dosages of antifungal drugs compared to OPC [11]. In this patient, replacement of a fluconazole-resistant C. albicans strain by a genetically different fluconazole-sensitive strain was observed; this finally caused further recurrences of OPC, which responded well to intermittent therapy with itraconazole solution. However, it is unclear whether the disappearance of the resistant genotype is due to the temporary increase of the CD4 cell count or because of therapy with itraconazole despite highlevel resistance to azoles in vitro. It may be concluded that serial DNA typing of C. albicans strains, together with antifungal susceptibility testing, may help us to monitor the susceptibility of clinical isolates to azole therapy.

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.

Respiratory coronavirus infections in children younger than two years of age.

Respiratory coronavirus infections in children younger than two years of age.

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.

Confirmation of Norwalk-like virus amplicons after RT-PCR by microplate hybridization and direct sequencing

A large number of Norwalk-like viruses (NLVs) have been identified from stool samples by RT-PCR by amplifying part of the polymerase-coding gene. A set of probes were selected based on sequence analysis of the viruses circulating in Finland during the years 1996–97 for confirmation of the findings by hybridization. A microplate hybridization test, which provides a rapid semi-automatic detection for PCR products, was designed and compared with agarose gel electrophoresis. From the material of 210 stool samples, mainly from diarrheal outbreaks during years 1997–1998, three probes, one for NLV genogroup GGI and one for each of the two GGII subgroups (Toronto-like and Lordsdale-like), were sufficient to detect 87.8% (36/41) of GGI and 89.0% (49/55) of GGII samples positive by gel electrophoresis. Amplicon sequencing of the strains not detected by the above probes revealed genetic variability in the sequences. Biotin-streptavidin binding was used both for microplate hybridization assays and for direct sequencing to identify the amplicons. Based on the sequences three more probes for the hybridization panel were added so that all the different NLVs of this study could be recognized.

Quantitative Detection of Cucumber Mosaic Cucumovirus RNA with Microplate Hybridization Using Digoxigenin-Labeled Oligo-Deoxyribonucleotide Probe

Quantitative Detection of Cucumber Mosaic Cucumovirus RNA with Microplate Hybridization Using Digoxigenin-Labeled Oligo-Deoxyribonucleotide Probe

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 and identification of human papilloma viral DNA, types 16, 18, and 33, by a combination of polymerase chain reaction and a colorimetric solid phase capture hybridisation assay

A colorimetric microplate hybridization assay was developed previously to simplify detection procedures of DNA fragments resulting from polymerase chain reactions (PCR). This format has now been adapted for the simultaneous detection and identification of three human papillomavirus (HPV), types 16, 18 and 33, associated frequently with cervical cancer. This post-PCR detection system uses three type-specific capture oligonucleotides linked covalently to a single microplate well and three type-specific multibiotinylated oligonucleotidic probes for detection. It therefore offers a double specificity; the first is conferred by pairs of primers, specific of each type of virus tested, and the second, by the sets of capture and detection probes which are complementary to internal regions of the amplified DNA fragments. The detection format outperformed agarose gel electrophoresis of amplified DNA products in sensitivity and specificity. The rapidity and simplicity of this hybridisation system would justify its use in routine diagnostic examination of cervical specimens (smears and biopsies).

PCR ELISA for the quantitative detection of Epstein–Barr virus genome

A highly sensitive and nonradioactive microplate hybridization assay for the detection of Epstein–Barr virus (EBV)-specific polymerase chain reaction (PCR) product was developed. The PCR product is labelled by adding digoxigenin-dUTP directly to the reaction mixture and, after denaturation, is captured by a microtitre plate coated with an extravidin-linked biotinylated probe. Captured products are reacted with a peroxidase-conjugated anti-digoxigenin antibody and detected using tetramethylbenzidine. The assay detected less than ten EBV genomes in a background EBV-negative DNA of 0.75 μg and, when tested on clinical samples, it was able to define the viral load in throat washings of patients with acute infectious mononucleosis, immunosuppressed patients with HIV infection, and rare normal individuals who shed the virus in the oropharynx.

Simplified reverse transcription polymerase chain reaction procedure with detection by microplate hybridization for routine screening of hepatitis A virus

Reverse transcription polymerase chain reaction, using either nested or seminested primers, is used extensively for the detection of viruses in small quantities. However, existing methods are prone to false positive reactions. We report here an improved polymerase chain reaction technique based on the use of longer primers (39 nucleotides) with single-step amplification, applied to the detection of hepatitis A in low quantities. While the sensitivity of this technique (10 x the 50% tissue culture infective dose) is equivalent to that of existing methods, it is a simpler procedure, less time consuming, and less susceptible to contamination and therefore provides a more reliable tool for routine diagnosis. Finally, the development of a DNA enzyme immunoassay detection technique and the complete automation of the procedure allow a large number of samples to be processed in clinical laboratories.

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.

Simplified reverse transcription polymerase chain reaction procedure with detection by microplate hybridization for routine screening of hepatitis A virus

Simplified reverse transcription polymerase chain reaction procedure with detection by microplate hybridization for routine screening of hepatitis A virus

Analysis of TCRAV and TCRBV Repertoires in Healthy Individuals by Microplate Hybridization Assay

ABSTRACT: We have developed an adaptor ligation PCR-based microplate hybridization assay (MHA) for analysis of T cell receptor α chain variable region (TCRAV) and T cell receptor β chain variable region (TCRBV) repertoires. Forty three TCRAV and thirty eight TCRBV-specific probes were immobilized onto microplate wells in water-soluble carbodiimide. After hybridization of 5′-biotinylated PCR products, quantitative ELISA was carried out and followed by automated colorimetric reading. The conditions for immobilization and hybridization were optimized using representative TCRBV-specific probes. The sensitivity of MHA allows us to detect as low as 40 pg of biotinylated PCR products. The frequencies of individual V segments obtained by MHA were consistent with those obtained by FACS analysis and reverse dot blot assays. Analysis of the entire TCRAV and TCRBV repertoires could be done using a single 96-well plate, and completed in less than 6 h. Simplicity and reproducibility of this method make it suitable for routine laboratory use. The expression of TCRAV and TCRBV segments was next studied in peripheral blood mononuclear cells (PBMC) of 14 healthy donors using the newly developed MHA method. TCRAV8S1, TCRAV23S1, TCRBV2S1, TCRBV3S1, TCRBV4S1, and TCRBV6S5 were highly expressed in PBMC. Further, the TCRAV repertoires among individuals were less variable compared to the TCRBV repertoires. Interestingly, considerable variations in the expression levels of BV3S1, BV4S1, and BV17S1 were observed among individuals. One polymorphic site was found at the coding region of BV4S1, and there were two alleles. These results suggest that variable expression among individuals may be associated with unknown allelic polymorphism in coding and/or regulatory regions of these TCRBV segments, or with disparity in HLA genes.

Microplate hybridization for Borna disease virus RNA in human peripheral blood mononuclear cells.

We developed a simple and sensitive microplate hybridization procedure with which to identify Borna disease virus cDNA in amplified products from human peripheral blood mononuclear cells. The mean values for the positive PCR products were significant compared with those for any of the negative products, indicating that this method can be applied to rapidly diagnose a large number of clinical specimens.