Nucleic Acid Hybridization Assay Research Articles

Synthetic oligonucleotide probes differentiate respiratory syncytial virus subgroups in a nucleic acid hybridization assay

A new approach to respiratory syncytial virus subgroup differentiation has been developed on the basis of the hybridization of subgroup-specific synthetic oligonucleotides with viral mRNA. Two oligonucleotide probes were designed from the nucleotide sequences of an A and a B subgroup respiratory syncytial virus glycoprotein G gene. All 28 virus isolates tested were correctly classified by subgroup with these probes.

The biotin-(strept)avidin system: principles and applications in biotechnology

The biotin-(strept)avidin system has been used for many years in a variety of different applications. Here we present a general overview of the system, describe its components and advantages, and show how the system is used in various applications, with emphasis on immunological and nucleic acid hybridization assays. This system is now considered a versatile independent technology with broad applications in many branches of biotechnology. Clearly, its use will continue to grow in the years to come.

Dipstick for nucleic acid hybridization assays — permits multiple target analyses : Microprobe Corp WO 9001-564; 22 February 1990

Dipstick for nucleic acid hybridization assays — permits multiple target analyses : Microprobe Corp WO 9001-564; 22 February 1990

Detection of Human Papillomavirus DNA on Routine Papanicolaou's Smears by In Situ Hybridization with the Use of Biotinylated Probes

Specific human papillomavirus (HPV) types have been implicated as playing a major role in the development of cervical neoplasias. HPV DNA types usually have been identified by nucleic acid hybridization assays with the use of radiolabeled probes. These techniques are sensitive and specific but are not suitable for large-scale clinical use. To detect specific HPV DNAs simply and rapidly, in situ hybridization (ISH) with the use of biotinylated HPV DNA 6/11 and 16/18 probes was done on destained Papanicolaou's (Pap) smears from 545 patients. All smears showed koilocytotic changes. HPV DNAs were demonstrated not only in cervical intraepithelial neoplasia (CIN) and atypia, but also in squamous cells with minimal nuclear changes (karyomegaly) and perinuclear halos. HPV DNA 16/18 was detected in 52% of the smears with CIN I, 44% of those with CIN II and III, 19% of those with atypia, and 4% of the minimal changes. HPV DNA 6/11 was detected in 27% of the smears with CIN I, 27% of those with atypia, and 6% of the minimal changes. HPV DNAs were also detected in smears without koilocytotic changes. Thus, ISH with the use of biotinylated probes can serve as an adjunct to Pap smears in detecting HPV infection.

Evaluation of Two Commercially Available Nucleic Acid Hybridization Assays for the Detection and Typing of Human Papillomavirus in Clinical Specimens

Southern blot (Oncor, Gaithersburg, MD) and dot blot (Life Technologies, Gaithersburg, MD) nucleic acid hybridization assays were compared for their ability to detect and type human papillomavirus (HPV) DNA in 50 cervical swab specimens and 11 biopsy specimens. Overall agreement between the two methods was 78.7%. With the use of Southern blot analysis, HPV 6, 11, 16, or 18 was detected in 22 specimens, however, 4 were untypable because of abnormal or smeared band patterns. Dot blot analysis detected HPV 6/11, 16/18, or 31/33/35 in those same 22 specimens and in 9 additional specimens. Eight of the 13 specimens in which HPV was not detected or untypable by Southern blot contained type 31/33/35 by dot blot. Based on convenience of specimen collection and transport, ease of performance and the ability to detect HPV types 31, 33, and 35, the authors are currently using the dot blot assay for the detection and typing of HPV in clinical specimens.

Prolactin messenger ribonucleic acid concentrations throughout the ovine estrous cycle: Assessment relative to prolactin serum and pituitary amounts

Prolactin (PRL) mRNA concentrations were assessed by nucleic acid hybridization assays in pituitaries of ewes representing the defined stages of the ovine estrous cycle. Concomitantly, pituitary and serum PRL concentrations were measured in these ewes using radioimmunoassays. It was observed that PRL serum, pituitary and mRNA concentrations tended to increase near the time of the gonadotropin preovulatory surge, particularly between 24 hrs before behavioral estrus to 5 hours after estrus. However, the changes in PRL mRNA, serum and pituitary concentrations were shown not to be statistically significant. These data suggest that PRL production during the sheep estrous cycle is maintained without dramatic changes in synthesis or secretion.

TIME-RESOLVED FLUOROMETRY WITH LANTHANIDE CHELATES AS LABELS-PRINCIPLES, APPLICATIONS AND NEW DEVELOPMENTS

time-resolved fluorometry with fluorescent lanthanide chelaters as labels is gaining acceptance as a high-sensitivity non-isotopic immunoassay technique. More recently, the same technique has been used for non-isotopic nucleic acid hybridization assays. In this paper, I discuss the basic principles of the technology, its applications in biotechnology and some recent developments and future directions.

A nonradioactive riboprobe assay for the detection of hepatitis B virus DNA in human sera

Sequences of hepatitis B virus DNA were detected specifically in the sera of infected individuals by a non-radioactive riboprobe nucleic acid hybridization assay. The nucleic acids contained in serum specimens were prepared by an overnight proteinase K-/SDS-digest, phenol/chloroform-extraction and ethanol-precipitation, and immobilized on nylon membranes by the dot-blot technique. Digoxigeninlabelled 1.4 kb RNA-probes were generated by the phage sp 6 RNA-polymerase from a linearized HBV DNA transcription template containing the appropriate promoter. Hybridized probes were detected by alkaline phosphatase-conjugated sheep anti-digoxigenin Fab-fragments, and 5-bromo-4-chloro-3-indolylphosphate (BCIP) and nitroblue tetrazolium salt (NET) as chromogenic substrates for the subsequent ELISA procedure. With a detection limit of 0.5–1.0 pg HBV DNA and a high specificity, the results were comparable to those obtained by the standard assay employing radioactively labeled DNA-probes.

Europium Chelate Labels in Time-Resolved Fluorescence Immunoassays and DNA Hybridization Assays

Like many analytical methodologies, immunoassays and nucleic acid hybridization assays rely on the reaction between an analyte of interest and a specific reagent. The analyte concentration is then deduced by measuring either the amount of analyte-reagent complex formed (product) or the amount of residual reagent. The authors describe the application of fluorescent rare-earth chelates to immunoassay and DNA probing.

Methodologies for in vitro nucleic acid amplification and their applications

The capability to detect the genetic elements (DNA or RNA) of a particular pathogen as a means of identifying the infectious agent has been the traditional function of nucleic acid hybridization assays. The low copy number of genetic material from several types of viral pathogens has fostered the development of in vitro nucleic acid amplification methods as a means to increase the copy number of the characteristic genetic elements of pathogenic agents. The polymerase chain reaction (PCR) and a transcription-based amplification system (TAS) are two amplification methods that have been developed to serve this function. Both methods have been employed to study both genetic and infectious disease problems. This review discusses the characteristics of these amplification methods and describes some of their applications, especially in the study of HIV-1.

Differentiation of respiratory syncytial virus subgroups with cDNA probes in a nucleic acid hybridization assay

A new approach to respiratory syncytial (RS) virus subgroup determination was developed by using a simple nucleic acid filter hybridization technique. By this method, virus-infected cells are bound and fixed in a single step, and the viral RNA in the fixed-cell preparation is characterized directly by its ability to hybridize to cDNA probes specific for either the A or B subgroups of RS virus. The subgroup-specific probes were constructed from cDNA clones that corresponded to a portion of the extracellular domain of the RS virus G protein of either a subgroup B RS virus (8/60) or a subgroup A RS virus (A2). The cDNA probes were labeled with 32P and used to analyze RS virus isolates collected over a period of three decades. Replicate templates of infected cell preparations were hybridized with either the subgroup A or B probe. The subgroup assignments of 40 viruses tested by nucleic acid hybridization were in agreement with the results of subgroup determinations based on their reactivities with monoclonal antibodies, which previously has been the only method available for determining the subgroup classification of RS virus isolates. The nucleic acid hybridization assay has the advantage of providing broad-based discrimination of the two subgroups on the basis of nucleic acid homology, irrespective of minor antigenic differences that are detected in assays in which monoclonal antibodies are used. The nucleic acid hybridization technique provides a reliable method for RS virus subgroup characterization.

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.

An assay for quantitative nucleic acid hybridization on membrane filters

A quantitative nucleic acid hybridization assay based on a 6-mm-diameter nitrocellulose membrane filter and only 20 μl of hybridization mixture per determination is described. As a consequence of the small ratio of hybridization volume to membrane surface, the hybridization rates reached in this system are higher than those obtained at volume to surface ratios of conventional protocols, allowing even RNA at very low concentrations to complete hybridization. This advantage, together with the high reproducibility and hybrid stability obtained with the assay, increases the ability of the filter hybridization technique to analyze quantitatively minute amounts of RNA.

Nucleic acid hybridization assays employing dA-tailed capture probes: II. Advanced multiple capture methods

A fourth capture is added to the reversible target capture procedure of the preceding paper. This results in an improved radioisotopic detection limit of 7.3 × 10 −21 mol of target. In addition, the standard triple capture method is converted into a nonradioactive format with a detection limit of under 1 amol of target. The principal advantage of nonradioactive detection is that the entire assay can be performed in about 1 h. Nucleic acids are released from cells in the presence of the (‘capture probe’) which contains a 3′-poly(dA) sequence and the (‘labeled probe’) which contains a detectable nonradioactive moiety such as biotin. After a brief hybridization in solution, the target is captured on oligo(dT) magnetic particles. The target is further purified from sample impurities and excess labeled probe by recapture either once or twice more on fresh magnetic particles. The highly purified target is then concentrated to 200 nl by recapture onto a poly(dT) nitrocellulose filter and rapidly detected with streptavidin-alkaline phosphatase using bromochloroindolyl phosphate and nitroblue tetrazolium. Using this procedure, as little as 0.25 amol of a target plasmid has been detected nonradioactively in crude samples in just 1 h without prior purification of the DNA and RNA. Finally, a new procedure called background capture is introduced to complement the background-reducing power of RTC.

Nucleic acid hybridization assays employing dA-tailed capture probes: I. Multiple capture methods

A quantitative hybridization assay termed “reversible target capture” is described. The technique is designed to extensively purify the target nucleic acid from crude cell lysates in about 1 h without phenol extraction. Simple, rapid methods are described that explain how each process in the assay is optimized. The procedure involves hybridizing the target nucleic acid in solution with a dA-tailed capture probe and a labeled probe. The capture probe-target-labeled probe “ternary complex” is then captured on magnetic beads containing oligo(dT). After the excess unhybridized labeled probe, cell debris, and other sample impurities are washed away, the intact ternary complex is further purified by chemical elution from the beads and recapture on fresh beads. The ternary complex is then eluted thermally and recaptured on a third set of beads or on poly(dT) filters. This triple capture method results in a detection limit of approximately 0.2 amol (100 fg) of target with 32P-labeled riboprobes. This is approximately 1000 times more sensitive than sandwich assays employing only a single capture step. The method is illustrated by detecting Listeria cells in the presence of heterologous bacteria. With three rounds of target capture, as few as six Listeria cells have been detected in the presence of 1.25 × 10 7 control cells.

Tissue-specific expression, developmental regulation, and genetic mapping of the gene encoding CCAAT/enhancer binding protein.

This paper presents the results of experiments that determine the chromosomal location of the mouse gene encoding CCAAT/enhancer binding protein (C/EBP) and measure its expression as a function of tissue type and temporal period of development in mice and rats. Three alleles of the C/EBP gene were identified according to restriction fragment length polymorphisms. The strain distribution pattern of the three alleles was determined in recombinant inbred mouse strains and compared to that of other mouse genes. These results mapped the gene to a position within 2.5 centimorgans (cM) of the structural gene encoding glucose phosphate isomerase on chromosome 7 of the mouse. The expression pattern of the C/EBP gene was studied by a combination of nucleic acid hybridization and antibody staining assays. High levels of C/EBP mRNA were observed in tissues known to metabolize lipid and cholesterol-related compounds at uncommonly high rates. These included liver, fat, intestine, lung, adrenal gland, and placenta. More detailed analysis of two of these tissues, liver and fat, showed that C/EBP expression was limited to fully differentiated cells. Moreover, analysis of the temporal pattern of expression of C/EBP mRNA in two tissues, liver and intestine, revealed a coordinated induction just prior to birth. These observations raise the possibility that the synthesis of C/EBP may be responsive to humoral factors and that modulation in C/EBP expression might mediate coordinated changes in gene expression that facilitate adaptive challenges met during development or during the fluctuating physiological states of adult life.

A new colorimetric nucleic acid hybridization assay for Listeria in foods

Non-isotopic colorimetric detection has been applied in a rapid nucleic acid dipstick hybridization assay for detection of Listeria spp. in food and environmental samples. The assay takes approximately 2.5–3 h following a two day broth and plate enrichment. Hybridization occurs between fluorescein labeled detector probes, poly(deoxyadenosine)-tailed capture probes, and Listeria-specific regions of 16 S ribosomal RNA. These target: probe complexes are captured on poly(deoxythymidine) coated plastic dipsticks. Detection is based on binding of horseradish peroxidase conjugated anti-fluorescein antibody to the hybridization complex, and enzyme-mediated color development. The colorimetric endpoint is read on a photometer at 450 nm. In these initial studies, 306 inoculated dairy, meat and seafood samples, and 200 environmental samples were tested. When compared with total culture results the hybridization assay had unconfirmed positive and false-negative rates of approximately 1.4–2.9% and 0.8–4.7%, respectively.

Nucleic acid hybridization assays employing dA-tailed capture probes. Single capture methods.

Several novel hybridization techniques are described. Cells or specimens are treated to release nucleic acids and a liquid phase hybridization is carried out with a dA-tailed capture probe and a reporter probe in chaotropic salts or in salts containing SDS/proteinase K. In another format the tailed capture probe is preimmobilized on polystyrene and used to capture target nucleic acids from the solution. No phenol extraction or centrifugation is required to prepare the nucleic acids. Capture of the target on the poly (dT)-solid supports is used to remove excess labelled probe and sample impurities prior to non-radioisotopic or radioisotopic detection. This paper shows the advantage of a single round of capture on polystyrene, including the ability to assay large numbers of samples manually, the ability to analyse each sample for many analytes simultaneously, the use of rapid non-radioisotopic detection, and the ability to readily adapt the assay for automation.

Nucleic acid amplification in vitro: detection of sequences with low copy numbers and application to diagnosis of human immunodeficiency virus type 1 infection.

The enzymatic amplification of specific nucleic acid sequences in vitro has revolutionized the use of nucleic acid hybridization assays for viral detection. With this method, the copy number of a pathogen-specific sequence is increased several orders of magnitude before detection is attempted. The sensitivity and specificity of detection are thus markedly improved. Mullis and Faloona devised the first method of sequence amplification in vitro, the polymerase chain reaction (K.B. Mullis and F.A. Faloona, Methods Enzymol. 155:355-350, 1987). By this method, synthetic oligonucleotide primers direct repeated, target-specific, deoxyribonucleic acid-synthetic reactions, resulting in an exponential increase in the amount of the specific target sequence. The application of sequence amplification to viral detection was initially performed with human immunodeficiency virus type 1 and human T-cell lymphoma virus type I. In principle, however, this approach can be applied to the detection of any deoxyribonucleic or ribonucleic acid virus; the only requirement is that sufficient nucleotide sequence data exist to allow the synthesis of target-specific oligonucleotide primers. The use of target amplification in vitro will permit a variety of studies of viral pathogenesis which have not been feasible because of the low copy number of the viral nucleic acids in infected material. This approach is particularly applicable to the study of human retroviral infections, which are chronic and persistent and are characterized by low titers of virus in tissues. In addition, target amplification in vitro will facilitate the development of new methods of sequence detection, which will be useful for rapid viral diagnosis in the clinical laboratory.

Experimental vesicular stomatitis virus infection of swine: Extent of infection and immunological response

Swine, a natural host species for infection by vesicular stomatitis virus (VSV), were infected with VSV-New Jersey (VSV-NJ) serotype virus obtained from a recent field isolate. Tissues collected from the infected pigs were examined for the presence of infective virus, for viral antigens, and/or for viral nucleic acid. Infective virus could be recovered from tissues near the site of infection for as long as 6 days after the primary infection with VSV. However, no infective virus was recovered following hypothermia induced 11 weeks after infection, or following a secondary challenge with virus 22 weeks after initial infection. Immunofluorescence tests for viral antigens and nucleic acid hybridization assays failed to detect viral antigens or nucleic acids in tissues from which no infective virus could be recovered. Titers of serum-neutralizing antibody peaked 3–5 weeks after infection and then fell slightly until the secondary infection which caused a rapid anamnestic response. Peripheral blood mononuclear cells (PBM) tested 3, 5, 8 or 18 weeks after primary infection all produced readily detectable antigen-specific proliferative responses when cultured with VSV. Thus, although direct tests failed to demonstrate persistence of virus after infection, the humoral and cellular immune response remained elevated for months. Infective VSV was not required to stimulate the proliferative response since UV-inactivated VSV was immunogenic in these in vitro tests. Following primary infection, antigen-specific proliferative responses could be stimulated by several strains of VSV-NJ, but not by VSV-Indiana (VSV-Ind) serotype virus. Secondary infection had relatively little effect on the proliferative response to VSV-NJ strains, but it did cause the PBM to gain responsiveness to VSV-Ind.