DNA Hybridization Method Research Articles

Molecular approaches to malaria and babesiosis diagnosis.

The development of additional methods for detecting and identifying Babesia and Plasmodium infections may be useful in disease monitoring, management and control efforts. The preliminary evaluate synthetic peptide-based serodiagnosis, a hydrophilic sequence (DDESEFDKEK) was selected from the published BabR gene of B. bovis. Immunization of rabbits and cattle with the hemocyanin-conjugated peptide elicited antibody responses that specifically detected both P. falciparum and B. bovis antigens by immunofluorescence and Western blots. Using a dot-ELISA with this peptide, antisera from immunized and naturally-infected cattle, and immunized rodents, were specifically detected. Reactivity was weak and correlated with peptide immunization or infection. DNA-based detection using repetitive DNA was species-specific in dot-blot formats for B. bovis DNA, and in both dot-blot and in situ formats for P. falciparum; a streamlined enzyme-linked synthetic DNA assay for P. falciparum detected 30 parasites/mm3 from patient blood using either colorimetric (2-15 h color development) or chemiluminescent detection (0.5-6-min exposures). Serodiagnostic and DNA hybridization methods may be complementary in the respective detection of both chronic and acute infections. However, recent improvements in the polymerase chain reaction (PCR) make feasible a more sensitive and uniform approach to the diagnosis of these and other infectious disease complexes, with appropriate primers and processing methods. An analysis of ribosomal DNA genes of Plasmodium and Toxoplasma identified Apicomplexa-conserved sequence regions. Specific and distinctive PCR profiles were obtained for primers spanning the internal transcribed spacer locus for each of several Plasmodium and Babesia species.

Identification of insecticidal activity of Bacillus thuringiensis against the silkworm by DNA hybridization method

Identification of insecticidal activity of Bacillus thuringiensis against the silkworm by DNA hybridization method

Dried gel hybridization in place of southern hybridization for detection of Listeria monocytogenes DNA fragments.

A simple, sensitive, dried gel DNA hybridization method for detection of Listeria monocytogenes DNA fragments is described. DNA samples were fractionated on an agarose gel. The gel was then denatured in NaOH-NaCl and neutralized in Tris-NaCl. The resulting agarose gel was dried and hybridized with 32P-labelled DNA probe. No transfer to nitrocellulose membranes was used.

A Comparison of an Enzyme Immunoassay, DNA Hybridization, Antibody Immobilization, and Conventional Methods for Recovery of Naturally Occurring Salmonellae from Processed Broiler Carcasses

Three hundred and ninety raw processed broiler carcasses were obtained from four processing plants and evaluated for the presence of salmonellae by the whole bird rinse procedure. Enzyme immunoassay (Salmonella-Tek™), colorimetric DNA hybridization (GENE-TRAKR), and antibody immobilization (1–2 Test™) methods were compared to the Food Safety Inspection Service (FSIS) culture method for detection of salmonellae.All samples were preenriched in buffered peptone water incubated at 37°C then transferred to and enriched in TT broth (Difco, Detroit, MI) incubated at 42°C for 24 h. After enrichment, manufacturer's instructions were then followed for “rapid” procedures and the FSIS recovery and confirmation scheme was followed for the cultural method. For each sample, if discrepant results were observed between the four procedures, selective plates were streaked from both the TT selective broth and the GN postenrichment broth (Difco). All samples which were positive after initial sampling of TT broth plus additional positive samples from TT selective and GN postenrichment broths were called confirmed cultural positives.Salmonellae were detected on 71% of the carcasses using confirmed cultural procedures and 65% with FSIS culture. Presumptive salmonellae positive samples were found 66, 71, and 76% of the time with the 1–2 Test, GENE-TRAK, and Salmonella-Tek, respectively. As compared to confirmed culture, only 1.4% false positives were observed with the 1–2 Test and GENE-TRAK, whereas 6% false positives were obtained using Salmonella-Tek. When the false negatives for the four methods were compared to confirmed culture, Salmonella-Tek, GENE-TRAK, 1–2 Test, and FSIS culture had 0.4, 2.5, 7.2, and 8.3%, respectively. The close correlation of these rapid procedures to the conventional procedures warrants consideration of these procedures for detection of salmonellae from broiler chickens.

A Colorimetric DNA Hybridization Method for the Detection of Escherichia coli in Foods

A colorimetric DNA hybridization assay has been developed for the rapid detection of Escherichia coli in foods. The method employs two oligonucleotide probes which are specific for the 16S ribosomal RNA of E. coli. Probes are added to lysates of test cultures and allowed to hybridize to target rRNA if present. The probe-target complex is captured via hybridization to a polystyrene dipstick. The immobilized target is detected using an antibody-horseradish peroxidase conjugate which binds to the immobilized probe-target complex. The probe-target-antibody complex generates a colorimetric signal when exposed to a substrate/chromogen mixture. A total of 233 E. coli isolates representing typical, toxigenic, invasive, hemorrhagic serotype 0157:H7, and other pathogenic strains all resulted in a positive assay signal. Dose-response experiments indicate the sensitivity of the assay is approximately 1 × 106 CFU/ml. Specificity of the assay was determined by testing 207 strains of non-E. coli species at 109 CFU/ml. All of the non-E. coli organisms tested were negative with the exception of Escherichia fergusonii and Shigella species. A total of 345 enriched samples including inoculated, uninoculated, and naturally-contaminated foods was tested for the presence of E. coli by the hybridization assay and a conventional cultural method. The false-negative rate for the hybridization assay was 1.2%. By comparison, the false-negative rate for the culture method in these studies was 23.4%. Based on these data, the DNA hybridization method is significantly more accurate than conventional methods for the detection of E. coli in foods.

Organelle analysis of symmetric and asymmetric hybrids between Lycopersicon peruvianum and Lycopersicon esculentum

The organelles of somatic hybrids obtained from symmetric and asymmetric fusions between the Lycopersicon species L. peruvianum and L. esculentum were analyzed by DNA hybridization methods. In the asymmetric fusions the L. peruvianum protoplasts were gamma-irradiated at a dose of 50, 300 and 1,000 Gy. The organelles were characterized using the Petunia chloroplast probe pPCY64 and the mitochondrial EcoRI-SalI fragment of the Pcf gene. In all symmetric and asymmetric hybrid plants, a total of 73 being analyzed, only one of the parental chloroplast genomes was present, except for one hybrid plant which harbored both parental chloroplast genomes. No recombination and/or rearrangement in the chloroplast genome could be identified with the pPCY64 probe. Irradiation of the L. peruvianum protoplasts did not significantly reduce the fraction of asymmetric hybrids with L. peruvianum chloroplasts. A novel mitochondrial restriction pattern was present in 5 out of 24 hybrids tested. In 9 hybrids novel combinations of chloroplasts and mitochondria were found, indicating that both organelle types sorted out independently.

Comparative study of a DNA hybridization method and two isolation procedures for detection of Yersinia enterocolitica O:3 in naturally contaminated pork products

We compared a DNA-DNA hybridization assay, using a synthetically produced oligonucleotide probe, and two conventional isolation procedures (methods A and B) with regard to their relative efficiency in detecting Yersinia enterocolitica O:3 in naturally contaminated pork products. Method A was as described by Wauters et al. (Appl. Environ. Microbiol. 54:851-854, 1988). Method B has been recommended by the Nordic Committee on Food Analysis (method no. 117, 1987). The genetic probe was used in a colony hybridization assay to detect virulent yersiniae at each of the isolation steps with composed methods A and B. A total of 50 samples of raw pork products obtained from 13 meat-processing factories in Norway were examined. Y. enterocolitica serogroup O:3, biovar 4, was isolated from altogether 9 (18.0%) of the samples by using the two isolation procedures. In contrast, colony hybridization using the genetic probe indicated that 30 (60.0%) of the samples contained virulent yersiniae. All samples which were positive on cultivation were also positive by hybridization. The results indicate that the occurrence of pathogenic Y. enterocolitica in Norwegian pork products is substantially higher than previously demonstrated and, therefore, reinforce our suggestion that pork products represent an important potential source of human infection in Norway. The results also indicate that the use of conventional isolation procedures may lead to considerable underestimation of pathogenic Y. enterocolitica in pork products.

Species composition shift of confined bacterioplankton studied at the level of community DNA

Using a new DNA hybridization technique that does not require culturing, we compared the species composition of natural planktonic bacterial assemblages before and after confinement in 20 1 containers for ca 2 d Although confinement is known to cause species shifts, possibly by stimulating growth of certain types of cells near the container wall, we found that such shifts were minor; 5 to 15 % of the communities had changed during confinement. The greatest shifts occurred in the samples that had the fastest bacterial growth rate measured by [Hlthymidine incorporation. Despite the minor changes in species composition, the fraction of the cultivable cells (colony forming units; CFU) increased 4to 23-fold, but amounted to 70 % DNA homology belong to one species (Wayne et al. 1987), strict interpretation of community DNA similarity to within a few percent is not possible, partly because there is a slight cross-hybridization between species (generally < 10 %). Details of the concept, tests and evaluations, and limitations of the method are presented in Lee & Fuhrman (1990). In this study, confinement effects on bacterial species compositions were examined by the community DNA hybridization method. An important difference from previous studies was the use of larger (20 1) volume containers, needed to obtain sufficient DNA for the hybridizations. We also investigated changes of CFU, cell concentrations, and growth rates under confinement. The community DNA hybridization showed ca 5 to 15 % of species composition shift while the CFU were always < 2 % of the total cell count. Materials and methods. Sampling: Naturally occurring marine planktonic bacteria were sampled (Table 1) from an oligotrophic Pacific station (550 km west of San Diego, USA) at 25 m (OpenPac25) and 100 m (OpenPaclOO) water depths, and from a jetty at Playa Del Rey, Santa Monica Bay, Los Angeles, CA (CoastalPac). Other samples included in the community DNA comparisons were from a beach at Crane Neck, Long Island, New York, USA, (LIS) and the Caribbean Sea (Carib; 100 km west of Dominica). To collect the seawater, we used Niskin bottles (OpenPac and Carib samples), or clean plastic buckets thoroughly rinsed with seawater (CoastalPac and LIS samples). The water collected with the buckets was transferred to clean (see below) 20 1 polyethylene containers for transport and storage (1.e. confinement). Confinement experiment: The seawater samples were transferred to and stored in 20 1 polyethylene containers (Cubitainer; Consolidated Plastics Co., Inc., Twinsburg, OH, USA) which were soaked overnight with 10 % HC1 and rinsed thoroughly with the sample seawater. No treatment was given except aging in 20 1 containers. The OpenPac25 sample was stored on the ship deck in the shade for 48 h, and the OpenPaclOO sample was stored in the ship's lab for 32 h. The CoastalPac sample was stored indoors in the lab at University of Southern California for 48 h. Storage temperatures were 14 to 18 'C (OpenPac) and 21 'C (CoastalPac), whereas in situ temperatures were 17 OC (OpenPac25), 13 OC (OpenPaclOO), and 20 'C (CoastalPac). Cell collection for DNA: Bacterial cells were collected by pressure filtration as described previously (Lee & Fuhrman 1990). In brief, the water was first pre-filtered through type AE glass fiber filters (142 mm diameter; Gelman Sciences Inc., Ann Arbor, MI, USA) to remove larger particles and eukaryotic cells, and bacterial cells were collected on 0.22 pm pore size Durapore filters (142 mm diameter; Millipore Corp., Bedford, MA, USA). The filtrations were done in a relatively short period of time (total of 1.5 to 3 h after sample collection, depending on the volume filtered). Subsamples were taken from unfiltered seawater, intermediate filtrate (between AE filter and Durapore filter) and final filtrate. Filtering efficiencies were monitored from subsamples by the acridine orange direct count (AODC) method (Hobbie et al. 1977). Filtration was done in the same way for stored seawater samples at the end of confinement. DNA extraction: We followed the protocol of Fuhrman et al. (1988) to extract DNA from the cells collected on Durapore filters, with one more step of purification with phenol/chloroform/isoamyl alcohol (24: 6: 1 by vol, pH 8.0). After final purification and redissolution in TE (10 mM Tris, 1 mM EDTA, pH 8.0) at the concentrations of 100 to 500 ng p l l , DNA was quantified by Hoechst 33258 dye (bisbenzimde; Sigma Chemical Co., St. Louis, MO, USA) fluorometry (Paul & Myers 1982). Probe and target DNA preparation: Probe DNA was labeled with [alpha-^S]dATP (DuPont, NEN Research Products, Boston, MA, USA) by nick translation and purified as described previously (Lee & Fuhrman 1990). Probes were dried by vacuum centrifugation Lee & Fuhrman: Species composition shift of confined microorganisms 197 Table 1. Dates and locations of natural bacterioplankton samples

Detection of human cytomegalovirus in cervicovaginal cells by culture, in situ DNA hybridization and DNA amplification methods

The presence of human cytomegalovirus (HCMV) was tested in 388 cervicovaginal cells specimens obtained from the same number of pregnant women. HCMV was detected in 5·41%, 11·6% and 13·9% of these specimens by conventional culture, in situ DNA hybridization and polymerase chain reaction (PCR) methods, respectively. The sensitivities of detecting HCMV by in situ hybridization and PCR methods were 76·2% and 90·5% and the specificities were 92·1% and 90·5%, respectively, when compared with conventional culture method. The PCR compared favourably with both conventional culture and in situ hybridization methods and it may become a valuable and useful tool for the early and rapid detection of HCMV in clinical specimens.

Mineralization of orthanilic acid is a plasmid-associated trait in Alcaligenes sp. O-1

A bacterial isolate (O-1) which degrades different substituted benzenesulphonates was identified as a strain of Alcaligenes sp. using chemotaxonomic, DNA:DNA hybridization and serolgical methods. Its ability to mineralize orthanilic acid (2-aminobenzenesulphonate) is correlated with the presence of a 117 MDa plasmid, designated pSAH. Selective curing of pSAH resulted in the abolition of the orthanilic acid minieralization phenotype, which could be fully restored by re-establishment of pSAH. A second plasmid of 6·8 MDa, designated pME1702, with no detectable functions, is also present in Alcaligenes sp. O-1. Upon conjugation with Alcaligenes sp. O-1 as donor, the plasmid-free recipient Pseudomonas putida PaW130 acquird the ability to utilize orthanilic Alcalignes sp. O-1. Growth characteristics, the inducibility of enzyme activity (desulphonation), and expression of characteristic proteins during mineralization of orthanilic acid indicate that a plasmid-encoded cluster of genes is involed in the biodegradation of orthanilic acid. Southern blot hybridizations did not reeal any relationship between pSAH and the archetypal TOL plasmid pWW0, which also mediates degradation of substituted aromatic compounds.

Detection of human papillomavirus-genomic DNA in oral epithelial dysplasias, oral smokeless tobacco-associated leukoplakias, and epithelial malignancies

Human papillomavirus (HPV) is an infectious agent that is increasingly associated with mucosal cancers, in particular cancer of the cervix. The present investigation was undertaken in an attempt to determine whether HPV could be easily detected in biopsies of oral tissues, specifically oral squamous cell carcinomas, oral epithelial dysplasias, smokeless tobacco keratoses, verrucous hyperplasia, and verrucous carcinoma. In situ DNA hybridization methods were used to isolate specific HPV genomes. Among 100 instances of benign leukoplakia, only 4% of non-tobacco-related and 10% of smokeless tobacco-related lesions harbored viral sequences. We were able to detect viral sequences in dysplastic lesions 3% of the time. Alternatively, 17% and 20% of the verrucous hyperplasias and verrucous carcinomas were positive for viral nucleic acids. Six percent of the squamous cell carcinomas harbored HPV. On the basis of these findings, it is concluded that HPV of known genotype can be identified in oral premalignant and malignant neoplasms.

Amplification of kinetoplast DNA as a tool in the detection and diagnosis of Leishmania

This paper demonstrates how the polymerase chain reaction can be used to increase the sensitivity of detection of Leishmania parasites by DNA hybridization methods through the amplification of the minicircle target sequence. The oligonucleotide primers used are able to direct the amplification of all Leishmania strains tested. In addition, the PCR products from L. mexicana and L. braziliensis strains can be distinguished by hybridization with kDNA probes. The method is sensitive enough to detect the kDNA from a single organism and this sensitivity allows the use of nonradioactive hybridization methods. This method can be used to detect Leishmania from human biopsy material.

Development of a Colorimetric, Second Generation Nucleic Acid Hybridization Method For Detection of Salmonella in Foods and a Comparison With Conventional Culture Procedure

ABSTRACTA new, highly specific colorimetric DNA hybridization method (cDNAH) for detection of Salmonella in foods was developed. It detected 355/362 strains representing 206/213 serovars of Salmonella, and showed no cross‐reactivity with 32 different non‐Salmonella species, including a variety of Enterobacter and Citrobacter species. The cDNAH method was compared with the BAM/AOAC culture method in two studies: (1) with 20 different inoculated and uninoculated foods overall agreement between the two methods was 99%. (2) with naturally contaminated foods at two independent sites agreement with culture was 99.8% where 9/404 samples were positive for Salmonella.

DNA Relatedness among Strains of Leptospira biflexa

The slot blot method of DNA hybridization was used to study 38 strains of Leptospira biflexa belonging to 38 serovars. Fifteen of these serovars were placed into six groups. The remaining 23 serovars were generally too diverse to show significant DNA relatedness either to these groups or to one another. Serovar thracia was related to Group 5, but it was not included in this group because its percent relatedness was too low. We found that genetically related organisms were antigenically dissimilar. The absence of any significant genetic relationship between Leptonema illini and the Leptospira biflexa serovars tested supports the placement of the former species in a separate genus.

Comparative Study of Colorimetric DNA Hybridization Method and Conventional Culture Procedure for Detection of Salmonella in Foods

A second generation nucleic acid hybridization assay has been developed and evaluated against the conventional culture method for detection of salmonellae in foods. The assay involves a liquid hybridization with Salmonella-specific oligonucleotide probes, capture of probe:target hybrids onto a solid support (plastic dipstick), and a colorimetric end point detection. The assay can be completed in 2.5 h, following approximately 44 h of culture enrichment. One thousand samples representing 20 food types were analyzed in parallel by both methods. Samples included uninoculated test product, and product inoculated with Salmonella at 2 levels. Eighteen Salmonella serotypes were used as inocula. The data demonstrate that the colorimetric hybridization method and the conventional culture method are equivalent in their ability to detect Salmonella contamination of foods.

Evaluation of sensitivity of different antigen and DNA-hybridization methods in African swine fever virus detection

ELISA, immunodot and DNA hybridization methods have been adapted to detect African swine fever virus (ASFV), and their sensitivities were compared using virus obtained from cell cultures. About 2.3 × 10 2 50% hemadsorbing doses (HAD 50) of virus were detected with ELISA sandwich using an anti-ASFV IgG biotinylated followed by avidin-peroxidase. The immunodot technique showed similar sensitivity, detecting about 4.6 × 10 2 HAD 50 of virus. ASFV-DNA was detected using radioactive DNA probes and molecular hybridization. The maximal viral detection capacity of this technique was about 1.8 × 10 3 HAD 50. The antigenic and DNA detection of ASFV during the infection of animals with virulent and attenuated viruses, was also studied. For this purpose, sera and red blood cells from several infected pigs were obtained at different days post-inoculation. The virus was detected at the third day after infection by the three methods. However, ASFV-DNA detection was more efficient than antigenic detection at nine days post-inoculation, when antigen detection failed, because immuno-complexes with circulating viruses were formed in the subacute infection.

Colorimetric Deoxyribonucleic Acid Hybridization Assay for Rapid Screening of Salmonella in Foods: Collaborative Study

A collaborative study was performed in 11 laboratories to validate a colorimetric DNA hybridization (DNAH) method for rapid detection of Salmonella in foods. The method was compared to the standard culture method for detection of Salmonella in nonfat dry milk, milk chocolate, soy isolate, dried whole egg, ground black pepper, and raw ground turkey. Samples inoculated with high (0.4-2 cells/g) and low (0.04-0.2 cells/g) levels of Salmonella and uninoculated control samples were included in each food group analyzed. There was no significant difference in the proportion of samples positive by DNAH and culture procedure for any of the 6 foods. The colorimetric DNA hybridization assay screening method has been adopted official first action as a rapid screening method for detection of Salmonella in all foods.

Comparison of two methods in the determination of the sensitivity of 84 herpes simplex virus (HSV) type 1 and 2 clinical isolates to acyclovir and alpha-interferon

Two methods, the colorimetric method (neutral red dye uptake), and DNA hybridization using a HSV thymidine kinase gene probe (TK) have been used to examine the sensitivity of 84 herpes simplex virus (HSV) type 1 and 2 clinical isolates to two antiviral drugs, acyclovir (ACV) and alpha-interferon (α-IFN). Using the colorimetric method, HSV isolates had ED 50s ranging from 0.03 ± 0.02 μg/ml to 0.164 ± 0.03 μg/ml for ACV and 6.3 ± 5.2 IU/ml to 55.0 ± 11.4 IU/ml for α-IFN. With the DNA hybridization method, ED 50s ranged from 0.033 ± 0.012 μg/ml to 0.190 ± 0.031 μg/ml for ACV and 8.5 ± 5.0 IU/ml to 43.5 ± 6.0 IU/ml for α-IFN. Two strains of HSV-1 were found to be resistant to very high concentrations of ACV (>50.0 μg/ml). The values obtained by the two methods showed good correlation ( r=0.724, P=0.002). Furthermore, our results demonstrate that the two methods are reproducible, reliable and the dye uptake assay is suitable for use in a diagnostic virology laboratory.

Detection of human papillomaviruses in exfoliated cervicovaginal cells by in situ DNA hybridization analysis

A total of 851 specimens of exfoliated cervicovaginal cells and 27 specimens of male urethral smears obtained from 706 individuals with various clinical findings were examined for the presence of human papillomavirus (HPV) types 6, 11, 16, 18, 31, and 33 by in situ DNA hybridization analysis. The nonradioactive DNA in situ hybridization method used in this study showed no detectable cross-hybridization either among different types of HPV (except between types 6 and 11) or between HPV DNA and human cellular DNA. Furthermore, this system was found to be more sensitive than the Southern blotting method in detecting HPV. HPV was found in 233 of 276 (84.4%) and in 34 of 47 (72.3%) samples of cervicovaginal cells from patients with urogenital condylomata and cervical dysplasia, respectively. HPV was also detected in 6 of 39 (15.4%) women with normal cytological findings who were also symptom-free. Young women who were at low risk but were infected with HPV showed significantly reduced ratios of helper-inducer T lymphocytes to suppressor-cytotoxic T lymphocytes compared with those of uninfected normal controls (1.28 +/- 0.31 versus 2.47 +/- 0.64; P less than 0.001). This in situ DNA hybridization method can have broad application to the screening of HPV in early lesions and in normal-looking tissues and may be used to identify patients at risk of more serious or possibly malignant progression.

DNA hybridization with oligodeoxyribonucleotide probes for identifying enterotoxin-producing Escherichia coli.

The DNA hybridization method using oligodeoxyribonucleotide probes was compared with the GM, ELISA and the infant mouse assay for determining production of LT and ST, respectively, by 2000 strains of Escherichia coli. Sensitivity and specificity by DNA hybridization for LT were 98.7 and 99.8%, and for ST were 78.8 and 99.6%.