Non-radioactive Hybridization Research Articles

Trypanosoma cruzi: Parasite Detection and Strain Discrimination in Chronic Chagasic Patients from Northeastern Brazil Using PCR Amplification of Kinetoplast DNA and Nonradioactive Hybridization

Blood samples from 172 individuals from northeastern Brazil were subjected to PCR amplification of Trypanosoma cruzi-specific kDNA sequences. This method enabled us to detect parasite DNA in 21 of 47 patients that were serologically positive, In addition, 1 patient that gave doubtful results with chagasic serology was confirmed as positive by PCR. We applied the same PCR detection method to the feces of wild triatomines captured in the same region, obtaining three positive results that were confirmed by microscopic examination. The 25 amplified products obtained in this study were then reamplified with primers that gave a final amplicon containing sequences from the most variable region of kDNA minicircles. These were used as probes in hybridization experiments aimed at defining the degree of relatedness between the strains infecting humans and insects based on kDNA homologies. We found that the amplification products from the three triatomines were related and showed no crosshybridization with those obtained from human infections. Eight amplified products from human infections showed no cross-hybridization and did not hybridize with products from other patients. This indicates that the strains of T. cruzi circulating in the region present a high level of genetic heterogeneity. Finally, a number of amplified products hybridized with amplicons that did not hybridize with each other, indicating that infections with a parasite population presenting a mixed kDNA content (either due to different strains of T. cruzi or to a hybrid parasite) are a more frequent event than previously thought.

Synthesis and evaluation of a non-radioactive gene probe for the detection of C. perfringens alpha toxin

The synthesis and evaluation of a non-radioactive hybridization probe is described, specific detecting the Clostridium perfringens alphatoxin gene (plc) by colony blot hybridization assay. A vector free digoxigenin-dUTP-labelled probe was generated by polymerase chain reaction (PCR) targeting the cloned plc gene of C.perfringens strain ATCC 13124. In a colony blot hybridization assay 296 strains of C.perfringens were tested for plc. None of the strains failed in hybridization. Presence of plc was even demonstrated in C.perfringens strains reported to lack lecithinase activity. Specificity of the probe was shown with various strains of other bacterial species. None different Clostridia sp. tested, e.g. C.bifermentans, C.tertium, C.novyi, C.chauvoei, C.sporogenes, C.difficile, C.putrifucum, C.sordellii, C.botulinum, C. septicum and C.histolyticum, hybridized with the plc specific probe. Strains expressing an enzymatically related phospholipase like Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus gave also negative results. Comparing the results of conventionally used egg yolk turbidity assay and those gained with DNA hybridization, the plc probe proved to be a much more sensitive and specific diagnostic tool for the detection of C.perfringens plc.

Non-radioactive multiplex-SSCP analysis: detection of a new type II procollagen gene (COL2A1) mutation.

We have developed a protocol that allows fast and efficient mutation screening of the 54 exons from the type II procollagen (COL2A1) gene. The protocol is based on the multiple non-radioactive hybridization of blotted single-strand conformation polymorphism gels. Using this screening procedure we have been able to identify a new (Gly895 to Ser) mutation in the COL2A1 gene of a patient with a mild form of spondyloepiphyseal dysplasia congenita.

Rapid detection ofSalmonellasubspecies I by PCR combined with non-radioactive hybridisation using covalently immobilised oligonucleotide on a microplate

A polymerase chain reaction (PCR)-based test was developed for the detection of Salmonella. One pair of oligonucleotide primers was designed to amplify a 93-bp fragment of a gene required for the invasion of HeLa cells by Salmonella ser Typhi strain Ty2. The amplified product was analysed by non-radioactive sandwich hybridisation in microtiter plates using two oligonucleotides. The capture oligonucleotide was covalently linked onto animated wells of microtiter plates. The detection oligonucleotide was labelled with biotine. The hybrid molecules were detected by avidine conjugated with alkaline phosphatase and chromogenic substrate. The described combination of microplate sandwich hybridisation and PCR seems to be a suitable method for rapid detection of Salmonella subspecies I. It only requires a thermal cycler and a conventional microtiter reader, and can be readily done on a large scale.

Rapid detection of Salmonella subspecies I by PCR combined with non-radioactive hybridisation using covalently immobilised oligonucleotide on a microplate

A polymerase chain reaction (PCR)-based test was developed for the detection of Salmonella. One pair of oligonucleotide primers was designed to amplify a 93-bp fragment of a gene required for the invasion of HeLa cells by Salmonella ser Typhi strain Ty2. The amplified product was analysed by non-radioactive sandwich hybridisation in microtiter plates using two oligonucleotides. The capture oligonucleotide was covalently linked onto aminated wells of microtiter plates. The detection oligonucleotide was labelled with biotine. The hybrid molecules were detected by avidine conjugated with alkaline phosphatase and chromogenic substrate. The described combination of microplate sandwich hybridisation and PCR seems to be a suitable method for rapid detection of Salmonella subspecies I. It only requires a thermal cycler and a conventional microtiter reader, and can be readily done on a large scale.

Determining relative abundance of specific DNA sequences in flow cytometrically sorted maize nuclei

A wide breadth of DNA content variation has been reported among maize lines. While the extent of this variation has been well documented, few studies have focused on its cause. Some of the nuclear DNA content variation has been explained by the presence of B chromosomes or knobs. However, variation in these two structures does not account for all of the observed variation. In order to identify other fluctuating DNA sequences, a rapid and reliable method of estimating relative abundance of DNA sequences needed to be developed. The potential of flow cytometry in conjunction with spot hybridization for determining relative abundance of specific DNA sequences in maize was studied. Different numbers of G1 phase nuclei were sorted on nitrocellulose filters and non-radioactive hybridization and signal detection performed. Results from these experiments revealed a significant, positive linear correlation between the amount of target sequence and signal density using both knob (R = 0.98) and ribosomal spacer (R = 0.99) DNA sequences. In addition, G1 phase nuclei of eight inbred lines differing in the amount of knob heterochromatin, were sorted on to filters and the non-radioactive hybridization and signal detection performed. A significant, positive linear correlation between C-band number and signal density (R = 0.83; P = 0.0051) as well as between per cent heterochromatin and signal density (R = 0.96; P = 0.0002) was observed. These results indicate the usefulness of flow cytometry for spot hybridization in determining the relative abundance of DNA sequences in the maize genome

Detection of Microbial Cells in Aerosols Using Nucleic Acid Probes

Non-radioactive hybridization methods were evaluated for the identification of microorganisms in mixed bioaerosols. A cultivation-dependent method, colony hybridization, was compared to a direct, cultivation-independent approach, whole cell hybridization with fluorescently labeled oligonucleotides. After sampling of the aerosols by filtration, special processing of filters (cells) preceded hybridization with fluorescently, digoxigenin- or enzyme-labeled oligonuculeotide probes. Group, genus, or species affiliation of collected cells was analyzed with rRNA-targeted probes. Using nucleic acid probes directed against the multiple cloning site, plasmid bearing Escherichia coli colonies could be differentiated from wild-type colonies. The microbial composition of aerosols ranging from less than one to greater than 109 cells/m3 air could be analyzed with appropriate hybridization formats: whole cell hybridization was only applicable to dense aerosols, colony hybridization yielded best results with lower concentrations. After a short incubation period (several hours), a combination of both formats could be used to rapidly determine the fraction of culturable cells within a bioaerosol. When applying these techniques for the monitoring of aerosols generated by standard microbiological laboratory procedures, low concentrations of airborne Escherichia coli cells (1–450 m−3) could be detected. Compared to conventional air monitoring techniques, hybridization with nucleic acid probes should allow more rapid and reliable detection of airborne microorganisms including genetic engineered microorganisms.

Detection and identification of Clavibacter michiganensis subsp. sepedonicus and Clavibacter michiganensis subsp. michiganensis by nonradioactive hybridization, polymerase chain reaction, and restriction enzyme analysis

To develop a rapid and reliable detection and identification method for Clavibacter michiganensis subsp. sepedonicus and C. michiganensis subsp. michiganensis, two biotinylated probes and derived primer sets were evaluated for specificity using a large number of bacterial strains. Detection in dot blot analysis using the Diagen probe against C. michiganensis subsp. sepedonicus was possible with all 32 C. michiganensis subsp. sepedonicus strains tested. Cross-hybridization occurred with all nine C. michiganensis subsp. insidiosus strains tested. No hybridization occurred with any of 54 other related and unrelated bacterial strains including C. michiganensis subsp. michiganensis, C. michiganensis subsp. nebraskensis, C. michiganensis subsp. tessellarius, C. iranicus, C. rathayi, and C. tritici and potato saprophytes. Hybridization of the MIC 1 probe against C. michiganensis subsp. michiganensis was obtained with 22 out of 24 C. michiganensis subsp. michiganensis strains. A weak hybridization signal occurred only with two strains of C. michiganensis subsp. insidiosns. No hybridization occurred with any of the 71 other related and unrelated bacterial strains tested including tomato saprophytes. Restriction fragment length polymorphisms detected with the Diagen probe allowed differentiation between C. michiganensis subsp. sepedonicus and the related C. michiganensis subsp. insidiosus. Restriction fragment length polymorphism analysis using the MIC 1 probe and BamH1 showed at least two groups of patterns within C. michiganensis subsp. michiganensis. By using a primer set derived from the Diagen probe, a DNA sequence could be amplified with all C. michiganensis subsp. sepedonicus strains tested. Only the nontarget organism C. michiganensis subsp. insidiosus yielded a similar polymerase chain reaction product. Restriction enzyme analysis of the polymerase chain reaction product enabled rapid distinction between the subspecies. With a CMM primer set derived from the MIC 1 probe a DNA sequence was amplified from the same 22 out of 24 C. michiganensis subsp. michiganensis strains that showed hybridization with the MIC 1 probe. The polymerase chain reaction product could be verified by restriction enzyme analysis. The Diagen and MIC 1 probes and derived primer sets were shown to be useful for the detection and identification of C. michiganensis subsp. sepedonicus and C. michiganensis subsp. michiganensis. The MIC 1 probe, however, failed to detect two strains of the latter subspecies.Key words: biotin, PCR, REA, potato bacterial ring rot, bacterial canker of tomato, RFLP, Clavibacter michiganensis subsp. insidiosus.

Comparison of polymerase chain reaction and nonradioactive hybridization techniques for the identification of Mycobacterium avium strains

A polymerase chain reaction able to amplify specifically a 205-base-pair DNA fragment of Mycobacterium avium genome was used compared to a nonradioactive hybridization assay (AccuProbe) and to conventional biochemical identification. The PCR approach to diagnosis of M. avium infection is a valid diagnostic alternative to the more classical procedures.

Comparison of polymerase chain reaction with standard methods in the diagnosis ofMycobacterium tuberculosis infection

The polymerase chain reaction (PCR) method was evaluated in the detection of Mycobacterium tuberculosis in comparison with direct microscopy and culture procedures including the standard radiometric system (Bactec). Amplified DNA fragments from clinical samples were analyzed by dot-blot and non-radioactive oligonucleotide hybridization techniques or by agarose gel electrophoresis. The results revealed nested PCR to be the method of choice. The combination of three culture methods could detect Mycobacterium tuberculosis in only 79% of PCR-positive cases. The mean time required to achieve a positive culture result was 17 days in contrast to the PCR method requiring only two to three days. The nested PCR assay provided rapid and reliable results allowing a definitive diagnosis particularly in a number of samples which were negative on culture.

Identification of Clostridium perfringens by 16S and 23S rRNA Oligonucleotide Probes

The published 16S rRNA sequence of Clostridium perfringens [ Gamier et al. (1991] was compared to the 16S rRNA database of Clostridium species and two C. perfringens-specific oligonucleotide probes were developed. In addition, the 23S rDNA of C. perfringens was partially sequenced and used to design a third oligonucleotide probe. The specificity of the probes was determined by dot blot hybridisation using crude rRNA from several strains of each C. perfringens type, Gram-positive representatives of various genera and Escherichia coli . All probes were specific for C. perfringens but one 16S rRNA probe failed to detect all of the C. perfringens strains tested. Using the polymerase chain reaction technology in combination with a nonradioactive hybridisation assay the identification of isolated DNA from pure cultures of C. perfringens that express the whole range of C. perfringens toxin types was achieved within a few hours.

Use of oligonucleotide-alkaline phosphatase conjugates as non-radioactive probes for rapid analysis of a proteinase inhibitor gene fromZea mays

Conjugates of oligonucleotides and alkaline phosphatase have been prepared and used as nonradioactive hybridization probes for the study ofPis3 (=MPI) a gene encoding a proteinase inhibitor fromZea mays. Attachment of the alkaline phosphatase was carried out either at the 5′ or 3′ end of two 25-bp oligonucleotides. Sensitivity of each alkaline phosphatase-oligonucleotide probe was assessed using a chemiluminescent substrate for detection of alkaline phosphatase activity. This sensitive method allows the rapid analysis of genomic clones isolated from aZea mays library and the subsequent characterization of the completePis3 gene without the need for construction of restriction maps for the cloned DNA fragments. This general strategy may be valuable for the identification of any gene for which a limited sequence is known and for location of specific DNA sequences that represent a small region within a larger DNA fragment.

Detection of herpes simplex virus DNA from cerebrospinal fluid by PCR and a rapid, nonradioactive hybridization technique

A molecular assay for the detection of herpes simplex virus (HSV), including a novel, nonradioactive hybridization technique, was evaluated with a total of 123 cerebrospinal fluid specimens. After DNA extraction, specific HSV DNA sequences were amplified with digoxigenin-labeled primers derived from the DNA polymerase gene-coding region from HSV. Amplified products were detected by the Enzymun-Test DNA detection assay (Boehringer, Mannheim, Federal Republic of Germany), which uses biotinylated probes. Amplification with nonlabeled primers and then Southern blotting and nonradioactive detection of hybrids by the digoxigenin technique was the reference system. The sensitivities of the molecular assays were determined with 10-fold dilutions of plasmid pS4 with the SalI restriction fragment of the DNA polymerase gene obtained from the HSV type 1 strain Angelotti. The Enzymun assay was able to detect all of the 16 positive samples, giving 100% agreement with the Southern blot hybridization results. Optical density values were widely separated for the positive and negative groups of specimens. Ten copies of plasmid pS4 per microliter could be distinctly detected by the Enzymun assay. The cutoff was determined for the hybridization assay, and an equivocal zone was defined. The whole molecular assay including the Enzymun-Test DNA detection proved to be sensitive and easy to use. It may contribute to the rapid and safe detection of HSV DNA in cerebrospinal fluid.

Development of 23S rDNA-oligonucleotide Probes for the Identification of Salmonella species

Summary Partial 23S rDNA sequences from Salmonella typhimurium ACM 3598 and Salmonella bongori ACM 3478 were determined, compared to published 23S rRNAs and two oligonucleotide probes developed. These probes were tested against nucleic acids from several species of Salmonella , 63 strains of Salmonella and eleven additional members of the family Enterobacteriaceae by dot blot and colony hybridization. While one probe detected strains of S. choleraesuis, S. enteritidis, S. typhimurium and, with slightly less intensity, S. typhi , the second probes detected strains of S. bongori and additional strains of DNA homology group V only. Using a rapid lysis method and application of the polymerase chain reaction technology to amplify rRNA genes a mixture of both probes allowed the detection of rDNA of pure cultures of Salmonella strains within 10 h in a non-radioactive hybridization assay, thus showing a great potential for a simple, reliable and rapid identification.

Chemiluminescent hybridization assay for hepatitis delta virus RNA in serum

A nonradioactive hybridization assay for the detection of hepatitis delta virus RNA in serum is described. This assay utilizes a digoxigenin-labeled RNA hybridization probe and chemiluminescent immunodetection. The probe can detect as little as 0.4 pg of cDNA with a 60-min exposure. Results obtained are in agreement with serological indicators of hepatitis delta virus infection and are comparable to those obtained by hybridization assays employing radioactively labeled RNA.

Identification of Bacillus licheniformis by Colony Hybridization with 23S rRNA-targeted Oligonucleotide Probes

Summary A rapid method for the identification of bacteria was developed which can be performed even in a routine laboratory. The sequence of 23S rRNA genes of Bacillus licheniformis was determined and two complementary oligonucleotide probes were synthesized. The specifity of the probes was checked by non-radioactive dot blot and colony hybridization experiments with Bacillus type strains typically occurring in food and with original isolates from milk and cream. One oligonucleotide probe hybridized to target nucleic acids from all Bacillus licheniformis strains tested, whereas the second probe was specific for only part of the strains studied. The probes can be used for the specific and fast detection and identification of Bacillus licheniformis .

Adenosine A 2a receptor mRNA is expressed by enkephalin cells but not by somatostatin cells in rat striatum: a co-expression study

The cellular co-expression of adenosine A 2a receptor mRNA and preproenkephalin A (PPE A) mRNA and A 2a receptor mRNA and prosomatostatin (pSRIF) mRNA in rat striatum was studied using a combination of radioactive and non-radioactive in situ hybridization techniques. Cells containing adenosine A 2a receptor mRNA were visualised using an 35S-labelled oligonucleotide whilst those containing PPE A mRNA and pSRIF mRNA were detected using alkaline phosphatase-labelled antisense oligonucleotides; both radioactive and non-radioactive hybridization signals were visualized on the same tissue section. Bright field examination of striatal sections hybridized with both the [ 35S]adenosine A 2a receptor probe and the alkaline phosphatase-labelled PPE A probe revealed dense clusters of silver grains overlying cells containing alkaline phosphatase reaction product demonstrating that the two gene transcripts were expressed by the same medium-sized nerve cells. The cellular expression of the two mRNAs was consistently found to be concordant demonstrating that adenosine A 2a receptor mRNA is expressed by medium-sized striatal enkephalin cells. In contrast, clusters of silver grains were never detected overlying striatal cells containing pSRIF mRNA indicating that this population of interneurones do not express the adenosine A 2a receptor sub-type. The expression of adenosine A 2a receptors by enkephalin cells in striatum suggests that adenosine may play a role in modulating the activity of GABA/enkephalin striatopallidal neurones through interaction with A 2a receptors.

Detection of human papillomavirus by nonradioactive hybridization.

The presence of the human papillomavirus (HPV) DNA sequence in cervical smears of women with cervical intraepithelial neoplasia (CIN) was investigated through dot blot using a nonradioactive method based on the principle of enhanced chemiluminiscence. From 154 samples examined, 59 (38.3%) had HPV-related sequences determined by dot-blot hybridization; among these, 18 samples (11.68%) reacted either to HPV 6/11 or to HPV 16/18 probes, 20 (12.98%) reacted to only HPV 16/18, and 21 (13.63%) reacted exclusively to HPV 6/11. The relative sensitivity of the method was controlled comparing the results of both tests with those obtained by Southern blot in 32 samples. The risk of HPV positivity was increased in women who first had intercourse at an early age and in patients with CIN II or III. Both associations were significant at the 95% confidence interval (CI). An increased risk of HPV positivity was also associated with patient's age at diagnosis, education levels, number of sexual partners, and use of oral contraceptives, but none of these factors were significant at 95% CI. Other risk factors for cervical cancer, such as the number of pregnancies or cigarette smoking, were not found to be associated with HPV infection and indicate that the association of these factors with cervical cancer probably is independent of HPV infection. Even if HPV is the major risk factor for CIN, this also supports a multifactorial model of cervical cancer etiology with an increased risk of HPV infection.

The development and assessment of DNA and oligonucleotide probes for the specific detection of Bacillus anthracis

Two DNA probes and a number of oligonucleotide probes were designed from the virulence factor genes of Bacillus anthracis. These probes were tested for specificity against 52 B. anthracis strains and 233 Bacillus strains encompassing 23 other species. A rapid slot blotting technique was used for screening the large numbers of isolates involved. All probes tested appeared to be specific for B. anthracis under high stringency conditions. These probes could differentiate between virulent and avirulent strains. The probes were also applied to the detection of B. anthracis in routine environmental and clinical samples. A non-radioactive hybridization and detection system based on digoxigenin-11-dUTP was developed.

PCR product quantification by non-radioactive hybridization procedures using an oligonucleotide covalently bound to microwells

Oligonucleotides derived from IS6110, an insertion sequence from Mycobacterium tuberculosis , have been covalently immobilized on polystyrene Covalink NH microwells to develop a sandwich and a competitive non-radioactive hybridization assay for the quantitative determination of the DNA fragments obtained by polymerase chain reaction (PCR). Using the appropriate standard DNA, the method can be employed for the quantitative analysis of PCR fragments. The sandwich assay can detect as little as 3 fmol of target DNA per well and the standard curve may be used with quantities ranging from 3 to 300 fmol per well. The competitive hybridization assay is less sensitive since it is quantitative between 100 and 8000 fmol per well. We show here that both kinds of assays can be used to identify M. tuberculosis strains isolated from clinical samples. The non-radioactive hybridization procedures using an oligonucleotide covalently bound to microwells involve few and simple operations, and are thus suitable for routine diagnosis. Moreover, when stored at 5°C, precoated strips can still be used for hybridization up to at least 10 months.